Discovery of M5049: A Novel Selective Toll-Like Receptor 7/8 Inhibitor for Treatment of Autoimmunity.

Toll-like receptor (TLR) 7 and TLR8 are transmembrane receptors that recognize single-stranded RNA. Activation of these receptors results in immune cell stimulation and inflammatory cytokine production, which is normally a protective host response. However, aberrant activation of TLR7/8 is potentially pathogenic and linked to progression of certain autoimmune diseases such as lupus. Thus, we hypothesize that an inhibitor that blocks TLR7/8 would be an effective therapeutic treatment. Prior efforts to develop inhibitors of TLR7/8 have been largely unsuccessful as a result of the challenge of producing a small-molecule inhibitor for these difficult targets. Here, we report the characterization of M5049 and compound 2, molecules which were discovered in a medicinal chemistry campaign to produce dual TLR7/8 inhibitors with drug-like properties. Both compounds showed potent and selective activity in a range of cellular assays for inhibition of TLR7/8 and block synthetic ligands and natural endogenous RNA ligands such as microRNA and Alu RNA. M5049 was found to be potent in vivo as TLR7/8 inhibition efficaciously treated disease in several murine lupus models and, interestingly, was efficacious in a disease context in which TLR7/8 activity has not previously been considered a primary disease driver. Furthermore, M5049 had greater potency in disease models than expected based on its in vitro potency and pharmacokinetic/pharmacodynamic properties. Because of its preferential accumulation in tissues, and ability to block multiple TLR7/8 RNA ligands, M5049 may be efficacious in treating autoimmunity and has the potential to provide benefit to a variety of patients with varying disease pathogenesis. SIGNIFICANCE STATEMENT: This study reports discovery of a novel toll-like receptor (TLR) 7 and TLR8 inhibitor (M5049); characterizes its binding mode, potency/selectivity, and pharmacokinetic and pharmacodynamic properties; and demonstrates its potential for treating autoimmune diseases in two mouse lupus models. TLR7/8 inhibition is unique in that it may block both innate and adaptive autoimmunity; thus, this study suggests that M5049 has the potential to benefit patients with autoimmune diseases.

Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Efficacy and Pharmacodynamic Modeling of the BTK Inhibitor Evobrutinib in Autoimmune Disease Models.

Because of its role in mediating both B cell and Fc receptor signaling, Bruton's tyrosine kinase (BTK) is a promising target for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that potently inhibits BCR- and Fc receptor-mediated signaling and, thus, subsequent activation and function of human B cells and innate immune cells such as monocytes and basophils. We evaluated evobrutinib in preclinical models of RA and SLE and characterized the relationship between BTK occupancy and inhibition of disease activity. In mouse models of RA and SLE, orally administered evobrutinib displayed robust efficacy, as demonstrated by reduction of disease severity and histological damage. In the SLE model, evobrutinib inhibited B cell activation, reduced autoantibody production and plasma cell numbers, and normalized B and T cell subsets. In the RA model, efficacy was achieved despite failure to reduce autoantibodies. Pharmacokinetic/pharmacodynamic modeling showed that mean BTK occupancy in blood cells of 80% was linked to near-complete disease inhibition in both RA and SLE mouse models. In addition, evobrutinib inhibited mast cell activation in a passive cutaneous anaphylaxis model. Thus, evobrutinib achieves efficacy by acting both on B cells and innate immune cells. Taken together, our data show that evobrutinib is a promising molecule for the chronic treatment of B cell-driven autoimmune disorders.

Protective effect of TSLP delivered at the gut mucosa level by recombinant lactic acid bacteria in DSS-induced colitis mouse model.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is a cytokine known to mature dendritics cells, lower pro-inflammatory IL-12 secretion, induce differentiation of anti-inflammatory FoxP3+ regulatory T cells (Treg). Moreover, Crohn's disease patients have shown a reduction of intestinal TSLP expression. To understand the role of TSLP in inflammation, we constructed Lactococcus lactis strain producing TSLP (LL-TSLP) and investigated the effect of its administration on dextran sulfate sodium (DSS)-induced colitis model in mice. RESULTS: LL-TSLP secrete an active molecule which lowers secretion of IL-12 by dendritic cells. Treatment with LL-TSLP, increases the amount of TGF-ß secreted by T cells in Mesenteric Lymph Node in healthy mice. In acute DSS-induced colitis, LL-TSLP delayed the Disease Activity Index and lowered histological score and colonic INF-γ production. In a DSS-recovery model, LL-TSLP induced a better protective effect if the strain was administered at the beginning of the colitis. At Day 4 of colitis we observed an induction of Treg by LL-TSLP. CONCLUSIONS: TSLP showed an anti-inflammatory protective role in DSS-induced colitis. We have demonstrated that a short and early administration of LL-TSLP is more efficient than a long lasting treatment.

