Combination Of R848 Research Articles

The Combination of R848 with Sorafenib Enhances Antitumor Effects by Reprogramming the Tumor Immune Microenvironment and Facilitating Vascular Normalization in Hepatocellular Carcinoma.

Novel promising strategies for combination with sorafenib are urgently needed to enhance its clinical benefit and overcome toxicity in hepatocellular carcinoma (HCC). the molecular and immunomodulatory antitumor effects of sorafenib alone and in combination with the new immunotherapeutic agent R848 are presented. Syngeneic HCC mouse model is presented to explore the antitumor effect and safety of three sorafenib doses alone, R848 alone, or their combination in vivo. R848 significantly enhances the sorafenib antitumor activity at a low subclinical dose with no obvious toxic side effects. Furthermore, the combination therapy reprograms the tumor immune microenvironment by increasing antitumor macrophages and neutrophils and preventing immunosuppressive signaling. Combination treatment promotes classical M1 macrophage-to-FTH1high M1 macrophage transition. The close interaction between neutrophils/classical M1 macrophages and dendritic cells promotes tumor antigen presentation to T cells, inducing cytotoxic CD8+ T cell-mediated antitumor immunity. Additionally, low-dose sorafenib, alone or combined with R848, normalizes the tumor vasculature, generating a positive feedback loop to support the antitumor immune environment. Therefore, the combination therapy reprograms the HCC immune microenvironment and normalizes the vasculature, improving the therapeutic benefit of low-dose sorafenib and minimizing toxicity, suggesting a promising novel immunotherapy (R848) and targeted therapy (tyrosine kinase inhibitors) combination strategy for HCC treatment.

MuSyC dosing of adjuvanted cancer vaccines optimizes antitumor responses.

With the clinical approval of T-cell–dependent immune checkpoint inhibitors for many cancers, therapeutic cancer vaccines have re-emerged as a promising immunotherapy. Cancer vaccines require the addition of immunostimulatory adjuvants to increase vaccine immunogenicity, and increasingly multiple adjuvants are used in combination to bolster further and shape cellular immunity to tumor antigens. However, rigorous quantification of adjuvants’ synergistic interactions is challenging due to partial redundancy in costimulatory molecules and cytokine production, leading to the common assumption that combining both adjuvants at the maximum tolerated dose results in optimal efficacy. Herein, we examine this maximum dose assumption and find combinations of these doses are suboptimal. Instead, we optimized dendritic cell activation by extending the Multidimensional Synergy of Combinations (MuSyC) framework that measures the synergy of efficacy and potency between two vaccine adjuvants. Initially, we performed a preliminary in vitro screening of clinically translatable adjuvant receptor targets (TLR, STING, NLL, and RIG-I). We determined that STING agonist (CDN) plus TLR4 agonist (MPL-A) or TLR7/8 agonist (R848) as the best pairwise combinations for dendritic cell activation. In addition, we found that the combination of R848 and CDN is synergistically efficacious and potent in activating both murine and human antigen-presenting cells (APCs) in vitro. These two selected adjuvants were then used to estimate a MuSyC-dose optimized for in vivo T-cell priming using ovalbumin-based peptide vaccines. Finally, using B16 melanoma and MOC1 head and neck cancer models, MuSyC-dose–based adjuvating of cancer vaccines improved the antitumor response, increased tumor-infiltrating lymphocytes, and induced novel myeloid tumor infiltration changes. Further, the MuSyC-dose–based adjuvants approach did not cause additional weight changes or increased plasma cytokine levels compared to CDN alone. Collectively, our findings offer a proof of principle that our MuSyC-extended approach can be used to optimize cancer vaccine formulations for immunotherapy.

Toll-like receptor 7/8 agonist R848 alters the immune tumor microenvironment and enhances SBRT-induced antitumor efficacy in murine models of pancreatic cancer

BackgroundStereotactic body radiotherapy (SBRT) has been increasingly used as adjuvant therapy in pancreatic ductal adenocarcinoma (PDAC), and induces immunogenic cell death, which leads to the release of tumor antigen and...

Blood DCs activated with R848 and poly(I:C) induce antigen-specific immune responses against viral and tumor-associated antigens

Monocyte-derived Dendritic cells (DCs) have successfully been employed to induce immune responses against tumor-associated antigens in patients with various cancer entities. However, objective clinical responses have only been achieved in a minority of patients. Additionally, generation of GMP-compliant DCs requires time- and labor-intensive cell differentiation. In contrast, Blood DCs (BDCs) require only minimal ex vivo handling, as differentiation occurs in vivo resulting in potentially better functional capacities and survival. We aimed to identify a protocol for optimal in vitro activation of BDCs including the three subsets pDCs, cDC1s, and cDC2s. We evaluated several TLR ligand combinations and demonstrated that polyinosinic:polycytidylic acid [poly(I:C)] and R848, ligands for TLR3 and TLR7/8, respectively, constituted the optimal combination for inducing a positive co-stimulatory profile in all BDC subsets. In addition, TLR3 and TLR7/8 activation led to high secretion of IFN-α and IL-12p70. Simultaneous as opposed to separate tailored activation of pDCs and cDCs increased immunostimulatory capacities, suggesting that BDC subsets engage in synergistic cross-talk during activation. Stimulation of BDCs with this protocol resulted in enhanced migration, high NK-cell activation, and potent antigen-specific T-cell induction.We conclude that simultaneous activation of all BDC subsets with a combination of R848 + poly(I:C) generates highly immunostimulatory DCs. These results support further investigation and clinical testing, as standalone or in conjunction with other immunotherapeutic strategies including adoptive T-cell transfer and checkpoint inhibition.

