TLR7 agonist, DSP-0509, with radiation combination therapy enhances anti-tumor activity and modulates T cell dependent immune activation.

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.

In situ chemoimmunotherapy hydrogel elicits immunogenic cell death and evokes efficient antitumor immune response.

BACKGROUND: Chemoimmunotherapy has shown promising advantages of eliciting immunogenic cell death and activating anti-tumor immune responses. However, the systemic toxicity of chemotherapy and tumor immunosuppressive microenvironment limit the clinical application. METHODS: Here, an injectable sodium alginate hydrogel (ALG) loaded with nanoparticle albumin-bound-paclitaxel (Nab-PTX) and an immunostimulating agent R837 was developed for local administration. Two murine hepatocellular carcinoma and breast cancer models were established. The tumor-bearing mice received the peritumoral injection of R837/Nab-PTX/ALG once a week for two weeks. The antitumor efficacy, the immune response, and the tumor microenvironment were investigated. RESULTS: This chemoimmunotherapy hydrogel with sustained-release character was proven to have significant effects on killing tumor cells and inhibiting tumor growth. Peritumoral injection of our hydrogel caused little harm to normal organs and triggered a potent antitumor immune response against both hepatocellular carcinoma and breast cancer. In the tumor microenvironment, enhanced immunogenic cell death induced by the combination of Nab-PTX and R837 resulted in 3.30-fold infiltration of effector memory T cells and upregulation of 20 biological processes related to immune responses. CONCLUSIONS: Our strategy provides a novel insight into the combination of chemotherapy and immunotherapy and has the potential for clinical translation.

Supported liquid extraction combined with liquid chromatography tandem mass spectrometry for the quantitative analysis of a TLR7 agonist imiquimod LFX453 in plasma at low picograms per milliliter: Method validation and its application to a pharmacokinetic study in minipig.

Sample preparation is essential for low-level compound determination. In the present work, supported liquid extraction (SLE) was used as sample preparation for the low-level determination of a new TLR7 agonist imiquimod compound, LFX453. Samples were extracted on ISOLUTE® SLE 96-well plates using tert-butyl-methyl ether followed by evaporation and dry residue reconstitution with 150 µl of a mixture of 0.1% formic acid in acetonitrile-water (50/50, v/v). Samples were eluted using a flow rate of 0.750 ml/min on a C18 column (50 × 2.1 mm, 2.7 µm) with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Tandem mass spectrometry was used to analyze the samples in positive mode. The method run time was 6.5 min, and the low limit of quantification was 1.00 pg/ml with 0.100 ml of minipig plasma. Intra-run and inter-run precision and accuracy were within the acceptance criteria at four concentration levels over a concentration ranging from 1.00 to 200 pg/ml. There was no matrix effect and recovery, three freeze-thaw cycles and incurred samples reanalysis were validated. The method was successfully applied for measuring LFX453 in minipig plasma after application on minipig skin.

A translational strategy employing physiologically based modelling to predict the pharmacological active dose of RO7119929, an oral prodrug of a targeted cancer immunotherapy TLR7 agonist.

RO7119929 is being developed as an orally administered prodrug of the TLR7-specific agonist and active drug, RO7117418, for the treatment of patients with solid tumours.In this publication, we present a case study wherein the human pharmacokinetics and pharmacological active dose were prospectively predicted following oral administration of the prodrug.A simple translational pharmacokinetic-pharmacodynamic strategy was applied to predict the pharmacological active dose of the prodrug in human. In vivo studies in monkey showed that an unbound plasma exposure of active drug of 1.5 ng/mL elicited secretion of key serum pharmacodynamic cytokine and chemokine biomarkers in monkey. This threshold of 1.5 ng/mL was close to the minimum effective concentration of active drug required to induce cytokine secretion in human peripheral blood mononuclear cells (3 ng/mL).Measured in vitro physicochemical and biochemical properties of the prodrug and active drug were applied as input parameters in physiologically based pharmacokinetic models to predict the pharmacokinetics of active drug after oral dosing of the prodrug in humans. Then, using the PBPK model, a dose which delivered an unbound plasma Cmax in line with the target pharmacodynamic threshold of 1.5 ng/mL was found. This defined the lowest pharmacologically active dose as 3 mg.The prodrug entered the clinic in 2020 in patients with primary or secondary liver cancers. Clear pharmacodynamic, transient, and dose-dependent cytokine induction was observed at prodrug doses > 1 mg.