Food allergy in mice is modulated through the thymic stromal lymphopoietin pathway.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is involved in the pathogenesis of allergic reactions in the skin and the lung. Nevertheless, data on the role of TSLP in food allergy are scarce. We explored the role of TSLP in a mouse model with oral sensitization and oral challenge eliciting food allergy. METHODS: TSLP receptor (TSLPR)-/- mice and wild type mice were orally sensitized to ß-lactoglobulin in presence of cholera toxin (CT) or CT alone. The elicited immune response was characterized in vitro and the mice were subsequently challenged with the antigen. Lymphocytes from various locations in the gut were activated either by the antigen or by CT and assayed for cytokine secretion. RESULTS: Here we report that TSLPR-/- are less prone to generate food-induced reactions in conjunction with a decreased antigen-specific IgG1, but not IgE response. In addition, mesenteric lymphnode lymphocytes of TSLPR-/- mice were secreting lower quantities of IL-4, IL-5 and IL-10 after in vivo Ag activation, whereas higher numbers of IL-17 secreting cells were observed. Similarly, activation by the Th2-type adjuvant cholera toxin resulted in an increased frequency of IL-12 and IL-17 secreting lamina propria and mesenteric lymphocytes, together with increased production of IL-12 by activated dendritic cells in TSLPR-/- mice. CONCLUSIONS: TSLP can be considered as an essential, but not exclusive, mediator for elicitation of food allergy in mice, as well as a potential target for future therapeutic interventions.

Discovery of potent, selective, and orally bioavailable alkynylphenoxyacetic acid CRTH2 (DP2) receptor antagonists for the treatment of allergic inflammatory diseases.

New phenoxyacetic acid antagonists of CRTH2 are described. Following the discovery of a hit compound by a focused screening, high protein binding was identified as its main weakness. Optimization aimed at reducing serum protein binding led to the identification of several compounds that showed not only excellent affinities for the receptor (41 compounds with K(i) < 10 nM) but also excellent potencies in a human whole blood assay (IC(50) < 100 nM; PGD2-induced eosinophil shape change). Additional optimization of the PK characteristics led to the identification of several compounds suitable for in vivo testing. Of these, 19k and 19s were tested in two different pharmacological models (acute FITC-mediated contact hypersensitivity and ovalbumin-induced eosinophilia models) and found to be active after oral dosing (10 and 30 mg/kg).

Dibutyl phthalate-induced thymic stromal lymphopoietin is required for Th2 contact hypersensitivity responses.

Thymic stromal lymphopoietin (TSLP) is an IL-7-related cytokine, produced by epithelial cells, that has been linked to atopic dermatitis and asthma; however, it remains unclear how TSLP shapes the adaptive immune response that causes these allergic disorders. In this study, we demonstrate a role for TSLP in a Th2 model of contact hypersensitivity in mice. TSLP is required for the development of Th2-type contact hypersensitivity induced by the hapten FITC in combination with the sensitizing agent dibutyl phthalate. TSLPR-deficient mice exhibited a dramatically reduced response, including markedly reduced local infiltration by eosinophils, Th2 cytokine production, and serum IgE levels, following FITC sensitization and challenge. The reduced response by TSLPR-deficient mice is likely due to decreased frequency and reduced T cell stimulatory function of skin-derived Ag-bearing FITC(+)CD11c(+) dendritic cells in draining lymph nodes following FITC sensitization. These data suggest that skin-derived dendritic cells are direct or indirect targets of TSLP in the development of type 2 immune responses in the skin, where TSLP drives their maturation, accumulation in skin draining lymph nodes, and ability to induce proliferation of naive allergen-specific T cells.

Human epidermal Langerhans cells express the tight junction protein claudin-1 and are present in human genetic claudin-1 deficiency (NISCH syndrome).

Claudin-1 (CLDN1) is a structural tight junction (TJ) protein and is expressed in differentiating keratinocytes and Langerhans cells in the epidermis. Our objective was to identify immunoreactive CLDN1 in human epidermal Langerhans cells and to examine the pattern of epidermal Langerhans cells in genetic human CLDN1 deficiency [neonatal ichthyosis, sclerosing cholangitis (NISCH) syndrome]. Epidermal cells from healthy human skin labelled with CLDN1-specific antibodies were analysed by confocal laser immunofluorescence microscopy and flow cytometry. Skin biopsy sections of two patients with NISCH syndrome were stained with an antibody to CD1a expressed on epidermal Langerhans cells. Epidermal Langerhans cells and a subpopulation of keratinocytes from healthy skin were positive for CLDN1. The gross number and distribution of epidermal Langerhans cells of two patients with molecularly confirmed NISCH syndrome, however, was not grossly altered. Therefore, CLDN1 is unlikely to play a critical role in migration of Langerhans cells (or their precursors) to the epidermis or their positioning within the epidermis. Our findings do not exclude a role of this TJ molecule once Langerhans cells have left the epidermis for draining lymph nodes.

Cutting edge: Lack of evidence for connexin-43 expression in human epidermal Langerhans cells.

A provocative study has shown that viral peptides may be transferred in vitro from epithelial cells to APC through connexin-43 gap junction channels. In support of this cross-presentation pathway, the study also reported that human dendritic cells, including Langerhans cells of skin, express connexin-43. In this report we show that if this was the case, the levels of connexin-43 are below those detectable by immunofluorescence, flow cytometry, quantitative PCR of purified CD1a+ cells, and electron microscopy, raising questions about the relevance of the connexin-43-dependent mechanism for Langerhans cells of noninflamed human skin.

