Treatment of pancreatic cancer with irreversible electroporation and intratumoral CD40 antibody stimulates systemic immune responses that inhibit liver metastasis in an orthotopic model.

BACKGROUND: Pancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically in locally advanced PC, but most patients eventually develop distant recurrence. We have previously shown that IRE alone is capable of generating protective, neoantigen-specific immunity. Here, we aim to generate meaningful therapeutic immune effects by combining IRE with local (intratumoral) delivery of a CD40 agonistic antibody (CD40Ab). METHODS: KPC46 organoids were generated from a tumor-bearing male KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) mouse. Orthotopic tumors were established in the pancreatic tail of B6/129 F1J mice via laparotomy. Mice were randomized to treatment with either sham laparotomy, IRE alone, CD40Ab alone, or IRE followed immediately by CD40Ab injection. Metastatic disease and immune infiltration in the liver were analyzed 14 days postprocedure using flow cytometry and multiplex immunofluorescence imaging with spatial analysis. Candidate neoantigens were identified by mutanome profiling of tumor tissue for ex vivo functional analyses. RESULTS: The combination of IRE+CD40 Ab improved median survival to greater than 35 days, significantly longer than IRE (21 days) or CD40Ab (24 days) alone (p<0.01). CD40Ab decreased metastatic disease burden, with less disease in the combination group than in the sham group or IRE alone. Immunohistochemistry of liver metastases revealed a more than twofold higher infiltration of CD8+T cells in the IRE+CD40 Ab group than in any other group (p<0.01). Multiplex immunofluorescence imaging revealed a 4-6 fold increase in the density of CD80+CD11c+ activated dendritic cells (p<0.05), which were spatially distributed throughout the tumor unlike the sham group, where they were restricted to the periphery. In contrast, CD4+FoxP3+ T-regulatory cells (p<0.05) and Ly6G+myeloid derived cells (p<0.01) were reduced and restricted to the tumor periphery in the IRE+CD40 Ab group. T-cells from the IRE+CD40 Ab group recognized significantly more peptides representing candidate neoantigens than did T-cells from the IRE or untreated control groups. CONCLUSIONS: IRE can induce local tumor regression and neoantigen-specific immune responses. Addition of CD40Ab to IRE improved dendritic cell activation and neoantigen recognition, while generating a strong systemic antitumor T-cell response that inhibited metastatic disease progression.

Factors related to white thrombi in acute ischemic stroke in cancer patients.

OBJECTIVES: Thrombi in cerebral large vessel occlusion associated with active cancer are often fibrin and platelet-rich white thrombi. However, evaluating the thrombus composition in a short time before thrombectomy is often ineffective. We sought to determine factors related to white thrombi in acute ischemic stroke due to large vessel occlusion in cancer patients. METHODS: Consecutive cancer patients undergoing thrombectomy for acute ischemic stroke due to large vessel occlusion between January 2018 and May 2022 were retrospectively reviewed. The patients were classified into white thrombus and red thrombus groups on the basis of the pathological findings of retrieved thrombi. Patient characteristics and laboratory findings were compared between the two groups. RESULTS: There were 12 patients in the white thrombus group and 11 patients in the red thrombus group. Active cancer was significantly more in the white thrombus group than in the red thrombus group (91.7% vs. 36.3%, p = 0.0094). Internal carotid artery occlusion was significantly less in the white thrombus group than in the red thrombus group (0% vs. 36.4%, p = 0.037). Among laboratory findings, D-dimer levels were an independent factor associated with white thrombi (odds ratio 8.97 [95% confidence interval 1.71-368.99], p < 0.0001). The cutoff value of D-dimer levels for predicting white thrombi was 3.5 µg/mL (83.3% sensitivity and 100% specificity). CONCLUSIONS: In acute ischemic stroke in cancer patients, active cancer, no internal carotid artery occlusion, and higher D-dimer levels (≥3.5 µg/mL) may be associated with occlusion with fibrin and platelet-rich white thrombi.

Moyamoya Syndrome in a Patient with Williams Syndrome: A Case Report.