Photothermal immunotherapy of melanoma using TLR-7 agonist laden tobacco mosaic virus with polydopamine coat.

Photothermal therapy (PTT) is a promising cancer treatment that debulks tumors locally while priming immune responses. However, PTT as a standalone treatment approach often has limited systemic efficacy, motivating the development of synergistic combination approaches. Toward this goal, herein, the tobacco mosaic virus (TMV) was loaded with a small molecule immunomodulator, toll-like receptor 7 agonist (1V209), and its surface was coated with photothermal biopolymer polydopamine (PDA). The resulting 1V209-laden and PDA-coated TMV was used to treat B16F10 dermal melanoma in C57BL/6 mice. 1V209-TMV-PDA was intratumorally injected and irradiated using an 808-nm near infrared laser. 60 % of the mice receiving PTT with intratumoral 1V209-TMV-PDA + laser remained alive at the end point - in contrast to only 20 % survivors were observed in the control group. Immunological analysis indicates systemic anti-tumor immunity being induced by the combination therapy with a greater number of tumor-specific T cells (as determined by a splenocyte assay). This study highlights the potential of TMV versatility as a multifunctional nano-platform for combined PTT-immunotherapy.

Lymph-Node-Targeted Cholesterolized TLR7 Agonist Liposomes Provoke a Safe and Durable Antitumor Response.

Toll-like receptor (TLR) agonists as the potent stimulants of an innate immune system hold promises for applications in anticancer immunotherapy. However, most of them are limited in the clinical translation due to the uncontrolled systemic inflammatory response. In the current study, 1V209, a small molecule TLR7 agonist, was conjugated with cholesterol (1V209-Cho) and prepared into liposomes (1V209-Cho-Lip). 1V209-Cho-Lip exerted minimal toxic effects and enhanced the transportation ability in lymph nodes (LNs) compared with 1V209. 1V209-Cho-Lip treatment inhibited tumor progression in CT26 colorectal cancer, 4T1 breast cancer, and Pan02 pancreatic ductal cancer models through inducing effective DC activation and eliciting CD8+ T cell responses. Furthermore, 1V209-Cho-Lip induced tumor-specific memory immunity to inhibit cancer recurrence and metastasis. These results indicate that cholesterol conjugation with 1V209 is an effective approach to target lymph nodes and to reduce the adverse effects. This work provides a rational basis for the distribution optimization of TLR agonists for potential clinical use.

Vesatolimod, a Toll-like Receptor 7 Agonist, Induces Immune Activation in Virally Suppressed Adults Living With Human Immunodeficiency Virus-1.

