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Osteoarthritis (OA) is a common disease worldwide with large unmet medical needs. To bring innovative treatments to OA patients, we at Merck have implemented a comprehensive strategy for drug candidate evaluation. We have a clear framework for decision-making in our preclinical pipeline, to design our clinical proof-of-concept trials for OA patients. We have qualified our strategy to define and refine dose and dosing regimen, for treatments administered either systemically or intra-articularly (IA). We do this through preclinical in vitro and in vivo studies, and by back-translating results from clinical studies in OA patients.

The rational targeting of a transcription factor, interferon regulatory factor 5 (IRF5), inhibits downstream functions., The transcription factor interferon regulatory factor 5 (IRF5) plays essential roles in pathogen-induced immunity downstream of Toll-, nucleotide-binding oligomerization domain–, and retinoic acid–inducible gene I–like receptors and is an autoimmune susceptibility gene. Normally, inactive in the cytoplasm, upon stimulation, IRF5 undergoes posttranslational modification(s), homodimerization, and nuclear translocation, where dimers mediate proinflammatory gene transcription. Here, we report the rational design of cell-penetrating peptides (CPPs) that disrupt IRF5 homodimerization. Biochemical and imaging analysis shows that IRF5-CPPs are cell permeable, noncytotoxic, and directly bind to endogenous IRF5. IRF5-CPPs were selective and afforded cell type– and species-specific inhibition. In plasmacytoid dendritic cells, inhibition of IRF5-mediated interferon-α production corresponded to a dose-dependent reduction in nuclear phosphorylated IRF5 [p(Ser462)IRF5], with no effect on pIRF5 levels. These data support that IRF5-CPPs function downstream of phosphorylation. Together, data support the utility of IRF5-CPPs as novel tools to probe IRF5 activation and function in disease.

Background: The Phase 2/3 APRIL-SLE study evaluated the safety and efficacy of atacicept, a dual inhibitor of the B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), in systemic lupus erythematosus (SLE). Objectives: The goal of this post-hoc analysis was to use cell-based gene signatures on gene expression data from the APRIL-SLE study to identify clusters of patients with potential to flare and to assess clusters for differences in treatment effects of atacicept vs placebo. Methods: A published immune cell deconvolution algorithm (Abbas et al, 2009) was applied to whole-blood gene expression data from APRIL-SLE patients to identify relative proportions of 17 immune cell types. Patients were then grouped into clusters based on these immune cell profiles using a k-medoid clustering algorithm and were compared to each other based on patient characteristics, biomarkers and clinical efficacy. In addition, baseline expression and change in expression of putative APRIL-responder genes were compared among clusters. APRIL-responder genes were identified by combining differential expression results from the APRIL-SLE study (Week 52 vs Day 1 randomization) and tabalumab (targets BLyS only) Phase 3 studies (Week 52 vs baseline; GSE88887). Results: Patient gene expression data (N=105; placebo, n=30; atacicept 75 mg, n=40; atacicept 150 mg, n=35) were used to group patients into five main clusters (P1-P5) by predominant characteristic cells: P1, T helper cells; P2, plasma cells; P3, neutrophils and B cells; P4, B cells; P5, activated dendritic cells. Patients in P2 and P5 were more likely to have positive anti-dsDNA antibodies (≥30 IU/ml), elevated BLyS; ≥1.6 ng/ml, and high interferon gene signature in the blood than other those in other clusters. Patients in P2 were most likely to have low complement C3 and C4 levels. Placebo-group flare rates in P2 (100%), P4 (100%) and P5 (83%) were markedly higher than in P1 (33%) and P3 (29%). In P2, P4, and P5 the median time-to-flare was much lower with placebo (85, 98.5, and 115.5 days, respectively) than with atacicept 150 mg (over 364 days for all three clusters). A comparison of differentially-expressed genes from clinical studies of SLE patients treated with atacicept and tabalumab revealed possible APRIL-responder genes: SDC1, PARM1 and MZB1. These genes had a higher baseline expression in P2 and P4 compared with other clusters. SDC1 was reduced from baseline more in P2, P4, and P5 after atacicept treatment, while PARM1 and MZB1 decreased after atacicept treatment in P2 and P4. Conclusion: These post-hoc analyses revealed different subsets of SLE patients based on their molecular profiles. Atacicept may have different treatment effects in the identified patient subsets vs placebo, providing insights into potential mechanisms of flare in SLE. References: [1] Abbas AR, Wolslegel K, Seshasayee D, Modrusan Z, Clark HF. Deconvolution of blood microarray data identifies cellular activation patterns in systemic lupus erythematosus. PLoS ONE. 2009;4(7):e6098. Disclosure of Interests: Eileen Samy Employee of: Current employees of EMD Serono, Matthew Studham Employee of: Current employees of EMD Serono, Amy Kao Employee of: Current employees of EMD Serono, Philipp Haselmayer Employee of: Current employee of Merck KGaA, Peter Chang Employee of: Current employees of EMD Serono, P. Alexander Rolfe Employee of: Current employees of EMD Serono, David Wofsy Consultant for: GlaxoSmithKline – Member, data safety monitoring board Novartis – Member, data safety monitoring board Celgene – member, scientific advisory board, Julie DeMartino Shareholder of: Shares in Merck & Co, Employee of: Current employees of EMD Serono; Past employee of Merck & Co. & Roche, Robert Townsend Employee of: Current employees of EMD Serono