INTRODUCTION: Moyamoya syndrome associated with Williams syndrome is very rare but has been reported to have severe outcomes. Here, we reported a case of Williams syndrome with moyamoya syndrome that was confirmed by the presence of an RNF213 mutation. CASE PRESENTATION: A 6-year-old boy with Williams syndrome presented with right hemiparesis induced by hyperventilation. Magnetic resonance angiography and cerebral angiography showed severe stenosis of the bilateral internal carotid arteries and development of moyamoya vessels. Genetic analysis identified a heterozygous c.14576G>A (p.R4859K) mutation in RNF213. Moyamoya syndrome was diagnosed, and bilateral indirect revascularization surgery was conducted without complications and with a good postoperative course. In moyamoya syndrome associated with Williams syndrome, adequate perioperative management of both the moyamoya arteries and the cardiovascular abnormalities is important to prevent complications. CONCLUSION: This was the first report on a case in which moyamoya syndrome associated with Williams syndrome was confirmed by the presence of a heterozygous RNF213 mutation. Similar to the workup of moyamoya disease, confirmation of RNF213 mutation in Williams syndrome may be useful in predicting the development of moyamoya syndrome that can lead to severe complications.

Glyco-Nanoadjuvants: Impact of Linker Length for Conjugating a Synthetic Small-Molecule TLR7 Ligand to Glyco-Nanoparticles on Immunostimulatory Effects.

Immunotherapy has become a powerful clinical strategy for treating infectious diseases and cancer. Synthetic small-molecule toll-like receptor 7 (TLR7) ligands are attractive candidates as immunostimulatory agents for immunotherapy. TLR7 is mainly localized in intracellular endosomal compartments so that the formulation of their small-molecule ligands with macromolecules enhances endocytic uptake of TLR7 ligands and improves the pharmaceutical properties. Previously, we demonstrated that gold nanoparticles co-immobilized with a TLR7 ligand derivative, that is, a conjugate of synthetic small-molecule TLR7 ligand (1V209) and thioctic acid (TA) via 4,7,10-trioxa-1,13-tridecanediamine, and α-mannose (1V209-αMan-GNPs: glyco-nanoadjuvants) significantly enhances immunostimulatory effects. In the present study, we designed a second-generation glyco-nanoadjuvant that possesses a poly(ethylene glycol) (PEG) chain as a spacer between 1V209 and GNPs and investigated the impact of linker length in 1V209 derivatives on the immunostimulatory activities. We used different chain lengths of PEG (n = 3, 5, 11, or 23) as spacers between 1V209 and thioctic acid to prepare four 1V209-αMan-GNPs. In the in vitro study using primary mouse bone-marrow-derived dendritic cells, 1V209-αMan-GNPs that immobilized with longer 1V209 derivatives, especially the 1V209 derivative possessing PEG23 (1V209-PEG23-TA), showed the highest potency toward induction both for interleukin-6 and type I interferon production than those derivatives with shorter PEG chains. Furthermore, 1V209-αMan-GNPs that immobilized with 1V209-PEG23-TA showed significantly higher adjuvant effects for inducing both humoral and cell-mediated immune responses against ovalbumin in the in vivo immunization study. These results indicate that the linker length for immobilizing small-molecule TLR7 ligand on the GNPs significantly affects the adjuvant activity of 1V209-αMan-GNPs and that 1V209-αMan-GNPs immobilized with 1V209-PEG-23-TA could be superior adjuvants for immunotherapies.

Neuroendoscopic fenestration for intracranial unilocular cysts and isolated lateral ventricles: four pediatric cases.

The purpose of treatment for unilocular intracranial cysts (UICs) is to release elevated intracranial pressure. Neuroendoscopic fenestration (NF) is one of the most effective and minimally invasive options for treating UICs, especially in young children; however, the optimal location and number of fenestrations, the necessity of using endoscopic third ventriculostomy (ETV) in combination with fenestration, and the course of treatment are not well known. We retrospectively reviewed the hospital records between 2012 and 2019. The patients were studied in terms of sex, age at surgery, preoperative symptoms, cyst localization and size, course of treatment, ventricular diameter, developmental assessment, anatomical location, and the number of fenestrations. There were four eligible patients in the relevant period: two boys and two girls. The median age at the time of surgery was 16 months. With regard to the location of the cysts, there were two cases of cavum velum interpositum (CVI), one case of quadrigeminal cistern, and one case of an isolated lateral ventricle. The most common preoperative finding was an enlarged head circumference. All the patients were treated with NF, including one case of reoperation after open head surgery. Postoperatively, we used the frontal and occipital horn ratio (FOHR) to evaluate the ventricular size. The average reduction in the FOHR was 0.003. In the most recent developmental assessment or examination during the follow-up period, two patients showed normal development, and two patients showed developmental delay. Based on our past experience and reports, we believe that it is recommended to perform two fenestrations for a single cyst. This is because it creates a flow of cerebrospinal fluid (CSF) within the cyst into normal CSF reflux. For lesions with obstruction of the aqueduct, such as cysts in the quadrigeminal cistern, ETV should be considered if it can be performed safely, in preparation for the worsening of hydrocephalus due to obstruction by enlargement of the cyst.

