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Background:Janus kinase (JAK) inhibitors (JAKinibs) show similar efficacy in rheumatoid arthritis (RA). However, in vitro studies have shown differences in JAK selectivity profiles for baricitinib (BARI), tofacitinib (TOFA), upadacitinib (UPA) and filgotinib (FIL).1,2 These lead to distinct pharmacologic profiles in cellular signaling assays that may impact clinical efficacy or safety1. NK cells are innate lymphocytes important in anti-pathogen responses and immune surveillance, which function via production of cytokines and cell killing3. NK cell proliferation and IFNγ production are JAK-dependent pathways and may be modulated by JAKinibs. Clinical findings show transient decreases in NK cell numbers in patients treated with JAKinibs, but the link to safety is unclear4Objectives:To extend upon findings in proximal cell signaling assays, we compared the selectivity and potency of clinical JAKinibs on NK cell function by assessing proliferation mediated by IL-15 (JAK1/3) and IFN-γ production driven by IL-12 (JAK2/TYK2)+IL-18.Methods:NK cells were isolated from healthy donor PBMC, incubated in vitro with 8 concentrations of each evaluated JAKinib (TOFA, BARI, FIL, FIL metabolite, UPA) and stimulated with IL-15 for proliferation or IL-12/18 for IFNγ production. Proliferation was assessed by Cell Trace dye dilution after 6 days and IFNγ production by intracellular flow cytometry 4hrs post-stimulation. Half maximal inhibitory concentration (IC50) values were calculated for CD56bright, CD56dim, and total NK cells. Steady-state pharmacologic profile over a clinical dosing interval was modeled using concentration-time profiles from JAKinib population pharmacokinetic data in RA subjects under the therapeutic dose5-7. For each JAKinib, the time above IC50 and average daily inhibition of IFNγ or proliferation were calculated for each NK cell population in each donor.Results:Cellular assays in purified NK cells showed dose-dependent inhibition of IL-15-induced proliferation by all JAKinibs with TOFA showing the highest average inhibition and time above IC50 (35-60% inhibition for 8-15 hrs; TOFA>UPA>BARI≈FIL). The differences between JAKinibs are in line with differences in pSTAT inhibition downstream of IL-151. Interestingly, IL-12/18-induced production of IFNγ, which is mediated via JAK2/TYK2 (IL-12) and non-JAK dependent pathways (IL-18), showed weaker inhibition for all compounds. Moreover, all JAKinibs showed 50 for IFNγ production or pSTAT4 inhibition at clinical doses. CD56dim and CD56bright sub-populations of NK cells are proposed to have distinct functions and unique expression of surface receptors. Analysis of the IC50 for pSTAT4 and IFNγ production showed ~2-10-fold weaker inhibition by JAKinibs in CD56bright NK cells, suggesting less dependence on JAK-dependent signals in CD56bright NK cells than CD56dim NK cells.Conclusion:NK cell proliferation depends on JAK1 and JAK3-mediated signaling and is differentially inhibited at clinical doses of distinct JAKinibs. In contrast, functional responses downstream of JAK2/TYK2-dependent IL-12/18 were not substantially inhibited by any of the JAKinibs studied. Inhibition of functional and proliferative responses in purified NK cells aligned well with proximal pSTAT inhibition. JAKinib modulation of NK cell proliferation, but not response to IL-12, reflects unique pharmacologic profiles of the drugs studied and could be one component underlying clinical safety observations, including increased risk of viral infections or malignancy4.References:[1]Traves PG et al. Ann Rheum Dis 2021 (in press)[2]McInnes IB, et al. Arthritis Res Ther 2019;21:183.[3]Cooper MA, Fehniger TA, Caligiuri MA. Trends Immunol 2001 Nov;22(11):633-40.[4]Winthrop KL. Nat Rev Rheumatol 2017; 13(4):234-243[5]Zhang X, et al. CPT Pharmacometrics Syst Pharmacol 2017;6(12):804-13.[6]CDER. Application Number: 203214Orig1s000. NDA 203214: Tofacitinib.[7]Klunder B et al. Clin Pharmacokinet 2019;58(8):1045-58.Disclosure of Interests:Paqui Gonzalez-Traves Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Laura Simpson Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Bernard Murray Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Amy Meng Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Julie A. Di Paolo Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Ethan Grant Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Gundula Min-Oo Shareholder of: Gilead Sciences, Employee of: Gilead Sciences

