Toll-like Receptor 4 Ligands Down-regulate Fcγ Receptor IIb (FcγRIIb) via MARCH3 Protein-mediated Ubiquitination.

Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Responses to antibody-coated tumor cells are largely mediated by Fcγ receptors (FcγRs), which become activated upon binding to immune complexes. FcγRIIb is an inhibitory FcγR that negatively regulates these responses, and it is expressed on monocytes and macrophages. Therefore, deletion or down-regulation of this receptor may substantially enhance therapeutic outcomes. Here we screened a panel of Toll-like receptor (TLR) agonists and found that those selective for TLR4 and TLR8 could significantly down-regulate the expression of FcγRIIb. Upon further examination, we found that treatment of monocytes with TLR4 agonists could lead to the ubiquitination of FcγRIIb protein. A search of our earlier microarray database of monocytes activated with the TLR7/8 agonist R-848 (in which FcγRIIb was down-regulated) revealed an up-regulation of membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase. Therefore, we tested whether LPS treatment could up-regulate MARCH3 in monocytes and whether this E3 ligase was involved with LPS-mediated FcγRIIb down-regulation. The results showed that LPS activation of TLR4 significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the decrease in FcγRIIb following LPS treatment. These data suggest that activation of TLR4 on monocytes can induce a rapid down-regulation of FcγRIIb protein and that this involves ubiquitination.

Granzyme B expression is enhanced in human monocytes by TLR8 agonists and contributes to antibody-dependent cellular cytotoxicity.

FcγRs are critical mediators of mAb cancer therapies, because they drive cytotoxic processes upon binding of effector cells to opsonized targets. Along with NK cells, monocytes are also known to destroy Ab-coated targets via Ab-dependent cellular cytotoxicity (ADCC). However, the precise mechanisms by which monocytes carry out this function have remained elusive. In this article, we show that human monocytes produce the protease granzyme B upon both FcγR and TLR8 activation. Treatment with TLR8 agonists elicited granzyme B and also enhanced FcγR-mediated granzyme B production in an additive fashion. Furthermore, monocyte-mediated ADCC against cetuximab-coated tumor targets was enhanced by TLR8 agonist treatment, and this enhancement of ADCC required granzyme B. Hence we have identified granzyme B as an important mediator of FcγR function in human monocytes and have uncovered another mechanism by which TLR8 agonists may enhance FcγR-based therapies.

Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects ß-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate ß-glucan polymers, Dectin-1 signalling is only activated by particulate ß-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.

Effective innate and adaptive antimelanoma immunity through localized TLR7/8 activation.

Intratumoral immune activation can induce local and systemic antitumor immunity. Imiquimod is a cream-formulated, TLR7 agonist that is Food and Drug Administration approved for the treatment of nonmelanoma skin cancers, but it has limited activity against melanoma. We studied the antitumor activity and mechanism of action of a novel, injectable, tissue-retained TLR7/8 agonist, 3M-052, which avoids systemic distribution. Intratumoral administration of 3M-052 generated systemic antitumor immunity and suppressed both injected and distant, uninjected wild-type B16.F10 melanomas. Treated tumors showed that an increased level of CCL2 chemokines and infiltration of M1 phenotype-shifted macrophages, which could kill tumor cells directly through production of NO and CCL2, were essential for the antitumor activity of 3M-052. CD8(+) T cells, B cells, type I IFN, IFN-γ, and plasmacytoid dendritic cells were contributed to efficient tumor suppression, whereas perforin, NK cells, and CD4 T cells were not required. Finally, 3M-052 therapy potentiated checkpoint blockade therapy with anti-CTLA-4 and anti-programmed death ligand 1 Abs, even when checkpoint blockade alone was ineffective. Our findings suggest that intratumoral treatment with 3M-052 is a promising approach for the treatment of cancer and establish a rational strategy and mechanistic understanding for combination therapy with intratumoral, tissue-retained TLR7/8 agonist and checkpoint blockade in metastatic cancer.

Differential regulation of oxidative burst by distinct ß-glucan-binding receptors and signaling pathways in human peripheral blood mononuclear cells.