Background The Phase II/III APRIL-SLE study evaluated the safety and efficacy of atacicept in systemic lupus erythematosus (SLE). The goal of this post-hoc analysis was to use cell-based gene signatures on the APRIL-SLE gene expression data to identify clusters of patients with potential to flare and to assess for difference in treatment effect of atacicept vs placebo. Methods A published immune cell deconvolution algorithm was applied to whole-blood gene expression data from APRIL-SLE to identify relative proportions of 17 immune cell types. Patients were then grouped into clusters based on these immune cell profiles using a k-medoid clustering algorithm, and were compared to each other based on patient characteristics, biomarkers and clinical efficacy. In addition, the baseline expression and change in expression of putative APRIL-responder genes were compared among the clusters. APRIL-responder genes were identified by combining differential expression results from the APRIL-SLE study (Week 52 vs. Day 1 randomization) and tabalumab Phase III studies (Week 52 vs. Baseline; GSE88887). Results Patient gene expression data (N=105; Placebo: N=30; atacicept 75 mg: N=40; atacicept 150 mg N=35) was used to group patients into 5 main clusters (P1-P5) by predominant characteristic cells: P1, T helper cells; P2, plasma cells; P3, neutrophils and B cells; P4, B cells; P5, activated dendritic cells. Patients in P2 and P5 were more likely to have positive anti-dsDNA antibodies (≥30 IU/ml) and elevated BLyS (≥1.6 ng/ml), as well as high IFN gene signature in the blood. Patients in P2 were more likely to have low complement C3 and C4 levels. In P2, P4, and P5 clusters the flare rate in the placebo group was significantly higher than in P1 and P3. In P2 and P4, atacicept 150mg treatment group showed delayed time to flare and reduced flare rate as compared with the placebo group. A comparison of differentially-expressed genes from clinical studies of SLE patients on atacicept (targets BLyS & APRIL) vs tabalumab (targets BLyS) revealed possible APRIL-responder genes: SDC1, PARM1 and MZB1. These genes have a higher baseline expression in the P2 and P4 compared to other clusters. SDC1 was reduced more in P2, P4, and P5 after atacicept treatment, while PARM1 and MZB1 decreased after atacicept treatment in P2 and P4. Conclusions These post-hoc analyses revealed different subsets of SLE patients based on their molecular profiles, which identified patient subsets that might have differential treatment effect of atacicept vs placebo, and provided insights into potential mechanisms of flare. Funding Source(s): Merck KGaA, Darmstadt, Germany