Background:Patients showing inadequate or no response to current therapies represent a key unmet need in rheumatoid arthritis (RA). To address this, novel or combination therapies are of high clinical interest. Identification of novel therapeutic targets requires a greater understanding of the pathogenic molecular drivers in the RA synovium. However, our current knowledge of human molecular patterns that emerge as a result of disease progression is complicated by patient-to-patient heterogeneity and access to synovial tissue.Objectives:Here we use the current knowledge of human synovial heterogeneity to conduct a longitudinal study of global molecular responses in the rat collagen-induced arthritis (CIA) model to better understand synovial biology, improve the preclinical modeling of human disease, and discover novel targets for RA.Methods:A rat CIA model was performed as previously described.1RNA-Seq was performed on 56 knee synovial tissues collected at multiple time points throughout the course of disease. Differential gene expression was determined at each individual time point and longitudinally with disease progression. Published human synovial datasets were used to categorize these genes into myeloid, lymphoid, fibroid, and low inflammatory signatures.2Differentially expressed genes (DEGs) at each time point were compared to human synovial datasets of RA patients before and after treatment. In addition, we compared disease-driven genes in CIA to genes in RA patients that are unchanged following therapy to identify possible combination therapies.Results:Disease pathology in the rat CIA natural history study progressed as expected: significant decreases were seen in body weight, as well as increases in ankle diameter, paw weight, and histopathology scores of joints in collagen-injected vs noninjected rats. There were 1900 DEGs identified between diseased and naïve rats over the course of disease, representing disease-induced gene signatures (Fig. 1). Comparing these DEGs to reported human RA synovial signatures, both the lymphoid and myeloid signatures were found to be highly upregulated. Interestingly, there were no significant DEGs representing the human fibroid and low inflammatory synovial signatures identified in the CIA rat model. This suggests that the rat CIA model most closely models RA patients with an immune synovial phenotype. In addition, we examined the overlap between disease-driven genes in CIA and genes in RA patients that are unchanged following therapy to identify signaling pathways that may be of utility in combination therapy. Of genes that were upregulated in CIA, 94% of genes that mapped to extracellular matrix-receptor pathways remained unchanged in the synovial tissue of RA patients following tocilizumab treatment.Conclusion:Previous studies have shown that nearly 30% of treatment-naïve early RA patients exhibit a strong fibroid phenotype that correlates with less severe disease and a relatively poor response to disease-modifying anti-rheumatic drugs.3These data indicate that the synovial biology associated with such patients (fibroid or pauci-immune) is not well captured in CIA, the most common preclinical RA model. To assess potential new therapies targeting these patients, it will be necessary to develop alternative animal models with more intact fibroid signatures. In addition to these findings, we also characterized the global molecular changes that occur with disease progression in the CIA rat and made a comparison to RA patients on treatment, providing an overall understanding of disease-relevant pathways in the synovium that may point to possible combination therapies.References:[1]Trentham DE, et al.J Exp Med. 1977;146(3):857-868.[2]Dennis G Jr, et al.Arthritis Res Ther. 2014;16(2):R90.[3]Humby F, et al.Ann Rheum Dis. 2019;78(6):761-772.Disclosure of Interests:Debbie Ruelas Employee of: Gilead, Ruidong Li Employee of: Gilead, Christian Franci Employee of: Gilead, Victor Lira Employee of: Gilead, David Lopez Employee of: Gilead, Li Li Employee of: Gilead, Gundula Min-Oo Employee of: Gilead, Julie A. Di Paolo Employee of: Gilead