ß-Glucans possess broad immunomodulatory properties, including activation of innate immune functions such as oxidative burst activity. The differential roles of complement receptor type 3 (CR3) and Dectin-1, the known ß-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae-derived ß-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble ß-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively. Oxidative burst as measured by the formation of superoxide (SO) was detected in PBMC in response to WGP and ISG. SO induction with WGP was concluded to be Dectin-1-mediated and required Src family kinases, phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, the SO induction generated by ISG was CR3-mediated and required focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The study results support the hypothesis that human PBMC, specifically monocytes, utilize distinct receptors and overlapping, but distinct, signaling pathways for the oxidative burst in response to challenge by different physical forms of ß-glucan.

Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN.

Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10-20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)gamma production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNgamma, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity.

Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies.

BACKGROUND: Immune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically "cold" tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated. METHODS: The response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models. RESULTS: Targeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models. CONCLUSION: Localized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.

Comparison of human B cell activation by TLR7 and TLR9 agonists.

BACKGROUND: Human B cells and plasmacytoid dendritic cells (pDC) are the only cells known to express both TLR7 and TLR9. Plasmacytoid dendritic cells are the primary IFN-alpha producing cells in response to TLR7 and TLR9 agonists. The direct effects of TLR7 stimulation on human B cells is less understood. The objective of this study was to compare the effects of TLR7 and TLR9 stimulation on human B cell function. RESULTS: Gene expression and protein production of cytokines, chemokines, various B cell activation markers, and immunoglobulins were evaluated. Purified human CD19+ B cells (99.9%, containing both naïve and memory populations) from peripheral blood were stimulated with a TLR7-selective agonist (852A), TLR7/8 agonist (3M-003), or TLR9 selective agonist CpG ODN (CpG2006). TLR7 and TLR9 agonists similarly modulated the expression of cytokine and chemokine genes (IL-6, MIP1 alpha, MIP1 beta, TNF alpha and LTA), co-stimulatory molecules (CD80, CD40 and CD58), Fc receptors (CD23, CD32), anti-apoptotic genes (BCL2L1), certain transcription factors (MYC, TCFL5), and genes critical for B cell proliferation and differentiation (CD72, IL21R). Both agonists also induced protein expression of the above cytokines and chemokines. Additionally, TLR7 and TLR9 agonists induced the production of IgM and IgG. A TLR8-selective agonist was comparatively ineffective at stimulating purified human B cells. CONCLUSION: These results demonstrate that despite their molecular differences, the TLR7 and TLR9 agonists induce similar genes and proteins in purified human B cells.

TLR7 agonist 852A inhibition of tumor cell proliferation is dependent on plasmacytoid dendritic cells and type I IFN.

Antitumor effects of the toll-like receptor 7 (TLR7) agonist, 852A, were evaluated. Supernatants from human peripheral blood mononuclear cells (PBMC) stimulated with 852A inhibited the proliferation of tumor cell lines Hs294T and 769-P but had no effect on others (786-O and Caki-1). Because addition of 852A directly to the Hs294T cells did not inhibit their proliferation, the mechanism(s) of inhibition of tumor cell proliferation was investigated. Low nanomolar concentrations of 852A stimulated the production of interferon-alpha (IFN-alpha), IFN-inducible protein-10 (IP-10), interleukin-1 receptor antagonist (IL-1Ra), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from human PBMCs. Cytokines stimulated by submicromolar concentrations of 852A were sufficient to inhibit Hs294T proliferation. At higher concentrations (3-30 microM), 852A induced the production of IL-12p70, IL-18, and IFN-gamma. PBMC cultures depleted of plasmacytoid dendritic cells (pDC) did not produce IFN-alpha, and their conditioned medium did not inhibit Hs294T proliferation. Anti-IFN-alpha/beta receptor (IFNAR) and anti-IFN-alpha antibodies partially abrogated Hs294T proliferation inhibition by 852A-stimulated PBMC supernatants, whereas separate neutralization of TRAIL, tumor necrosis factor-alpha (TNF-alpha, IFN-gamma, IFN-beta, or IFN-omega had no effect. In vivo, six doses of 852A administration significantly delayed the onset of lung colonies in a B16 melanoma model. Thus, the results demonstrate that the TLR7 agonist 852A inhibits in vitro proliferation of some tumor cells in a pDC-dependent and IFN-alpha-dependent manner and can delay tumor growth in vivo.

