Sally T. Ishizaka

Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria

Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.

E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant

Safe and cost-effective adjuvants are a critical component to enhance the efficacy of subunit vaccines. Studies have demonstrated that modified natural lipid As derived from enterobacterial lipopolysaccharides, which are agonists of Toll-like receptor 4, are beneficial to vaccine performance. The synthetic phospholipid dimer, E6020, mimics the physicochemical and biological properties of many of the natural lipid As derived from Gram-negative bacteria. Similar to its natural counterparts, E6020, which was discovered and developed by Eisai, agonizes Toll-like receptor 4, albeit in an attenuated fashion, eliciting an immunostimulatory response that is conducive to use as a vaccine adjuvant. The derivation of E6020, along with physicochemical properties and in vitro and in vivo studies of immunostimulation and adjuvant activity, are reviewed as a background to its imminent assessment in the clinic.

Novel synthetic LPS receptor agonists boost systemic and mucosal antibody responses in mice.

Safe and cost-effective adjuvants are a critical requirement for subunit vaccine development. We report here the in vivo activity of a series of fully synthetic LPS receptor agonists that have been shown to activate NF-kappaB signaling through the Toll-like receptor 4 (TLR4). These compounds boost antibody responses to protein antigens when coadministered at microgram doses in mice. At these dosage levels no adverse effects are observed. Antibody responses are largely IgG1, with enhanced IgG2a, and down-regulated IgE as compared to alum adjuvanted immunization. Stimulation of Th1 is confirmed by enhanced gamma-interferon production after in vitro antigen restimulation of spleen cells from mice immunized with the synthetic adjuvants. The adjuvants are active by both subcutaneous and intranasal routes of vaccine administration, and in the latter case can amplify both serum IgG and serum and mucosal IgA responses. The compounds must be administered at the same site with antigen to boost anti-vaccine antibody. These fully synthetic ligands of the innate immune system offer the potential for use as effective, safe, and nonbiologically-derived adjuvants.

Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo

The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)–containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti–double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.

Synthetic Toll-like receptor 4 agonist enhances vaccine efficacy in an experimental model of toxic shock syndrome.

ABSTRACT The development of new protein subunit vaccines has stimulated the search for improved adjuvants to replace traditional aluminum-containing products. We investigated the adjuvant effects of a synthetic Toll-like receptor 4 (TLR4) agonist on vaccine efficacy in an experimental model of toxic shock syndrome. The TLR4 agonist E6020 has a simplified structure consisting of a hexa-acylated acyclic backbone. The vaccine examined is a recombinantly attenuated form of staphylococcal enterotoxin B (STEBVax). Using cells stably transfected with TLRs, E6020 transduced signals only through TLR4, suggesting monospecificity, while Escherichia coli 055:B5 lipopolysaccharide activated both the TLR2/6 heterodimer and TLR4. Coadministration of E6020 with STEBVax, by the intramuscular or intranasal route, induced significant levels of immunoglobulin G (IgG) in BALB/c mice. Further, increased IgG production resulted from the combination of E6020 with aluminum hydroxide adjuvant (AH). The antibody response to the vaccine coadministered with E6020 was a mixed Th1/Th2 response, as opposed to the Th2-biased response obtained with AH. Mice vaccinated with STEBVax coadministered with AH, TLR4 agonists, or a combination of both adjuvants were protected from toxic shock. Our data demonstrate the effectiveness of the synthetic TLR4 agonist E6020 as an alternative adjuvant for protein subunit vaccines that may also be used in combination with traditional aluminum-containing adjuvants.

Effective antibody therapy induces host protective antitumor immunity that is augmented by TLR4 agonist treatment

Toll-like receptors are potent activators of the innate immune system and generate signals leading to the initiation of the adaptive immune response that can be utilized for therapeutic purposes. We tested the hypothesis that combined treatment with a Toll-like receptor agonist and an antitumor monoclonal antibody is effective and induces host-protective antitumor immunity. C57BL/6 human mutated HER2 (hmHER2) transgenic mice that constitutively express kinase-deficient human HER2 under control of the CMV promoter were established. These mice demonstrate immunological tolerance to D5-HER2, a syngeneic human HER2-expressing melanoma cell line. This human HER2-tolerant model offers the potential to serve as a preclinical model to test both antibody therapy and the immunization potential of human HER2-targeted therapeutics. Here, we show that E6020, a Toll-like receptor-4 (TLR4) agonist effectively boosted the antitumor efficacy of the monoclonal antibody trastuzumab in immunodeficient C57BL/6 SCID mice as well as in C57BL/6 hmHER2 transgenic mice. E6020 and trastuzumab co-treatment resulted in significantly greater inhibition of tumor growth than was observed with either agent individually. Furthermore, mice treated with the combination of trastuzumab and the TLR4 agonist were protected against rechallenge with human HER2-transfected tumor cells in hmHER2 transgenic mouse strains. These findings suggest that combined treatment with trastuzumab and a TLR4 agonist not only promotes direct antitumor effects but also induces a host-protective human HER2-directed adaptive immune response, indicative of a memory response. These data provide an immunological rationale for testing TLR4 agonists in combination with antibody therapy in patients with cancer.