Background:The rheumatoid arthritis (RA) synovium is characterized by an overabundance of fibroblast-like synoviocytes (FLS), which play a central role in the initiation and perpetuation of disease via multiple mechanisms.1FLS promote disease progression by producing high levels of proinflammatory factors, migrating to and invading cartilage and bone, and promoting self-proliferation and resistance to apoptosis. Our current understanding of the molecular mechanisms that govern FLS-mediated pathology in the synovial joint remains incomplete. Importantly, almost 30% of treatment-naïve early RA patients exhibit a strong fibroid phenotype that correlates with relatively poor response to disease-modifying anti-rheumatic drugs.2Yet, current therapies in RA are not directly aimed at FLS pathology, creating an opportunity for novel therapeutic target discovery.Objectives:Our aim is to develop a broad suite of screening-amenable assays in RA patient-derived FLS for the discovery of target pathways that control multiple pathological properties, including cytokine secretion, migration, and invasion.Methods:A sensitive high-throughput RA-FLS secretion assay was developed to examine the ability of small-molecule inhibitors to block the production of interleukin (IL)-6 and matrix metalloproteinase (MMP)-3 in response to stimuli. To create a physiologically relevant stimulus, a surrogate synovial fluid cocktail (composed of 12 factors) was defined and titrated for optimal concentration selection. Small-molecule inhibitors (N=170) of diverse biological pathways were screened using the full cocktail or individual stimulation (TNFα, IL-1α, or IL-17) to characterize assay performance. In addition, an FLS platelet-derived growth factor (PDGF)-mediated migration screening assay was developed using a live cell imaging system (IncuCyte) to quantify real-time FLS migration.Results:Due to the variability and limited volume of synovial fluid, we developed a surrogate synovial fluid cocktail to mimic the relevant stimulation of RA-FLS in the inflamed joint. The surrogate cocktail was composed of 12 factors: TNFα, IL-1α, IL-17, IFNγ, OSM, LIF, GM-CSF, IP-10, VEGF, PDGF, AREG, and FGF2. Individual titration of these factors demonstrated that only 3 stimulatory factors (TNFα, IL-1α, and IL-17) resulted in a robust increase of IL-6 production. Importantly, when all 12 factors were combined, a synergistic increase in IL-6 and MMP-3 production by FLS was observed. Screening results identified several reference compounds, including an inhibitor of transforming growth factor-b–activated kinase 1 (TAK1), that was previously reported to block cytokine secretion in FLS.3Treatment with this compound showed complete inhibition of IL-6 and MMP-3 secretion. In addition to the cytokine secretion assay, treatment of FLS with this TAK1 inhibitor resulted in almost complete inhibition of migration (Fig. 1).Conclusion:Novel FLS assays were developed to discover new targets and interrogate pathways involved in multiple disease-driving mechanisms of FLS in RA. In order to mimic the inflammatory environment present in the RA synovium, we developed a 12-factor surrogate synovial fluid cocktail. A synergistic release of both IL-6 and MMP-3 was demonstrated following cocktail stimulation compared to individual cytokines. This points to the important contribution that multiple factors play in the FLS pathogenic processes and will allow us to uncover pathway interactions that may not be captured with single stimuli. In addition, the development of a real-time, 96-well, imaging-based assay to interrogate FLS migration will allow us to identify targets that control this critical pathological function of FLS.References:[1] Bartok B, et al.J Immunol. 2014;192(5):2063-2070.[2] Humby F, et al.Ann Rheum Dis. 2019;78(6):761-772.[3] Jones DS, et al.Nat Chem Bio. 2017;13(1):38-45.Disclosure of Interests:Debbie Ruelas Employee of: Gilead, Chen Chen Employee of: Gilead, Hoa Truong Employee of: Gilead, Victor Lira Employee of: Gilead, Yasamin Moazami Employee of: Gilead, Kevin Currie Employee of: Gilead, Julie A. Di Paolo Employee of: Gilead, Helen Yu Employee of: Gilead, Gundula Min-Oo Employee of: Gilead

Background:Janus kinase 1 (JAK1) inhibitors are efficacious in rheumatoid arthritis (RA). Despite having similar efficacy, in vitro studies have shown differences in JAK selectivity profiles for the small-molecule JAK inhibitors (JAKi) baricitinib (BARI), tofacitinib (TOFA), and upadacitinib (UPA).1For example, BARI and UPA are JAK1/JAK2 selective, while TOFA is JAK1/JAK3 selective, but each JAKi has some activity against other JAKs. As JAKs form signaling pairs, differences in selectivity could lead to distinct pharmacologic profiles that may impact clinical efficacy and safety.Objectives:As a first step to understand the basis of potential differences at therapeutic doses, we compared the selectivity and potency of filgotinib (FIL) and its major metabolite (MET) to those of BARI, TOFA, and UPA in cytokine-stimulated peripheral blood mononuclear cells (PBMCs) and whole blood (WB).Methods:PBMCs and WB from healthy donors were incubated in vitro with 8 doses of each JAKi, and levels of signal transducer and activator of transcription phosphorylation (pSTAT) were measured following cytokine stimulation. Half