BACKGROUND: Treatment with vesatolimod, an investigational, oral, toll-like receptor 7 (TLR7) agonist, leads to sustained viral remission in some non-human primates when combined with anti-envelope antibodies or therapeutic vaccines. We report results of a Phase Ib study evaluating safety, pharmacokinetics, and pharmacodynamics of vesatolimod in adults living with human immunodeficiency virus (HIV)-1. METHODS: In this double-blind, multicenter, placebo-controlled trial, participants on antiretroviral therapy with screening plasma HIV-1 RNA levels <50 copies/mL were randomized (6:2) to receive 6-10 doses of vesatolimod (1-12 mg) or matching placebo orally every other week in sequential dose-escalation cohorts. The primary study objectives included establishing the safety and virologic effects of vesatolimod (change from baseline in plasma HIV-1 RNA). Pharmacokinetics and pharmacodynamic/immunologic activity were assessed as secondary objectives. RESULTS: A total of 48 individuals were randomly assigned to vesatolimod (n = 36) or placebo (n = 12). Vesatolimod was generally well tolerated, with no study drug-related serious adverse events or adverse events leading to study drug discontinuation. There were no statistically significant changes from baseline in plasma HIV-1 RNA in the vesatolimod groups, compared to placebo.Vesatolimod plasma exposures increased dose proportionally; consistent responses in cytokines, interferon-stimulated gene expression, and lymphocyte activation were observed with increasing dose levels above 4 mg. Peak elevations 24 hours after receipt of a 6 mg dose were >3.9-fold higher for interferon gamma-induced protein 10 (IP-10), interleukin-1 receptor antagonist (IL-1RA), interferon-inducible T-cell alpha chemoattractant (ITAC) when compared to baseline values. CONCLUSIONS: Vesatolimod was well tolerated at doses ranging from 1 to 12 mg. Immune stimulation was observed at doses above 4 mg, providing rationale for future combination trials in people living with HIV. CLINICAL TRIALS REGISTRATION: NCT02858401.

A Double-blind, Randomized Phase 2 Controlled Trial of Intradermal Hepatitis B Vaccination With a Topical Toll-like Receptor 7 Agonist Imiquimod, in Patients on Dialysis.

BACKGROUND: Patients on dialysis are hyporesponsive to the hepatitis B virus vaccines (HBVv). We examined intradermal (ID) HBVv Sci-B-Vac, with topical Toll-like receptor 7 (TLR7) agonist imiquimod pretreatment in dialysis patients. METHODS: We enrolled and prospectively followed adult patients on dialysis between January 2016 and September 2018. Eligible patients were randomly allocated (1:1:1) into 1 treatment group, topical imiquimod cream followed by ID HBVv (IMQ + ID); and 2 control groups: topical aqueous cream (placebo) followed by ID HBVv (AQ + ID) or topical aqueous cream followed by intramuscular HBVv (AQ + IM). The primary endpoint was the seroprotection rate (hepatitis B surface antibody ≥10 mIU/mL) at 52 weeks. RESULTS: Ninety-four patients were enrolled, among which 57.4% were previous nonresponders. Seroprotection rate was significantly better at week 52 for the IMQ + ID group with 96.9% compared to 74.2% and 48.4% for AQ + ID and AQ + IM groups, respectively (P < .0001). The geometric mean concentration was significantly higher at week 52 for the IMQ + ID group: 1135 (95% confidence interval [CI], 579.4-2218.2) mIU/mL, compared to 86.9 (95% CI, 18.5-409.3) mIU/mL and 7.2 (2.0-26.5) mIU/mL for the AQ + ID and AQ + IM groups, respectively (P < .0001). IMQ + ID vaccination (odds ratio, 3.70 [95% CI, 1.16-11.81]; P = .027) was the only factor independently associated with higher 52-week seroprotection rate. Adverse reaction was infrequent. CONCLUSIONS: Pretreatment with topical imiquimod before ID HBVv Sci-B-Vac was safe with favorable seroprotection in dialysis patients. CLINICAL TRIALS REGISTRATION: NCT02621112.

Efficacy of TLR7 agonistic imidazoquinoline as immunochemotherapeutic agent against P. Berghei ANKA infected rodent host.

Malaria is a serious disease and is one of the most alarming public health issues. Plasmodium is being resistant to various antimalarials including Chloroquine (CQ) which was the first-line therapy for malaria treatment. WHO recommended several combination therapies but declining efficacy was reported to many of these therapies. Despite a great amount of research, efficient malaria vaccine still seems to be a distant dream. Immunochemotherapy could be an alternate strategy to deal with malaria. Based on the differential activity of various cytokines in the pathogenesis of and protection against malaria, the efficacy of highly active TLR7 agonistic imidazoquinoline (BBIQ) in combination with a suboptimal dose of CQ against P. berghei ANKA (PbA) in vivo was investigated. In mice treated with CQ alone, parasite appeared on Day 17 and all mice of this group died by Day 21. Whereas, mice treated with BBIQ along with CQ exhibited no appearance of parasite till Day 23. Frequencies of T cells (CD3+, CD4+and CD8+) and T regulatory cells (CD4+, CD25 +and FoxP3+) were found to be lower in brain of BBIQ + CQ treated mice as compared to BBIQ alone and CQ alone treated mice on Day 10. Inhibition of infiltration of inflammatory T cells and activation of T helper and T cytotoxic cells against the parasite was observed in the mice treated with this combination therapy. Serum levels of IFN-γ and IL-12 were found to be higher on same day in mice treated with BBIQ + CQ which revealed the generation of strong Th1 immune response in mice against the infection. Overall, TLR7 agonist acted as an efficient partner when combined with potent antimalarial drug.