Gradual dilatation of an occluded transverse sinus associated with dural arteriovenous fistula after balloon angioplasty with sinus packing: A case report.

BACKGROUND: There is no consensus as to whether balloon angioplasty alone or stent placement is effective for sinus occlusion associated with dural arteriovenous fistula (DAVF). Herein, we first report a case of transverse sinus occlusion associated with DAVF in which gradual sinus dilatation was observed after balloon angioplasty with embolization of the affected sinus with shunt flow. CASE PRESENTATION: A 69-year-old man presented with executive dysfunction. Magnetic resonance imaging revealed left transverse sinus-sigmoid sinus DAVF with occlusion of the left jugular vein and right transverse sinus. Before endovascular treatment, the patient had symptomatic epilepsy and subarachnoid hemorrhage. Retrograde leptomeningeal venous drainage disappeared with packing of the left transverse sinus-sigmoid sinus. Subsequently, balloon angioplasty of the right occluded transverse sinus was performed to maintain the normal venous drainage and remaining shunt outflow. Dilatation of the right transverse sinus was poor immediately after surgery. However, angiography after 10 days and 6 months revealed gradual dilatation of the right transverse sinus. CONCLUSION: Sinus occlusion, which is thought to be caused by sinus hypertension associated with DAVF rather than chronic organized thrombosis or thrombophilia, may dilate over time after balloon angioplasty and shunt flow reduction if occluded sinus is necessary for facilitating normal venous drainage.

Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z' factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.

Immunostimulatory TLR7 agonist-nanoparticles together with checkpoint blockade for effective cancer immunotherapy.

Mono- or dual-checkpoint inhibitors for immunotherapy have changed the paradigm of cancer care; however, only a minority of patients responds to such treatment. Combining small molecule immuno-stimulators can improve treatment efficacy, but they are restricted by poor pharmacokinetics. In this study, TLR7 agonists conjugated onto silica nanoparticles showed extended drug localization after intratumoral injection. The nanoparticle-based TLR7 agonist increased immune stimulation by activating the TLR7 signaling pathway. When treating CT26 colon cancer, nanoparticle conjugated TLR7 agonists increased T cell infiltration into the tumors by > 4× and upregulated expression of the interferon γ gene compared to its unconjugated counterpart by ~2×. Toxicity assays established that the conjugated TLR7 agonist is a safe agent at the effective dose. When combined with checkpoint inhibitors that target programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a 10-100× increase in immune cell migration was observed; furthermore, 100 mm3 tumors were treated and a 60% remission rate was observed including remission at contralateral non-injected tumors. The data show that nanoparticle based TLR7 agonists are safe and can potentiate the effectiveness of checkpoint inhibitors in immunotherapy resistant tumor models and promote a long-term specific memory immune function.

Synthesis and immunostimulatory activity of sugar-conjugated TLR7 ligands.

Toll-like receptors (TLRs) are a type of pattern recognition receptors (PRRs), which are activated by recognizing pathogen-associated molecular patterns (PAMPs). The activation of TLRs initiates innate immune responses and subsequently leads to adaptive immune responses. TLR agonists are effective immuomodulators in vaccine adjuvants for infectious diseases and cancer immunotherapy. In exploring hydrophilic small molecules of TLR7 ligands using the cell-targeted property of a vaccine adjuvant, we conjugated 1V209, a small TLR7 ligand molecule, with various low or middle molecular weight sugar molecules that work as carriers. The sugar-conjugated 1V209 derivatives showed increased water solubility and higher immunostimulatory activity in both mouse and human cells compared to unmodified 1V209. The improved immunostimulatory potency of sugar-conjugates was attenuated by an inhibitor of endocytic process, cytochalasin D, suggesting that conjugation of sugar moieties may enhance the uptake of TLR7 ligand into the endosomal compartment. Collectively our results support that sugar-conjugated TLR7 ligands are applicable to novel drugs for cancer and vaccine therapy.

Delivery of Immunotherapeutic Nanoparticles to Tumors via Enzyme-Directed Assembly.