First in human phase I trial of 852A, a novel systemic toll-like receptor 7 agonist, to activate innate immune responses in patients with advanced cancer.

PURPOSE: Recent advances in the understanding of innate immunity suggest that an orchestrated sequence of events is required to elicit a productive immune response against cancer. We studied the systemic administration of the Toll-like receptor 7 agonist 852A, a small-molecule imidazoquinoline, in patients with advanced cancer. Preclinical studies showed that 852A stimulates plasmacytoid dendritic cells to produce multiple cytokines, such as IFN-alpha, interleukin-1 receptor antagonist, and IFN-inducible protein-10. Our goal was to define the tolerated dose, pharmacokinetics, pharmacodynamics, and immunologic effects of 852A in humans. EXPERIMENTAL DESIGN: Eligible adult patients with refractory solid organ tumors received i.v. 852A thrice weekly for 2 weeks. Patients who had responses or stable disease were eligible for additional cycles. RESULTS: Twenty-five patients (median age, 55.0 years; 72% male) were enrolled in six cohorts at dose levels of 0.15 to 2.0 mg/m(2). Serum drug levels showed dose proportionality and no evidence of drug accumulation. The maximum tolerated dose was 1.2 mg/m(2); higher doses were limited by fatigue and constitutional symptoms. Increases in IFN-alpha, interleukin-1 receptor antagonist, and IFN-inducible protein-10, immunologic activity, and clinical symptoms were observed in all patients receiving dose levels > or =0.6 mg/m(2). Significant correlations were found between pharmacodynamic biomarkers and pharmacokinetic variables, and an objective clinical response was seen. CONCLUSIONS: 852A was safely administered i.v. at doses up to 1.2 mg/m(2) thrice weekly for 2 weeks with transient or reversible adverse effects. This novel Toll-like receptor 7 agonist is biologically active and holds promise for stimulating innate immune responses. Future trials are warranted to assess its therapeutic role in patients with cancer.

Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists.

BACKGROUND: Plasmacytoid Dendritic Cells (pDC) comprise approximately 0.2 to 0.8% of the blood mononuclear cells and are the primary type 1 interferon (IFN), producing cells, secreting high levels of IFN in response to viral infections. Plasmacytoid dendritic cells express predominantly TLRs 7 & 9, making them responsive to ssRNA and CpG DNA. The objective of this study was to evaluate the molecular and cellular processes altered upon stimulation of pDC with synthetic TLR 7 and TLR 7/8 agonists. To this end, we evaluated changes in global gene expression upon stimulation of 99.9% pure human pDC with the TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011. RESULTS: Global gene expression was evaluated using the Affymetrix U133A GeneChip(R) and selected genes were confirmed using real time TaqMan(R) RTPCR. The gene expression profiles of the two agonists were similar indicating that changes in gene expression were solely due to stimulation through TLR7. Type 1 interferons were among the highest induced genes and included IFNB and multiple IFNalpha subtypes, IFNalpha2, alpha5, alpha6, alpha8, alpha1/13, alpha10, alpha14, alpha16, alpha17, alpha21. A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC. Induction of an antiviral state was shown by the expression of several IFN-inducible genes with known anti-viral activity. Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted. The analysis revealed transcription networks that show increased expression of signaling components in TLR7 and TLR3 pathways, and the cytosolic anti-viral pathway regulated by RIG1 and MDA5, suggestive of optimization of an antiviral state targeted towards RNA viruses. The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program. CONCLUSION: Thus this study demonstrates that as early as 4 hr post stimulation, synthetic TLR7 agonists induce a complex transcription network responsible for activating pDC for innate anti-viral immune responses with optimized responses towards RNA viruses, increased co-stimulatory capacity, and increased survival.

Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells.

Previously, we have successfully targeted the mannose receptor (MR) expressed on monocyte-derived dendritic cells (DCs) using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGbeta). Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR)-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGbeta-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C) and DC TLR 7/8 with Resiquimod (R-848), respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.