Background Atacicept is an antagonist of the B cell regulatory factors BLyS (B lymphocyte stimulator) and APRIL (a proliferation inducing ligand). It is capable of binding to all known conformations of BLyS and APRIL and is thus expected to modulate the maturation, differentiation, and effector function of B cells. We aimed to define the relationship between atacicept drug exposure and pharmacodynamic (PD) effects in vivo. Here, we report results of our study examining the effects of atacicept on the immune response in a keyhole limpet hemocyanin (KLH) challenge model in cynomolgus monkeys. Methods Cynomolgus monkeys (Macaca fascicularis) were injected with KLH on Day −33, and on Day 1 received the first dose of either atacicept (0.3, 3, or 30 mg/kg) or vehicle followed by a KLH challenge. Animals were dosed weekly (Day 1, 8, 15) and were subsequently monitored for 2 weeks before the study ended on Day 29. Clinical signs, total and free drug levels, peripheral blood B and T cell subpopulations, and total and KLH-specific immunoglobulin (Ig) levels were monitored. Additionally, gene expression in blood was analysed using the NanoString nCounter platform. Results Atacicept was well-tolerated at all dose levels tested. All animals in the treatment arms had quantifiable levels of atacicept in serum throughout the study. Reduction of serum IgM and IgG was detected 7 days after treatment, with a continuous reduction in the mean serum IgM and IgG levels observed until Day 29 (table 1). In animals treated with 3 and 30 mg/kg atacicept, a significant decrease in serum anti-KLH IgG levels was observed versus vehicle-treated controls, beginning at Day 11 (table 2). A minor reduction in absolute CD3-CD20+, IgD +B cell numbers was seen in response to treatment, but no changes in T cell subsets were detected. Changes in gene expression following atacicept treatment were predominantly observed in B cell-related and Ig genes. Conclusions This study showed that atacicept modulates B cell responses, IgM and IgG levels, and Ig isotype switching in a KLH-antigen-challenged cynomolgus monkey model, thus supporting its use in the treatment of antibody-mediated diseases. Additionally, gene expression PD markers identified in this study will be used in subsequent clinical trials as exploratory PD readouts. Disclosure of Interest E. Samy Employee of: EMD Serono Research and Development Institute, Inc. (a business of Merck KGaA, Darmstadt, Germany), A. Bender Employee of: EMD Serono Research and Development Institute, Inc. (a business of Merck KGaA, Darmstadt, Germany), Y. Wu Employee of: EMD Serono Research and Development Institute, Inc. (a business of Merck KGaA, Darmstadt, Germany), V. Castagna Employee of: Istituto di Ricerche Biomediche Antoine Marxer RBM SpA, an affiliate of Merck KGaA, E. Bertotti Employee of: Istituto di Ricerche Biomediche Antoine Marxer RBM SpA, an affiliate of Merck KGaA, R. Boggio Employee of: Istituto di Ricerche Biomediche Antoine Marxer RBM SpA, an affiliate of Merck KGaA, A. Paoletti Employee of: Istituto di Ricerche Biomediche Antoine Marxer RBM SpA, an affiliate of Merck KGaA, S. Riva Employee of: Istituto di Ricerche Biomediche Antoine Marxer RBM SpA, an affiliate of Merck KGaA, P. Schneider Grant/research support from: EMD Serono Research and Development Institute, Inc. and Merck KGaA, P. Haselmayer Employee of: Merck KGaA, J. DeMartino Employee of: EMD Serono Research and Development Institute, Inc. (a business of Merck KGaA, Darmstadt, Germany)

Objectives Bruton9s tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). Materials and methods Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. Results Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. Conclusions Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases.

Objective Bruton's tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE. Methods Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age. Results The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti–double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138highB220low plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex–mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice. Conclusion Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of high unmet medical need. Although bromodomain (Brd) and extra terminal domain isoforms have recently been implicated in mediating inflammatory and oncologic indications, their roles in lung fibrosis have not been comprehensively assessed. We investigated the role of Brd on the profibrotic responses of lung fibroblasts (LFs) in patients with rapidly progressing IPF and a mouse bleomycin model of lung fibrosis. The enhanced migration, proliferation, and IL-6 release observed in LFs from patients with rapidly progressing IPF are attenuated by pharmacologic inhibition of Brd4. These changes are accompanied by enhanced histone H4 lysine5 acetylation and association of Brd4 with genes involved in the profibrotic responses in IPF LFs as demonstrated using chromatin immunoprecipitation and quantitative PCR. Oral administration of 200 mg/kg per day Brd4 inhibitor JQ1 in a therapeutic dosing regimen substantially attenuated lung fibrosis induced by bleomycin in C57BL/6 mice. In conclusion, this study shows that the Brd4 inhibitor JQ1, administered in a therapeutic dosage, is capable of inhibiting the profibrotic effects of IPF LFs and attenuates bleomycin-induced lung fibrosis in mice. These results suggest that Brd4 inhibitors may represent a novel therapy for the treatment of rapidly progressing IPF.