Amphiphilic diblock copolymers are prepared by ring opening metathesis polymerization, with one block containing hydrophobic Toll-like receptor 7 (TLR7) agonists and one block containing hydrophilic peptides as substrates for matrix metalloproteinases (MMPs). A fluorescent label is incorporated into the polymer chains for in vivo imaging. Upon dialysis against aqueous solution, polymers form 15 nm spherical micelles. Subsequent exposure to MMP-9 elicits a morphological change to yield immunostimulatory microscale assemblies. The intravenous (IV) administration of the formulation to mice bearing 4T1 breast cancer tumors results in nanoparticle accumulation in tumors, reduction in primary tumor growth, and inhibition of lung metastases, as compared to saline-treated animals. Mice administered the parent immunotherapeutic small molecule (1V209) experience significantly increased plasma levels of proinflammatory cytokines IL-6, IP-10, and MCP-1 at 2 h following IV administration, whereas the nanomaterial shows no increase over saline-treated controls. These data suggest that covalently packaging low molecular weight immunotherapeutics at high weight percent loadings in enzyme-responsive nanoparticles maintains drug efficacy while decreasing immunotoxicity, providing a platform for cancer immunotherapeutic delivery.

Gold Nanoparticles Coimmobilized with Small Molecule Toll-Like Receptor 7 Ligand and α-Mannose as Adjuvants.

Adjuvants enhance the immune response during vaccination. Among FDA-approved adjuvants, aluminum salts are most commonly used in vaccines. Although aluminum salts enhance humoral immunity, they show a limited effect for cell-mediated immune responses. Thus, further development of adjuvants that induce T-cell-mediated immune response is needed. Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns activate innate immunity, which is crucial to shape adaptive immunity. Using TLR ligands as novel adjuvants in vaccines has therefore attracted substantial attention. Among them a small molecule TLR7 ligand, imiquimod, has been approved for clinical use, but its use is restricted to local administration due to unwanted adverse side effects when used systematically. Since TLR7 is mainly located in the endosomal compartment of immune cells, efficient transport of the ligand into the cells is important for improving the potency of the TLR7 ligand. In this study we examined gold nanoparticles (GNPs) immobilized with α-mannose as carriers for a TLR7 ligand to target immune cells. The small molecule synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine (1V209), and α-mannose were coimmobilized via linker molecules consisting of thioctic acid on the GNP surface (1V209-αMan-GNPs). The in vitro cytokine production activity of 1V209-αMan-GNPs was higher than that of the unconjugated 1V209 derivative in mouse bone marrow-derived dendritic cells and in human peripheral blood mononuclear cells. In the in vivo immunization study, 1V209-αMan-GNPs induced significantly higher titers of IgG2c antibody specific to ovalbumin as an antigen than did unconjugated 1V209, and splenomegaly and weight loss were not observed. These results indicate that 1V209-αMan-GNPs could be useful as safe and effective adjuvants for development of vaccines against infectious diseases and cancer.

Irreversible Electroporation Combined with Checkpoint Blockade and TLR7 Stimulation Induces Antitumor Immunity in a Murine Pancreatic Cancer Model.

Irreversible electroporation (IRE) is a nonthermal ablation technique that is used clinically in selected patients with locally advanced pancreatic cancer, but most patients develop recurrent distant metastatic disease. We hypothesize that IRE can induce an in situ vaccination effect by releasing tumor neoantigens in an inflammatory context. Using an immunocompetent mouse model, we demonstrated that IRE alone produced complete regression of subcutaneous tumors in approximately 20% to 30% of mice. IRE was not effective in immunodeficient mice. Mice with complete response to IRE demonstrated prophylactic immunity and remained tumor free when rechallenged with secondary tumors on the contralateral flank. CD8+ T cells from IRE-responsive mice were reactive against peptides representing model-inherent alloantigens and conferred protection against tumor challenge when adoptively transferred into immunocompromised, tumor-naïve mice. Combining IRE with intratumoral Toll-like receptor-7 (TLR7) agonist (1V270) and systemic anti-programmed death-1 receptor (PD)-1 checkpoint blockade resulted in improved treatment responses. This combination also resulted in elimination of untreated concomitant distant tumors (abscopal effects), an effect not seen with IRE alone. These results suggest that the systemic antitumor immune response triggered by IRE can be enhanced by stimulating the innate immune system with a TLR7 agonist and the adaptive immune system with anti-PD-1 checkpoint blockade simultaneously. Combinatorial approaches such as this may help overcome the immunosuppressive pancreatic cancer microenvironment.