Peptides conjugated to 2-alkoxy-8-oxo-adenine as potential synthetic vaccines triggering TLR7.

Covalent linking of immunogenic oligopeptides with synthetic Toll-like receptor ligands is a useful approach to develop self-adjuvanting vaccines. In particular, small-molecule based agonists of Toll-like receptor 7 (TLR7) that are derived from 8-oxo-adenine core are potentially promising because these chemically robust TLR7 ligands can be connected to peptide T-cell epitopes via straightforward solid-phase peptide synthesis. In this contribution we present the synthesis of a Boc-protected 9-benzyl-2-alkoxy-8-oxo-adenine building block and its application in the online solid phase synthesis of three peptide conjugates that differ in the position of the TLR7 ligand within the peptide. The conjugates are able to induce dendritic cell maturation and T cell proliferation while the position of the ligand impacts T cell proliferation potency.

Emerging drugs for the treatment of hepatitis B.

INTRODUCTION: Nucleos(t)ide analogs and interferon-based compounds are currently approved for the treatment of chronic hepatitis B (CHB). Although these treatments are effective in suppressing viral replication, it is unable to completely eradicate the virus from the host. Therefore, CHB patients are at a life-long risk of developing complications, including hepatocellular carcinoma. AREAS COVERED: Drugs targeting novel sites of the hepatitis B virus (HBV) replication cycle and the host immune response in development are discussed. As current available drugs only target a small segment of the HBV life cycle, the development of new agents targeting different sites is an important step in eradicating HBV. The host immunological response is also vital in viral clearance. Newer agents in development include immunomodulatory agents and therapeutic vaccines. EXPERT OPINION: For any chance of eradication, a combination of drugs targeting both the host factors and different sites of the viral life cycle will be required. Two key components to achieving this goal include the removal of covalently closed circular DNA (cccDNA) together with restoration of the immune control against HBV.

Influence of PapMV nanoparticles on the kinetics of the antibody response to flu vaccine.

BACKGROUND: The addition of an adjuvant to a vaccine is a promising approach to increasing strength and immunogenicity towards antigens. Despite the fact that adjuvants have been used in vaccines for decades, their mechanisms of action and their influence on the kinetics of the immune response are still not very well understood. The use of papaya mosaic virus (PapMV) nanoparticles-a novel TLR7 agonist-was recently shown to improve and broaden the immune response directed to trivalent inactivated flu vaccine (TIV) in mice and ferrets. RESULTS: We investigated the capacity of PapMV nanoparticles to increase the speed of the immune response toward TIV. PapMV nanoparticles induced a faster and stronger humoral response to TIV that was measured as early as 5 days post-immunization. The addition of PapMV nanoparticles was shown to speed up the differentiation of B-cells into early plasma cells, and increased the growth of germinal centers in a CD4+ dependent manner. TIV vaccination with PapMV nanoparticles as an adjuvant protected mice against a lethal infection as early as 10 days post-immunization. CONCLUSION: In conclusion, PapMV nanoparticles are able to accelerate a broad humoral response to TIV. This property is of the utmost importance in the field of vaccination, especially in the case of pandemics, where populations need to be protected as soon as possible after vaccination.

Epicutaneous immune modulation with Bet v 1 plus R848 suppresses allergic asthma in a murine model.