Organ transplant patients are often treated with immunosuppressants, such as the calcineurin phosphatase inhibitor, cyclosporin A, to block T cell-mediated graft rejection. The calcium release-activated calcium (CRAC/ORAI) channels, which act upstream of calcineurin, are essential for calcium entry and CD4+ T-cell activation. Although cyclosporine A has also been shown to inhibit FoxP3+ Tregs both in vitro and in vivo, the role of ORAI channel inhibition in natural Tregs (nTregs) or inducible Tregs (iTregs) has not been investigated. We found that, despite inhibition of calcium influx through the ORAI channels, ORAI channel inhibitors were unable to repress FoxP3 expression in mouse and human nTregs, whereas FoxP3 expression was inhibited in iTregs. In contrast, cyclosporin A inhibited FoxP3 expression in both nTregs and iTregs. We also generated mice with a T cell-specific, conditional knockout of ORAI1 and found that the mice have normal nTreg development and suppressive activity. Moreover, iTregs derived from ORAI1 conditional knockout mice develop normally and are still susceptible to ORAI channel inhibition. Our data indicate that unlike CD4+ T cells and iTregs, nTregs are resistant to ORAI-mediated inhibition. Targeting ORAI channels potentially offers a novel way to inhibit pathologic T cells, while sparing nTreg-mediated tolerance.

Spleen Tyrosine Kinase (SYK) and Bruton's Tyrosine Kinase (BTK) are non-receptor cytoplasmic tyrosine kinases that are primarily expressed in cells of hematopoietic lineage. Both are key mediators in coupling activated immunoreceptors to downstream signaling events that affect diverse biological functions, from cellular proliferation, differentiation and adhesion to innate and adaptive immune responses. As such, pharmacological inhibitors of SYK or BTK are being actively pursued as potential immunomodulatory agents for the treatment of autoimmune and inflammatory disorders. Deregulation of SYK or BTK activity has also been implicated in certain hematological malignancies. To date, from a clinical perspective, pharmacological inhibition of SYK activity has demonstrated encouraging efficacy in patients with rheumatoid arthritis (RA), while patients with relapsed or refractory chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) have benefited from covalent inhibitors of BTK in early clinical studies. Here, we review and discuss recent insights into the emerging role of the SYK-BTK axis in innate immune cell function as well as in the maintenance of survival and homing signals for tumor cell progression. The current progress on the clinical development of SYK and BTK inhibitors is also highlighted.

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain-containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor-responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

Genetic mutation and pharmacological inhibition of Bruton9s tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2 H -isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fce receptor cross-linking-induced degranulation in mast cells (IC 50 = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC 50 = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC 50 = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Bruton's tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE.Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age.The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti-double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138(high) B220(low) plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex-mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice.Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A)/TNF superfamily member 15 (TNFSF15) is a proinflammatory cytokine and TNFα superfamily member that is linked preclinically and clinically to inflammatory bowel disease (IBD). By homology and function, TNFα is its closest family member. In this study, we investigated the mechanism of TL1A-induced inflammation in CD4+ T cells and compared it with the TNFα pathway. We found that TL1A induces proinflammatory cytokines, including TNFα, from isolated human CD4+CD161+ T cells, whereas these cells were resistant to TNFα treatment. Anti-TNFα failed to block TL1A-induced cytokine production, indicating that the effects of TL1A are direct. Lastly, CD161 and TL1A expression were significantly and selectively increased in gut tissue biopsies, but not in the peripheral blood, from IBD patients. Thus, TLIA not only functions upstream of TNFα, driving its expression from CD161+ T cells, but is also independent of TNFα. These findings may have therapeutic IBD implications.