Conjugation of a Small-Molecule TLR7 Agonist to Silica Nanoshells Enhances Adjuvant Activity.

Stimulation of Toll-like receptors (TLRs) and/or NOD-like receptors on immune cells initiates and directs immune responses that are essential for vaccine adjuvants. The small-molecule TLR7 agonist, imiquimod, has been approved by the FDA as an immune response modifier but is limited to topical application due to its poor pharmacokinetics that causes undesired adverse effects. Nanoparticles are increasingly used with innate immune stimulators to mitigate side effects and enhance adjuvant efficacy. In this study, a potent small-molecule TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), was conjugated to hollow silica nanoshells (NS). Proinflammatory cytokine (IL-6, IL-12) release by mouse bone-marrow-derived dendritic cells and human peripheral blood mononuclear cells revealed that the potency of silica nanoshells-TLR7 conjugates (NS-TLR) depends on nanoshell size and ligand coating density. Silica nanoshells of 100 nm diameter coated with a minimum of ∼6000 1V209 ligands/particle displayed 3-fold higher potency with no observed cytotoxicity when compared to an unconjugated TLR7 agonist. NS-TLR activated the TLR7-signaling pathway, triggered caspase activity, and stimulated IL-1ß release, while neither unconjugated TLR7 ligands nor silica shells alone produced IL-1ß. An in vivo murine immunization study, using the model antigen ovalbumin, demonstrated that NS-TLR increased antigen-specific IgG antibody induction by 1000× with a Th1-biased immune response, compared to unconjugated TLR7 agonists. The results show that the TLR7 ligand conjugated to silica nanoshells is capable of activating an inflammasome pathway to enhance both innate immune-stimulatory and adjuvant potencies of the TLR7 agonist, thereby broadening applications of innate immune stimulators.

Discovery of a Novel Microtubule Targeting Agent as an Adjuvant for Cancer Immunotherapy.

For an activating immunotherapy such as adjuvants, a compound that can prolong immune stimulation may enhance efficacy. We leveraged data from two prior high throughput screens with NF-κB and interferon reporter cell lines to identify 4H-chromene-3-carbonitriles as a class of compounds that prolonged activation in both screens. We repurchased 23 of the most promising candidates. Out of these compounds we found #1 to be the most effective agent in stimulating the release of cytokines and chemokines from immune cells, including murine primary bone marrow derived dendritic cells. Mechanistically, #1 inhibited tubulin polymerization, and its effect on immune cell activation was abolished in cells mutated in the beta-tubulin gene (TUBB) encoding the site where colchicine binds. Treatment with #1 resulted in mitochondrial depolarization followed by mitogen-activated protein kinase activation. Because tubulin polymerization modulating agents have been used for chemotherapy to treat malignancy and #1 activated cytokine responses, we hypothesized that #1 could be effective for cancer immunotherapy. Intratumoral injection of #1 delayed tumor growth in a murine syngeneic model of head and neck cancer. When combined with PD-1 blockade, tumor growth slowed in the injected tumor nodule and there was an abscopal effect in an uninjected nodule on the contralateral flank, suggesting central antitumor immune activation. Thus, we identified a new class of tubulin depolymerizing agent that acts as both an innate and an adaptive immune activating agent and that limits solid tumor growth when used concurrently with a checkpoint inhibitor.

Induction of oligoclonal CD8 T cell responses against pulmonary metastatic cancer by a phospholipid-conjugated TLR7 agonist.

Recent advances in cancer immunotherapy have improved patient survival. However, only a minority of patients with pulmonary metastatic disease respond to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T1 breast cancer models, as well as in B16 melanoma, and Lewis lung carcinoma models. In the 4T1 breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist compared with the unconjugated agonist. 1V270 exhibited a wide therapeutic window and minimal off-target receptor binding. The 1V270 therapy inhibited colonization by tumor cells in the lungs in an NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-specific CD8+ cell-dependent adaptive immune responses that suppressed late-stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analyses showed that 1V270 therapy induced oligoclonal T cells in the lungs and mediastinal lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy. Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor-specific adaptive immune responses. These results indicate that systemic 1V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers and that TCR repertoire analyses can be used to monitor, and to predict, the response to therapy.