BACKGROUND: Combining allergen(s) with an adjuvant is a strategy to improve the efficacy and safety of allergen-specific immunotherapy. Here, we aimed at investigating the adjuvant effects of polyadenylic-polyuridylic acid (poly(A:U)), a TLR3 agonist, and R848 (resiquimod), a TLR7 agonist, in epicutaneous immunotherapy with Bet v 1, the major birch pollen allergen, to intervene in birch pollen allergy. METHODS AND RESULTS: BALB/c mice received epicutaneous immunization (EPI) with recombinant Bet v 1 (rBet v 1) alone, or plus poly(A:U), or R848 on their depilated back using patches. Among the groups, EPI with rBet v 1 and R848 induced detectable levels of IFN-γ-producing CD4(+) T cells in lymph nodes and Bet v 1-specific IgG2a antibodies in the sera of mice. Before or after EPI, mice were sensitized with rBet v 1 plus aluminium hydroxide adjuvant and intranasally challenged with birch pollen extract. Prophylactic EPI with rBet v 1 plus R848 inhibited the production of biologically active Bet v 1-specific IgE antibodies in sensitization. Prophylactic and therapeutic EPI with rBet v 1 plus R848 suppressed lung inflammation upon challenges. Remarkably, only rBet v 1 plus R848 reduced the development of enhanced pause (PenH), a substituted parameter for airway hyper-reactivity, in challenged mice. In contrast to R848, poly(A:U) did not present adjuvant effect on the suppression of asthmatic features. CONCLUSION: Epicutaneous immunization with rBet v 1 plus R848 induced predominant Bet v 1-specific Th1 responses and efficiently suppressed asthmatic features elicited by birch pollen. R848 could be a promising adjuvant in epicutaneous immunotherapy for birch pollen-induced allergic asthma.

A phase I trial of LHC165 single agent and in combination with spartalizumab in patients with advanced solid malignancies.

BACKGROUND: LHC165 is a Toll-like receptor (TLR)-7 agonist that generates an effective tumor antigen-specific T-cell adaptive immune response as well as durable antitumor responses. We aimed to evaluate the safety, tolerability, efficacy, dose-limiting toxicities, and pharmacokinetics (PK) of LHC165 single agent (SA) ± spartalizumab [PDR001; anti-programmed cell death protein 1 (PD-1)] in adult patients with advanced solid tumors. MATERIALS AND METHODS: In this phase I/Ib, open-label, dose-escalation/expansion study, patients received LHC165 SA 100-600 µg biweekly through intratumoral (IT) injection and LHC165 600 µg biweekly + spartalizumab 400 mg Q4W through intravenous (IV) infusion. RESULTS: Forty-five patients were enrolled: 21 patients received LHC165 SA, and 24 patients received LHC165 + spartalizumab. The median duration of exposure was 8 weeks (range 2-129 weeks). No maximum tolerated dose was reached. Recommended dose expansion was established as LHC165 600 µg biweekly as SA and in combination with spartalizumab 400 mg Q4W. The most common drug-related adverse events (AEs) were pyrexia (22.2%), pruritus (13.3%), chills (11.1%), and asthenia (4.4%). The only serious AE (SAE) suspected to be related to the study drug was grade 3 pancreatitis (n = 1). Across all tumor types, overall response rate and disease control were 6.7% and 17.8%, respectively. Overall median progression-free survival (PFS) and immune-related PFS was 1.7 months. LHC165 serum PK demonstrated an initial rapid release followed by a slower release due to continued release of LHC165 from the injection site. CONCLUSIONS: LHC165 demonstrated acceptable safety and tolerability both as SA and in combination with spartalizumab, and evidence of limited antitumor activity was seen in adult patients with relapsed/refractory or metastatic solid tumors.

Exploration of 1-(indolin-1-yl)-2-(thiazol-2-yl)ethan-1-one derivatives as novel anti-HBV agent with potential TLR7-agonistic effect.

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.