Assessment of the translational value of mouse lupus models using clinically relevant biomarkers

Lupus is an autoimmune disease with a poorly understood etiology that manifests with a diverse pathology. This heterogeneity has been a challenge to clinical drug development efforts. A related difficulty is the uncertain translational power of animal models used for evaluating potential drug targets and candidate therapeutics, because it is unlikely that any 1 preclinical model will recapitulate the spectrum of human disease. Therefore, multiple models, along with an understanding of the immune mechanisms that drive them, are necessary if we are to use them to identify valid drug targets and evaluate candidate therapies successfully. To this end, we have characterized several different mouse lupus models and report their differences with respect to biomarkers and symptoms that are representative of the human disease. We compared the pristane-induced mouse lupus disease model using 3 different strains (DBA/1, SJL, BALB/c), and the spontaneous NZB x NZW F1(NZB/W) mouse model. We show that the models differ significantly in their autoantibody profiles, disease manifestations such as nephritis and arthritis, and expression of type I interferon-regulated genes. Similar to the NZB/W model, pristane-induced disease in SJL mice manifests with nephritis and proteinuria, whereas the pristane-treated DBA/1 mice develop arthritis and an interferon-driven gene signature that closely resembles that in human patients. The elucidation of each models strengths and the identification of translatable biomarkers yields insight for basic lupus research and drug development, and should assist in the proper selection of models for evaluating candidate targets and therapeutic strategies.

LPS binding protein does not participate in the pharmacokinetics of E5564

E5564, a lipid A analogue, is a potent antagonist of lipopolysaccharide (LPS). Clinically, E5564 was developed as a possible therapy for treatment of sepsis and septic shock. Surface plasmon resonance (SPR) analysis indicates that E5564 binds to LPS binding protein (LBP), in a manner similar to LPS. Gel-filtration radioactive chromatograms of [14C]-E5564 in plasma revealed that E5564 initially distributes to the lipoprotein fractions, separated from high-density lipoprotein (HDL); the bound fraction is then released and binds to HDL. Similar results were obtained by heparin-manganese precipitation. At doses of E5564 relevant to its clinical use (i.e. 6 µg/ml), antibodies against LBP did not influence either the distribution of E5564 to non-HDL lipoprotein fractions or the transfer of E5564 from non-HDLs to HDL. Under these conditions, transfer of E5564 to HDL occurs similarly in the plasma of LBP knockout (KO) mice as in the plasma from wild-type mice. In addition, plasma clearance of E5564 in LBP KO mice is similar to that of wild-type mice. Thus, LBP binds E5564 in a manner similar to LPS, but does not play a role in E5564 redistribution/binding to lipoprotein and plasma clearance.

Modulators of Toll-Like Receptor (TLR) Signaling

Publisher Summary This chapter deals with the modulators of toll-like receptor (TLR) signaling. The rate of TLR-related publications has risen consistently over the past decade. Despite this increasing wealth of information, relatively few TLR modulators have been addresses in the chapter. Among the pharmaceutically relevant concepts, TLR4, TLR7, and TLR9 have been featured most prominently. In terms of development, the most advanced compound is TLR4 antagonist E5564, which is in Phase III sepsis trials. Also of interest is the TLR7/8/9 antagonist CPG 52364, which is in early clinical development for SLE. TLR2 (as a heterodimer with TLR1 or TLR6), MyD88, and IRAK-4 may also prove to be attractive therapeutic targets for various inflammatory diseases; however, few agents have been reported and these have not yet advanced beyond early discovery. This situation may change as recent publications have disclosed valuable structural information for TLR1/2, TLR2/6, TLR3, MyD88, and IRAK-4, which may help stimulate additional structure-based drug design efforts in these areas.