Recent Advances and Perspectives in Small-molecule TLR Ligands and Their Modulators.

Activation of Toll-like receptors (TLRs) located on immune cells leads to induction of immune responses that can be useful in vaccines for infectious diseases, cancer immunotherapy, and autoimmune diseases. Novel TLR signaling pathway modulators can further enhance the efficacy of TLR ligands.

Combination immunotherapy with TLR agonists and checkpoint inhibitors suppresses head and neck cancer.

Checkpoint inhibitors have demonstrated efficacy in patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, the majority of patients do not benefit from these agents. To improve the efficacy of checkpoint inhibitors, intratumoral (i.t.) injection with innate immune activators, TLR7 and TLR9 agonists, were tested along with programmed death-1 receptor (PD-1) blockade. The combination therapy suppressed tumor growth at the primary injected and distant sites in human papillomavirus-negative (HPV-negative) SCC7 and MOC1, and HPV-positive MEER syngeneic mouse models. Abscopal effects and suppression of secondary challenged tumor suggest that local treatment with TLR agonists in combination with anti-PD-1 provided systemic adaptive immunity. I.t. treatment with a TLR7 agonist increased the ratio of M1 to M2 tumor-associated macrophages (TAMs) and promoted the infiltration of tumor-specific IFNγ-producing CD8+ T cells. Anti-PD-1 treatment increased T cell receptor (TCR) clonality of CD8+ T cells in tumors and spleens of treated mice. Collectively, these experiments demonstrate that combination therapy with i.t. delivery of TLR agonists and PD-1 blockade activates TAMs and induces tumor-specific adaptive immune responses, leading to suppression of primary tumor growth and prevention of metastasis in HNSCC models.

The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity.

Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.

Enhancement of the Immunostimulatory Activity of a TLR7 Ligand by Conjugation to Polysaccharides.

Toll-like receptors (TLRs) in the innate immune system recognize specific pathogen-associated molecular patterns derived from microbes. Synthetic small molecule TLR7 agonists have been extensively evaluated as topical agents for antiviral and anticancer therapy, and as adjuvants for vaccine. However, safe and reproducible administration of synthetic TLR7 ligands has been difficult to achieve due to undesirable pharmacokinetics and unacceptable side effects. Here, we conjugated a versatile low molecular weight TLR7 ligand to various polysaccharides in order to improve its water solubility, enhance its potency, and maintain low toxicity. The synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine, designated 1V209, was stably conjugated to primary amine functionalized Ficoll or dextran using benzoic acid functional groups. The conjugation ratios using specified equivalents of TLR7 ligand were dose responsive and reproducible. The zeta potential value of the polysaccharides was decreased in inverse proportion to the ratio of conjugated TLR7 ligand. These conjugates were highly water-soluble, stable for at least 6 months at room temperature in aqueous solution, and easy to lyophilize and reconstitute without altering potency. In vitro studies with murine mononuclear leukocytes showed that the TLR7 agonist conjugated to polysaccharides had 10- to 1000-fold higher potencies than the unconjugated TLR7 ligand. In vivo pharmacodynamics studies after injection indicate that the conjugates induced systemic cytokine production. When the conjugates were used as vaccine adjuvants, they enhanced antigen specific humoral and cellular immune responses to a much greater extent than did unconjugated TLR7 ligands. These results indicated that small molecule TLR7 ligands conjugated to polysaccharides have improved immunostimulatory potency and pharmacodynamics. Polysaccharides can be conjugated to a variety of molecules such as antigens, peptides, and TLR ligands. Therefore, such conjugates could represent a versatile platform for the development of vaccines against cancer and infectious diseases.

[Development of ICP-OES, ICP-MS and GF-AAS Methods for Simultaneous Quantification of Lead, Total Arsenic and Cadmium in Soft Drinks].

In this study, we developed methods to quantify lead, total arsenic and cadmium contained in various kinds of soft drinks, and we evaluated their performance. The samples were digested by common methods to prepare solutions for measurement by ICP-OES, ICP-MS and graphite furnace atomic absorption spectrometry (GF-AAS). After digestion, internal standard was added to the digestion solutions for measurements by ICP-OES and ICP-MS. For measurement by GF-AAS, additional purification of the digestion solution was conducted by back-extraction of the three metals into nitric acid solution after extraction into an organic solvent with ammonium pyrrolidine dithiocarbamate. Performance of the developed methods were evaluated for eight kinds of soft drinks.