Langerhans cells and lymph node dendritic cells express the tight junction component claudin-1.

Claudin-1 is a critical structural component of tight junctions that have an important role in adhesive properties, barrier function, and paracellular transport of epithelia and other nonhematopoietic tissues. We found claudin-1 in murine CD207+ Langerhans cells (LC) residing in epidermis. Claudin-1 was not detected in other skin dendritic cells (DC). LC expressed claudin-1 in steady state and inflamed skin. Claudin-1 was demonstrated further in lymph node LC under steady state and inflammatory conditions, including after direct tracking with tetramethylrhodamine-isothiocyanate (TRITC). All subsets of skin draining lymph node DC defined by CD205, CD11b, CD11c, and CD8, including a presumably blood-borne lymph node resident CD8+CD207+ LC population, were claudin-1+. TRITC tracking demonstrated claudin-1 in CD207- skin migrant DC in the lymph node, suggesting upregulation of this molecule during migration or once arrived in the lymph node. Claudin-1 expression in CD207+ cells was confirmed at the protein and mRNA levels. Transforming growth factor-beta, a factor critical for the induction of LC in vitro and in vivo, stimulated the accumulation of claudin-1 mRNA and protein when added to bone marrow cells cultured with GM-CSF and IL-4. Claudin-1 may thus have an important function in adhesion and/or migration of LC.

HIV-1-specific IFN-gamma/IL-2-secreting CD8 T cells support CD4-independent proliferation of HIV-1-specific CD8 T cells.

Functional and phenotypic characterization of virus-specific CD8 T cells against cytomegalovirus, Epstein-Barr virus, influenza (flu), and HIV-1 were performed on the basis of the ability of CD8 T cells to secrete IFN-gamma and IL-2, to proliferate, and to express CD45RA and CCR7. Two functional distinct populations of CD8 T cells were identified: (i) dual IFN-gamma/IL-2-secreting cells and (ii) single IFN-gamma-secreting cells. Virus-specific IFN-gamma/IL-2-secreting CD8 T cells were CD45RA-CCR7-, whereas single IFN-gamma CD8 T cells were either CD45RA-CCR7- or CD45RA+CCR7-. The proportion of virus-specific IFN-gamma/IL-2-secreting CD8 T cells correlated with that of proliferating CD8 T cells, and the loss of HIV-1-specific IL-2-secreting CD8 T cells was associated with that of HIV-1-specific CD8 T cell proliferation. Substantial proliferation of virus-specific CD8 T cells (including HIV-1-specific CD8 T cells) was also observed in CD4 T cell-depleted populations or after stimulation with MHC class I tetramer-peptide complexes. IL-2 was the factor responsible for the CD4-independent CD8 T cell proliferation. These results indicate that IFN-gamma/IL-2-secreting CD8 T cells may promote antigen-specific proliferation of CD8 T cells even in the absence of helper CD4 T cells.

Attenuated poxviruses expressing a synthetic HIV protein stimulate HLA-A2-restricted cytotoxic T-cell responses.

Efficient HIV vaccines have to trigger cell-mediated immunity directed against various viral antigens. However little is known about the breadth of the response induced by vaccines carrying multiple proteins. Here, we report on the immunogenicity of a construct harbouring a fusion of the HIV-1 IIIB gag, pol and nef genes (gpn) designed for optimal safety and equimolar expression of the HIV proteins. The attenuated poxviruses, MVA and NYVAC, harbouring the gpn construct, induced potent immune responses in conventional mice characterised by stimulation of Gpn-specific IFN-gamma-producing cells and cytotoxic T cells. In HLA-A2 transgenic mice, recombinant MVA elicited cytotoxic responses against epitopes recognised in most HLA-A2+ HIV-1-infected individuals. We also found that the MVA vaccine triggered the in vitro expansion of peripheral blood cells isolated from a HIV-1-seropositive patient and with similar specificity as found in immunised HLA-A2 transgenic mice. In conclusion, the synthetic HIV polyantigen Gpn delivered by MVA is immunogenic, efficiently processed and presented by human MHC class I molecules.

Cytomegalovirus (CMV)-specific cellular immune responses.

A large percentage of healthy individuals (50-90%) is chronically infected with Cytomegalovirus (CMV). Over the past few years, several techniques were developed in order to monitor CMV-specific T-cell responses. In addition to the identification of antigen-specific T cells with peptide-loaded MHC complexes, most of the current strategies to identify CMV-specific T cells are centered on the assessment of the functions of memory T cells including their ability to mediate effector function, to proliferate or to secrete cytokines following antigen-specific stimulation. The investigation of these functions has allowed the characterization of the CMV-specific T-cell responses that are present during different phases of the infection. Furthermore, it has also been shown that the combination of virus-specific CD4 and CD8 T-cell responses are critical components of the immune response in the control of virus replication.