The synergy of TLR and STING in cancer immunity

Abstract Adjuvants are essential components of cancer vaccine formulations to promote effective immune responses. We initially screened multiple immune-activating targets, including TLR and STING adjuvants using in-vitro assays to test their ability to activate bone marrow DC (BMDC) and showed STING had the most potent activation (i.e., MHC and co-stimulatory molecules) on DCs. Since STING and TLR signaling is non-redundant, TLR and STING adjuvants were combined to enhance DC activation. We utilized the multidimensional synergy of combinations (MuSyc), a novel synergy algorithm, to assess molecular synergy in combining STING and TLR adjuvants, and we noted that the combination of R848 (TLR7-8) plus STING agonists provided a synergistic efficacious and potent response on BMDCs. From this data-set, we generated a MuSYC synergy strategy where we will utilize max signal dose for STING adjuvant and ten times less of max signal dose for R848 (TLR7-8) to determine synergy for other assays. We tested this same MuSYC strategy for activation of human monocytic cell line THP-1 and determined that the synergy strategy induced similar or better activation effects compared to the max signal dose for both adjuvants. Finally, we tested the in-vivo anti-tumor immune response for the B16 melanoma and MOC2 head/neck tumor-bearing vaccination models that resulted in an improved anti-tumor response for the combinatorial STING-TLR adjuvanted vaccines compared to the single agents.

TLR7 Agonists Display Potent Antiviral Effects against Norovirus Infection via Innate Stimulation.

Norovirus infections are a significant health and economic burden globally, accounting for hundreds of millions of cases of acute gastroenteritis every year. In the absence of an approved norovirus vaccine, there is an urgent need to develop antivirals to treat chronic infections and provide prophylactic therapy to limit viral spread during epidemics and pandemics. Toll-like receptor (TLR) agonists have been explored widely for their antiviral potential, and several are progressing through clinical trials for the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and as adjuvants for norovirus viruslike particle (VLP) vaccines. However, norovirus therapies in development are largely direct-acting antivirals (DAAs) with fewer compounds that target the host. Our aim was to assess the antiviral potential of TLR7 agonist immunomodulators on norovirus infection using the murine norovirus (MNV) and human Norwalk replicon models. TLR7 agonists R-848, Gardiquimod, GS-9620, R-837, and loxoribine were screened using a plaque reduction assay, and each displayed inhibition of MNV replication (50% effective concentrations [EC50s], 23.5 nM, 134.4 nM, 0.59 μM, 1.5 μM, and 79.4 μM, respectively). RNA sequencing of TLR7-stimulated cells revealed a predominant upregulation of innate immune response genes and interferon (IFN)-stimulated genes (ISGs) that are known to drive an antiviral state. Furthermore, the combination of R-848 and the nucleoside analogue (NA) 2'C-methylcytidine elicited a synergistic antiviral effect against MNV, demonstrating that combinational therapy of host modulators and DAAs might be used to reduce drug cytotoxicity. In summary, we have identified that TLR7 agonists display potent inhibition of norovirus replication and are a therapeutic option to combat norovirus infections.

The synergistic effects of combining TLR ligand based adjuvants on the cytokine response are dependent upon p38/JNK signalling

Toll like receptor (TLR) ligands are important adjuvant candidates, causing antigen presenting cells to release inflammatory mediators, leading to the recruitment and activation of other leukocytes. The aim of this study was to define the response of human blood derived dendritic cells and macrophages to three TLR ligands acting singly or in combination, Poly I:C (TLR3), GLA (TLR4) and R848 (TLR7/8). Combinations of TLR agonists have been shown to have a synergistic effect on individual cytokines, here we look at the global inflammatory response measuring both cytokines and chemokines. Using a custom Luminex assay we saw dose responses in several mediators including CCL3 (MIP1α), IL-1α, IL-1β, IL-12, CXCL10 (IP-10) and IL-6, all of which were significantly increased by the combination of R848 and GLA, even when low dose GLA was added. The synergistic effect was inhibited by specific MAP kinase inhibitors blocking the kinases p38 and JNK but not MEK1. Combining TLR adjuvants also had a synergistic effect on cytokine responses in human mucosal tissue explants. From this we conclude that the combination of R848 and GLA potentiates the inflammatory profile of antigen presenting cells. Since the pattern of inflammatory mediators released can alter the quality and quantity of the adaptive immune response to vaccination, this study informs vaccine adjuvant design.