Aims: To characterize monocyte response in a delayed-type hypersensitivity reaction to intradermal tetanus toxoid (TT) injection. Materials & methods: Men with positive serum anti-tetanus titers were stratified by last TT vaccination. Subjects were administered three intradermal injections of TT and one saline control on the same side of the back. Skin biopsies were taken post-injection. After 2 weeks, the procedure was repeated on the contralateral side. Results: Men who received TT booster vaccination 1 month before the study showed greater reproducibility and lower variability in monocyte responses than those who were not revaccinated. Monocyte concentration in subjects re-vaccinated within 1 month of study start appeared maximal at 48 h post-injection. Conclusion: This assay represents a novel approach that allows for quantification of dermal monocyte/macrophage influx. This clinical methodology has potential utility in the pharmacodynamic evaluation of therapies targeting inflammatory disorders, which involve monocyte tissue recruitment, like the delayed-type hypersensitivity response.

Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.

Background Rheumatoid arthritis (RA) is a chronic and progressive autoinflammatory disorder characterized by the infiltration of inflammatory cells, including B-cells, T-cells and macrophages, in the synovial membrane ultimately leading to cartilage destruction and bone erosion. Clinical disease activity in RA correlates strongly with macrophage numbers in synovial tissue and expression of macrophage-derived cytokines. Bruton’s tyrosine kinase (Btk) is important not only in B cell activation, but also mediates immune complex-dependent activation of monocytes. Pharmacological inhibition of Btk has been shown to be effective in suppressing pathology in murine models of arthritis Objectives The objective of this study was to determine the potential role of Btk in the pathology of RA. Methods Btk expression was detected by immunohistochemistry and digital image analysis in synovial tissue from 16 RA and 12 PsA patients, naive to treatment with biologicals. Immunofluorescent double labelling confocal microscopy was performed to identify which cell types express Btk in RA synovial tissue. Validating qRT-PCR and immunoblotting experiments were performed on isolated relevant cell populations. Effects of a specific Btk inhibitor, RN486, on activation-dependent human macrophage IL-6 production (n=8) and RA synovial tissue explant cultures (n=6) were assessed by ELISA. RN486 influence on macrophage expression of genes related to angiogenesis, cellular adhesion, tissue remodelling and innate immune responses was determined using low density qPCR arrays. Results Btk was expressed at equivalent levels in patients with RA and PsA. No relationship was observed between expression levels and patient clinical characteristics (CRP, ESR, DAS28, RF-positivity). In RA, but not PsA, Btk expression was significantly related to numbers of synovial macrophages (R= 0.63, p Conclusions Btk is expressed in RA synovial tissue and macrophages would be prominent synovial targets of strategies aimed at inhibiting Btk in RA. Btk activity is needed to drive macrophage activation in response to multiple stimuli relevant to RA, and drives IL-6 production in RA synovial tissue. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA. Disclosure of Interest L. Hartkamp: None Declared, I. van Es: None Declared, J. Fine Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, M. Smith Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, J. Woods Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, S. Narula Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, J. DeMartino Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, P. Tak Consultant for: GlaxoSmithKline, Employee of: GlaxoSmithKline, K. Reedquist Grant/research support from: Hoffmann-La Roche

The p110δ isoform of PI3K is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which PI3K mediates inflammation are poorly understood. Here we describe a novel highly selective p110δ small molecule inhibitor, G-286, that allowed dissecting the contribution specifically of p110δ to cellular pathways that are implicated in arthritis disease pathogenesis in human and mice. Using G-286, we demonstrate that p110δ regulates B cell- but not myeloid-cell dependent inflammatory arthritis in mice. G-286 blocks B cell receptor dependent proliferation and reduces autoantibody levels in collagen-induced arthritis abrogating disease. However, G-286 does not significantly inhibit FcγR-mediated inflammatory cytokine production in murine macrophages. Accordingly, selective p110δ inhibition did not significantly affect disease progression in FcγR- and myeloid cell-dependent autoantibody-induced arthritis. Importantly, we provide evidence that human and mice diverge in their relative dependency on p110δ to regulate FcγR responses. In addition to B cells and myeloid cells, T cells and osteoclasts contribute to rheumatoid arthritis pathogenesis. Utilizing G-286, we dissect the relative impact of p110δ inhibition on human and mouse T cell and osteoclast function. These results provide new insight in the role of p110δ in arthritis disease processes and offer a compelling rationale for targeting p110δ for the treatment of human rheumatoid arthritis.