CaCO3-Encapsulated polydopamine with an adsorbed TLR7 agonist for improved tumor photothermal immunotherapy.

Because of the tumor's recurrence and significant metastasis, the standard single-therapy paradigm has failed to meet clinical requirements. Recently, researchers have focused their emphasis on phototherapy and immunogenic cell death (ICD) techniques. In response to the current problems of immunotherapy, a multifunctional drug delivery nanosystem (PDA-IMQ@CaCO3-blinatumomab, PICB) was constructed by using high physiological compatibility of polydopamine (PDA) and calcium carbonate (CaCO3). Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ) and bispecific antibody (BsAb) blinatumomab were loaded onto PDA-CaCO3 nanoparticles (NPs). The findings revealed that the system exhibited the advantages of good dispersion, high stability, excellent physiological compatibility, low toxicity, and high drug loading rate. Compared to the control group, it resulted in a 2.4-fold decrease in FOXP3+ regulatory T-cells within the tumor and a 5.0-fold increase in CD4+ effector T-cells, and promoted the production of damage-related molecular patterns to reinvigorate the ICD effect. PICB had a strong inhibitory effect on tumor growth in 4T1 tumor-bearing mice, and has no toxicity to other organs. Therefore, the multifunctional drug delivery nanosystem constructed in this study could effectively exert the properties of various components in vivo, fully demonstrate the synergistic effect between immunotherapy and photothermal therapy, thus significantly improving the tumor therapeutic efficacy, and has a promising clinical application.

The novel selective TLR7 agonist GY101 suppresses colon cancer growth by stimulating immune cells.

Toll-like receptor (TLR) 7, a transmembrane signal transduction receptor expressed on the surface of endosomes, has become an attractive target for antiviral and cancer immunotherapies. TLR7 can induce signal transduction by recognizing single-stranded RNA or its analogs, leading to the release of cytokines such as IL-6, IL-12, TNF-α and type-I IFN. Activation of TLR7 helps to enhance immunogenicity and immune memory by stimulating immune cells. Herein, we identified a novel selective TLR7 agonist, GY101, and determined its ability to activate TLR7. In summary, in vitro, compound GY101 significantly induced the secretion of IL-6, IL-12, TNF-α and IFN-γ in mouse splenic lymphocytes; in vivo, peritumoral injection of GY101 significantly suppressed colon cancer CT26, as well as poorly immunogenic B16-F10 and 4T1 cancer cell-derived tumor growth by activating the infiltration of lymphocytes and polarization of M2-like macrophages into M1-like macrophages. These results demonstrate that GY101, as a potent TLR7 agonist, holds great potential for cancer immunotherapy.

DSP-0509, a TLR7 agonist, exerted synergistic anti-tumor immunity combined with various immune therapies through modulating diverse immune cells in cancer microenvironment.

Toll-like receptor 7 (TLR7) acts as a crucial component of the innate immune system. Upon TLR7 binding to its ligand, myeloid cells, including dendritic cells (DCs) and macrophages, are activated and play vital roles in initiating adaptive immunity. Consequently, TLR7 agonists have been employed in cancer immunotherapy. We have synthesized DSP-0509, a systemic injectable TLR7 agonist, and in this investigation, we examined the effects of DSP-0509 on tumor-infiltrating lymphocytes (TILs) utilizing single-cell RNA sequencing (scRNA-seq) in a mouse model bearing tumors. Our results demonstrated that DSP-0509 induced an expansion of immune cell populations, such as Natural Killer (NK) cells, CD4+ T cells, and CD4+ regulatory T cells (Tregs). Subsequently, we combined an Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor with DSP-0509 to enhance the antitumor efficacy by reducing Tregs, as DSP-0509 led to an increase in Treg presence within tumors. Our findings demonstrated that this combination therapy effectively reduced Treg infiltration within the tumor, leading to enhanced antitumor activity. To further prevent CD8+ T cell exhaustion, we combined DSP-0509 with an anti-PD-1 antibody and assessed the alterations in TILs using scRNA-seq. Our results indicated that the combination treatment significantly increased the cluster of CD8+ T cells expressing Gzmb, Prf1, Ctla4, and Icos, when compared to the administration of DSP-0509 alone. Additionally, we observed a marked rise in the M1-like macrophage cluster in the combination treatment group compared to the group receiving only DSP-0509. To validate the potential of modulating myeloid cells within the tumor to enhance antitumor efficacy, we combined DSP-0509 with an inhibitor targeting the receptor tyrosine kinase AXL. In bone marrow derived macrophages (BMDMs), the AXL inhibitor further amplified DSP-0509-stimulated TNFα secretion while reducing IL-10 secretion. As a final step, we evaluated the antitumor activity by combining DSP-0509 and the AXL inhibitor in an in vivo tumor model, which demonstrated increased efficacy. In summary, our study elucidated the effects of DSP-0509 on immune activity within the tumor microenvironment. These findings provided valuable insights that pave the way for the development of novel combination immunotherapy strategies.