Administration of a dual toll-like receptor 7 and toll-like receptor 8 agonist protects against influenza in rats.

Toll-like receptors (TLR) detect conserved molecular patterns expressed by pathogens. Detection of the "molecular signature" for RNA viruses including influenza has been attributed to TLR3, TLR7, and TLR8. In the present study, compound 3M-011 was shown to be a synthetic human TLR7/8 agonist and cytokine inducer. 3M-011 was investigated as a stand-alone immune response modifier in a rat model of human influenza. Intranasal (IN) administration of 3M-011 significantly inhibited H3N2 influenza viral replication in the nasal cavity when administered from 72 h before IN viral inoculation to 6h after inoculation. Viral inhibition correlated with the ability of the TLR7/8 agonist to stimulate type I interferon (IFN) and other cytokines such as tumor necrosis factor-alpha, interleukin-12, and IFN-gamma from rat peripheral blood mononuclear cells. Prophylactic administration of TLR7/8 agonist also suppressed influenza viral titers in the lung, which corresponded with local IFN production. The activity of the TLR7/8 agonist resulted in greater inhibition of viral titers compared to rat recombinant IFN-alpha administered in a comparable dosing regimen. These studies indicate that TLR7/8 agonists may have prophylactic and therapeutic benefits in the treatment of respiratory viral infections, such as influenza, when administered prior to or shortly after viral inoculation.

TLR7/8 adjuvant overcomes newborn hyporesponsiveness to pneumococcal conjugate vaccine at birth.

Infection is the most common cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, newborns fail to respond optimally to most vaccines. Adjuvantation is a key approach to enhancing vaccine immunogenicity, but responses of human newborn leukocytes to most candidate adjuvants, including most TLR agonists, are functionally distinct. Herein, we demonstrate that 3M-052 is a locally acting lipidated imidazoquinoline TLR7/8 agonist adjuvant in mice, which, when properly formulated, can induce robust Th1 cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the alum-adjuvanted pneumococcal conjugate vaccine 13 (PCV13). When admixed with PCV13 and administered i.m. on the first day of life to rhesus macaques, 3M-052 dramatically enhanced generation of Th1 CRM-197-specific neonatal CD4+ cells, activation of newborn and infant Streptococcus pneumoniae polysaccharide-specific (PnPS-specific) B cells as well as serotype-specific antibody titers, and opsonophagocytic killing. Remarkably, a single dose at birth of PCV13 plus 0.1 mg/kg 3M-052 induced PnPS-specific IgG responses that were approximately 10-100 times greater than a single birth dose of PCV13 alone, rapidly exceeding the serologic correlate of protection, as early as 28 days of life. This potent immunization strategy, potentially effective with one birth dose, could represent a new paradigm in early life vaccine development.

Resiquimod as an immunologic adjuvant for NY-ESO-1 protein vaccination in patients with high-risk melanoma.

The Toll-like receptor (TLR) 7/8 agonist resiquimod has been used as an immune adjuvant in cancer vaccines. We evaluated the safety and immunogenicity of the cancer testis antigen NY-ESO-1 given in combination with Montanide (Seppic) with or without resiquimod in patients with high-risk melanoma. In part I of the study, patients received 100 µg of full-length NY-ESO-1 protein emulsified in 1.25 mL of Montanide (day 1) followed by topical application of 1,000 mg of 0.2% resiquimod gel on days 1 and 3 (cohort 1) versus days 1, 3, and 5 (cohort 2) of a 21-day cycle. In part II, patients were randomized to receive 100-µg NY-ESO-1 protein plus Montanide (day 1) followed by topical application of placebo gel [(arm A; n = 8) or 1,000 mg of 0.2% resiquimod gel (arm B; n = 12)] using the dosing regimen established in part I. The vaccine regimens were generally well tolerated. NY-ESO-1-specific humoral responses were induced or boosted in all patients, many of whom had high titer antibodies. In part II, 16 of 20 patients in both arms had NY-ESO-1-specific CD4⁺ T-cell responses. CD8⁺ T-cell responses were only seen in 3 of 12 patients in arm B. Patients with TLR7 SNP rs179008 had a greater likelihood of developing NY-ESO-1-specific CD8⁺ responses. In conclusion, NY-ESO-1 protein in combination with Montanide with or without topical resiquimod is safe and induces both antibody and CD4⁺ T-cell responses in the majority of patients; the small proportion of CD8⁺ T-cell responses suggests that the addition of topical resiquimod to Montanide is not sufficient to induce consistent NY-ESO-1-specific CD8⁺ T-cell responses.