maximal inhibitory concentration (IC50) values were calculated in phenotypically sorted leukocyte populations by flow cytometry. Therapeutic dose relevance of the in vitro analyses was assessed using calculated mean concentration-time profiles from JAKi population pharmacokinetic data in RA subjects. For each JAKi, the time above IC50and average daily pSTAT inhibition were calculated for each cytokine/STAT pair in B cells, CD4+ T cells, CD8+ T cells, monocytes, and/or NK cells.Results:Cellular assays in PBMCs and WB showed dose-dependent inhibition of cytokine-induced pSTATs with all JAKi (correlation between the protein-adjusted IC50values from PBMCs and IC50values from WB, r2=0.98). Among the most potently inhibited pathways were JAK1/TYK2-dependent cytokine, interferon alpha (IFNα), and the JAK1/2-dependent cytokine, interleukin (IL)-6. FIL and MET had weaker potencies against JAK2/TYK2 (G-CSF/pSTAT3), JAK1/2 (IFNƴ/pSTAT1), and JAK2/2 (granulocyte-macrophage colony-stimulating factor [GM-CSF])-dependent pathways compared to JAK1/TYK2 (IFNα/pSTAT5). FIL and MET showed the greatest selectivity vs the JAK2/2 pathway (GM-CSF/pSTAT3) in monocytes.The mean concentration-time profiles and time above IC50over 24 hr for each cytokine/STAT pathway showed that JAK1/2 (IL-6/pSTAT1) and JAK1/TYK2 (IFNα/pSTAT1) pathways were strongly modulated with all tested JAKi. FIL (200 mg) showed similar activity in average target coverage and time above IC50to the approved low doses of TOFA (5 mg) and UPA (15 mg); conversely, FIL had reduced mean average inhibition and time above IC50levels against JAK1/2 (IFNƴ/pSTAT1), JAK1/3-dependent cytokines (IL-2, -4, and -15), JAK2/TYK2 (G-CSF/pSTAT3), and JAK2/2 (GM-CSF/pSTAT5)-dependent pathways compared to TOFA and UPA, and in certain cases to BARI (2 mg).Conclusion:Different JAKi modulate distinct cytokine pathways to varying degrees, and no agent potently and continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. FIL (200 mg) showed a similar inhibition profile to TOFA, BARI, and UPA against the JAK1/TYK2- (IFNα/pSTAT1) or JAK1/2-dependent (IL-6/pSTAT1) responses, consistent with the role of these pathways in clinical efficacy.2However, FIL displayed a differentiated pharmacologic profile from the other JAKi, showing biologically reduced activity on the JAK1/2 (IFNγ)-, JAK1/3 (IL-2, -4 and -15)-, JAK2/TYK2 (G-CSF)-, and JAK2/2 (GM-CSF)-dependent pathways, which play important roles in hematopoiesis and immune function. These data suggest that FIL (200 mg) may have less impact on a subset of homeostatic immune functions signaling via JAK2 and JAK3 than those observed at the clinically approved doses of TOFA (5 mg and 10 mg), UPA (15 mg), and BARI (4 mg).References:[1]McInnes IB, et al. Arthritis Res Ther. 2019;21:183.[2]Banerjee S, et al. Drugs. 2017;77:521-546.Disclosure of Interests:Paqui Gonzalez-Traves Employee of: Gilead, Bernard Murray Employee of: Gilead, Federico Campigotto Employee of: Gilead, Amy Meng Shareholder of: Gilead Sciences, Employee of: Gilead, Julie A. Di Paolo Employee of: Gilead

Background Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterised by immune system hyperactivation leading to the production of autoantibodies and immune attack on multiple organs including the skin and kidneys. High levels of type I interferons (IFNa/b) alter the activation state of immune cell populations and have been identified as a risk factor for SLE. Antibody blockade of the interferon alpha receptor (IFNAR) has demonstrated clinical efficacy in a Phase 2b study in SLE, and validates targeting this pathway in SLE1. Janus kinase 1 (JAK1) is critical for mediating downstream signalling of type I IFNs. Inhibition of JAK1, therefore, is anticipated to reduce IFN signalling, activation of immune cell subsets, and SLE disease activity. The JAK1 selective inhibitor filgotinib (FIL) is currently being evaluated in three proof-of-concept Phase 2 studies in SLE-like diseases. Objectives To characterise the efficacy and mechanism-of-action of FIL in the NZB/W F1 spontaneous mouse model of lupus in a therapeutic setting. Methods FIL was tested in the NZB/W F1 model of lupus at two concentrations (0.05% and 0.1%) formulated in chow and administered ad libitum from weeks 28–40. Cyclophosphamide (CP) was used as a positive control at 5 mg/kg, once daily, i.p. Efficacy was determined by changes in proteinuria, renal histopathology, and survival. Splenic cell populations were analysed by flow cytometry at study termination at week 40 to measure changes in immune cell subsets in diseased versus non-diseased mice and following FIL treatment. An in vitro murine whole