A single, oral dose of the TLR7 agonist JNJ-64794964 induces transcriptomic and phenotypic changes in peripheral immune cells in healthy adults.

BACKGROUND: Chronic hepatitis B (CHB) is responsible for major disease burden worldwide. However, the number of available therapies is limited; cure remains an elusive goal. JNJ-64794964 (JNJ-4964) is an oral toll-like receptor-7 (TLR7) agonist being evaluated for the treatment of CHB. Here, we investigated the capacity of JNJ-4964 to induce transcriptomic and immune cell changes in peripheral blood in healthy volunteers. METHODS: Peripheral blood was collected in the JNJ-4964 first-in-human phase 1 trial at multiple time points to assess transcriptomics and changes in frequency and phenotype of peripheral-blood mononuclear cells. Correlation of changes to JNJ-4964 exposure (Cmax) and changes in cytokine levels (C-X-C motif chemokine ligand 10 [CXCL10] and interferon alpha [IFN-α]) were evaluated. RESULTS: Fifty-nine genes, mainly interferon-stimulated genes, were up-regulated between 6 hours and 5 days after JNJ-4964 administration. JNJ-4964 increased frequencies of CD69, CD134, CD137, and/or CD253-expressing natural killer (NK) cells, indicative of NK cell activation. These changes correlated with Cmax, increase of CXCL10, and induction of IFN-α and were observed at IFN-α levels that are associated with no/acceptable flu-like adverse events. JNJ-4964 administration resulted in increased frequencies of CD86-expressing B cells, indicative of B-cell activation. These changes were predominantly observed at high IFN-α levels, which are associated with flu-like adverse events. CONCLUSIONS: JNJ-4964 administration led to changes in transcriptional profiles and immune cell activation phenotype, particularly for NK cells and B cells. Together, these changes could represent a set of biomarkers for the characterization of the immune response in CHB patients receiving TLR7 agonists.

Novel sialoglycan linkage for constructing adjuvant-protein conjugate as potent vaccine for COVID-19.

Self-adjuvanting protein vaccines have been proved to be highly immunogenic with efficient codelivery of adjuvant and antigen. Current protein vaccines with built-in adjuvants are all modified at the peptide backbone of antigen protein, which could not achieve minor epitope interference and adjuvant multivalency at the same time. Herein, we developed a new conjugate strategy to construct effective adjuvant-protein vaccine with adjuvant cluster effect and minimal epitope interference. The toll-like receptor 7 agonist (TLR7a) is covalently conjugated on the terminal sialoglycans of SARS-CoV-2-S1 protein, leading to intracellular release of the small-molecule stimulators with greatly reduced risks of systemic toxicity. The resulting TLR7a-S1 conjugate elicited strong activation of immune cells in vitro, and potent antibody and cellular responses with a significantly enhanced Th1-bias in vivo. TLR7a-S1-induced antibody also effectively cross-neutralized all variants of concern. This sialoglycoconjugation approach to construct protein conjugate vaccines will have more applications to combat SARS-CoV-2 and other diseases.