Combination therapy targeting toll like receptors 7, 8 and 9 eliminates large established tumors.

BACKGROUND: The TLR7/8 agonist 3M-052 and the TLR9 agonist CpG ODN both trigger innate immune responses that support the induction of tumor-specific immunity. Previous studies showed that these agonists used individually could improve the survival of mice challenged with small tumors but were of limited therapeutic benefit against large/advanced tumors. METHODS: Normal mice were challenged with syngeneic tumors. Once these tumors reached clinically detectable size (500-800 mm(3)) they were treated by intra-tumoral injection with 3M-052 and/or CpG ODN. Anti-tumor immunity and tumor growth were evaluated. RESULTS: The co-delivery of agonists targeting TLRs 7, 8 and 9 increased the number and tumoricidal activity of tumor infiltrating CTL and NK cells while reducing the frequency of immunosuppressive MDSC. The combination of 3M-052 plus CpG ODN (but not each agent alone) eradicated large primary tumors and established long-term protective immunity. CONCLUSION: The combination of agonists targeting TLRs 7/8 and 9 represents a significant improvement in cancer immunotherapy.

The use of Toll-like receptor 7/8 agonists as vaccine adjuvants.

Small molecule Toll-like receptor (TLR) 7/8 agonists have demonstrated potential as vaccine adjuvants, since they directly activate APCs and can enhance both humoral and cellular immune responses, especially Th1 responses. Although the natural ligands for TLR7 and TLR8 are ssRNA, the vast majority of vaccine studies performed thus far have been performed with synthetic small molecule imidazoquinolines, such as imiquimod and resiquimod. Despite the approved clinical use of the topical TLR7 agonist, imiquimod (Aldara(®) Imiquimod 5% cream; 3M, MN, USA), for external genital warts, superficial basal cell carcinoma and actinic keratosis, no vaccines using TLR7, TLR8 or TLR7/8 agonists have progressed beyond early-phase clinical studies thus far. This review will highlight the nonclinical and clinical studies that indicate promise for TLR7/8 ligands as vaccine adjuvants, reasons for inconsistent results thus far, problems with current technology and potential paths forward for TLR7/8 agonists as vaccine adjuvants.

Binding of Soluble Yeast ß-Glucan to Human Neutrophils and Monocytes is Complement-Dependent.

The immunomodulatory properties of yeast ß-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate ß-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble ß-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast ß-glucan, this study evaluated and characterized the binding of soluble ß-glucan to human neutrophils and monocytes. The results demonstrated that soluble ß-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble ß-glucan in these cells. Binding of soluble ß-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble ß-glucan was demonstrated by detection of iC3b, the complement opsonin on ß-glucan-bound cells, as well as by the direct binding of iC3b to ß-glucan in the absence of cells. Binding of ß-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding.

Resiquimod and other immune response modifiers as vaccine adjuvants.

Synthetic immune response modifiers, such as resiquimod, are Toll-like receptor 7 and 8 agonists that act as vaccine adjuvants, enhancing antigen-specific antibody production and skewing immunity towards a Th1 response. These compounds stimulate dendritic cells to secrete cytokines, upregulate costimulatory molecule expression and enhance antigen presentation to T cells. The compounds have demonstrated vaccine adjuvant properties in a number of animal models. The adjuvant effects can be enhanced by measures that allow the drug to stay localized with the vaccine without quickly entering the systemic circulation. Clinical studies demonstrate that topical application of resiquimod and analogs is safe and effective at activating the local immune response. For injection, resiquimod or a similar compound may need to be formulated to allow for local immune activation without induction of systemic cytokines.