The Ultra-Potent and Selective TLR8 Agonist VTX-294 Activates Human Newborn and Adult Leukocytes

BackgroundNewborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed.MethodsTLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-κB reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry).ResultsVTX-294 was ∼100x more active on TLR8- than TLR7-transfected HEK cells (EC50, ∼50 nM vs. ∼5700 nM). VTX-294-induced TNF and IL-1β production were comparable in newborn cord and adult peripheral blood, while VTX-294 was ∼ 1 log more potent in inducing TNF and IL-1β production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1β responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs.ConclusionsVTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood.

Maturation by Toll Like Receptor Ligand R848 Improves the Uptake of Apoptotic Leukemic Cells by Monocyte Derived Dendritic Cells.

Abstract 2075Poster Board II-52 Introduction.Therapeutic vaccination with dendritic cells (DC) is currently considered as an investigational therapy in acute myeloid leukemia (AML) for eradication of minimal residual disease (MRD). Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g. monocyte derived DC (MoDC) loaded with leukemia associated antigens (LAA). Many sources of LAA, whether or not combined with various adjuvants and different methods of loading of antigen onto DC, have been used in an attempt to optimize anti-tumor responses. However, the optimal source of tumor antigen combined with adjuvants remains unclear. Leukemic cells harboring all unknown and known potential LAA are an attractive antigen source. Here we explore the use of leukemic blast derived apoptotic cells and lysates loaded onto MoDC combined with various maturation stimuli, such as the clinically applicable toll like receptor 7/8 ligand (TLR-L) R848 combined with conventional cytokine cocktail consisting of IL1β, IL6, TNFα and PGE2. Material & Methods.CD14+ monocytes isolated from peripheral blood of healthy donors were differentiated into MoDC with GM-CSF and IL-4 in serum free medium. Leukemic blasts were labeled with CFSE, followed by induction of apoptosis in serum deprived medium with heat shock (2h 42°C) or preparation of lysate by 3 freeze/thaw cycles. Apoptosis was confirmed by flow cytometry using Syto(62) and 7AAD. Subsequently, the CFSE-labeled apoptotic blasts and lysates were incubated in various ratios with immunofluorescently labeled MoDC in the presence of TLR-L (Poly-(I:C), LPS, PGN, Flagellin or R848) and/or the cytokine cocktail in different time frames. Uptake was defined by flow cytometry as percentage of CFSE positive MoDC. MoDC were analyzed for expression of co-stimulatory molecules, the chemokine receptor CCR7 and maturation-associated antigens. Migratory capacity towards CCL19 was measured in a transwell system. Mixed leukocyte reaction was performed to study T cell stimulation and IL12 secretion was quantified after CD40 ligation. Results.Apoptotic cells or lysates were taken up by MoDC in a dose dependent manner, up to 26% (12-49%) and 17% (median; range: 3-47%), respectively. Of all tested TLR-L, R848 most effectively enhanced the uptake of apoptotic cells, but not of lysates. Addition of the cytokine cocktail resulted in a decreased uptake by MoDC of both apoptotic cells and lysate (n=7), but most effectively induced phenotypic DC maturation as determined by up-regulation of co-stimulatory molecules, enhanced migratory capacity and enhanced ability to stimulate T cells. Both R848 and cytokine cocktail stimulated MoDC were able to produce IL-12 (n=6). To combine the enhanced uptake induced by R848 and the optimal maturation of MoDC by the cytokine cocktail, we co-incubated blasts and MoDC in the presence of R848 for 24h followed by a 48hr incubation with cytokine cocktail (n=9). The resulting uptake was superior to the cytokine cocktail alone, and migratory and co-stimulatory capacity was improved as compared to R848 alone. Conclusion.The TLR-L R848 significantly improved the uptake of leukemic blasts by MoDC. Combination of R848 and cytokines resulted in sufficient maturation induction and subsequent T cell priming. These data may add significantly to the development of new strategies for optimization of vaccine preparation in AML. Disclosures:No relevant conflicts of interest to declare.

Clonal dissection of the human memory B‐cell repertoire following infection and vaccination

The analysis of the human memory B-cell repertoire is of both fundamental and practical significance. We developed a simple method for the selective activation of memory B cells in total fresh or frozen PBMC using a combination of R848 and IL-2. In these conditions, 30-40% of memory B cells generated clones producing on average 200 ng IgG in 10 days. This method was used to measure the frequency of antigen-specific memory B cells as well as the fine specificity, cross-reactivity and neutralizing activity of the secreted antibodies. Following influenza vaccination, specific B cells expanded dramatically, reaching up to 50% of total clonable memory B cells on day 14. Specific B-cell expansions were detected also in individuals that did not show a significant serological response. Dynamic changes and persistence of B cells specific for a variety of pathogens were documented in serial PBMC samples collected over almost two decades. These results reveal novel aspects of memory B-cell kinetics and provide a powerful tool to monitor immune responses following infection and vaccination.