blood pSTAT1 assay and PK exposure data were used to assess target coverage in the model. Results In the NZB/W model, FIL showed a dose-dependent decrease in proteinurea with a concomitant reduction in BUN levels, renal inflammation, improved glomerular morphology, and increased survival (table 1). Diseased mouse spleens had decreased frequencies of naive T cells, increased frequencies of CD11+dendritic cells (DCs), and an increased ratio of memory:naive T cells versus non-diseased mice. FIL treatment showed a dose-responsive reversal of these populations toward non-diseased levels. FIL inhibited in vitro IFNa-stimulated pSTAT1 signalling in T cells. Calculated JAK target coverage of pSTAT1 inhibition was similar to that observed in human studies at clinical doses. Conclusions FIL demonstrated efficacy in the NZB/W F1 murine model of lupus with an alteration of splenic immune cell subsets affected by type I IFNs toward non-diseased levels. This data provides the basis for the evaluation of FIL as monotherapy in the currently ongoing Phase 2 studies in lupus-related diseases. Reference [1] Furie R, et al. Arthritis Rheumatol2017;69:376–386. Disclosure of Interest C. Pohlmeyer Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, Z.-H. Cui Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, P. Han Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, A. Clarke Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, R. Jones Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, N. Mollova Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, I. Mikaelian Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, S. Zaboli Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, A. Shauf Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc, J. Di Paolo Shareholder of: Gilead Sciences, Inc, Employee of: Gilead Sciences, Inc

Background Spleen Tyrosine Kinase (SYK) mediates signaling in a range of hematopoietic cells involved in the initiation and progression of RA including B cells, monocytes, macrophages, dendritic cells, and osteoclasts. There is strong preclinical validation for SYK as a therapeutic target for RA based on cellular data and animal models of disease. We have identified GS-9876 as a potent and selective SYK inhibitor with pharmaceutical properties compatible with once daily oral dosing in human. Objectives To characterize the cellular activity of GS-9876 on pathologically relevant pathways in RA, and establish target inhibition requireements for efficacy in rat models of arthritis. Methods The potency and selectivity of GS-9876 were characterized in biochemical and cellular assays. Effects in B cells were measured by inhibition of BCR-mediated protein phosphorylation, CD69 and CD86 expression, and in macrophages by inhibition of immune-complex (IC)-stimulated cytokine release. Cellular selectivity was demonstrated by comparing the inhibition of B cell proliferation versus T cell proliferation. GS-9876 potency in human blood was evaluated by inhibition of phosphorylated SYK (pSYK), CD63 expression on basophils, and CD69 expression on B cells. The in vivo efficacy of GS-9876 was tested in rat models of collagen-induced arthritis (CIA). Results GS-9876 is a potent SYK inhibitor (IC50=9.5±4.3 nM) and is highly selective against a panel of 456 other kinases. GS-9876 inhibited anti-IgM stimulated phosphorylation of AKT, BLNK, BTK, ERK, MEK, and PKCδ in human B cells with EC50 values of 24–51 nM. Functionally, GS-9876 inhibited anti-IgM mediated CD69 and CD86 expression on B-cells (EC50=112±10 nM and 164±15 nM, respectively) and anti-IgM /anti-CD40 co-stimulated B cell proliferation (EC50=108±55 nM). In human macrophages, GS-9876 inhibited IC-stimulated TNFα and IL-1β release (EC50=121±77 nM and 9±17 nM, respectively). Anti-CD3/anti-CD28 stimulated T cell proliferation was weakly inhibited (EC50=1291±398 nM), with selectivity >10-fold versus the inhibition of B cell proliferation. In human blood, GS-9876 blocked SYK phosphorylation, CD69 expression on B cells, and CD63 expression in basophils. GS-9876 demonstrated a dose-dependent improvement in clinical score and histopathology parameters with once-daily dosing in short and long term rat CIA models. Significant efficacy could be achieved with GS-9876 doses that produced trough pSYK inhibition of Conclusions GS-9876 is a novel SYK inhibitor that potently inhibits multiple cellular events implicated in RA pathogenesis and displays excellent in vivo efficacy in rat CIA models after once-daily dosing. GS-9876 has markedly improved selectivity over competitor SYK programs. Our data support the development of GS-9876 in inflammatory diseases, with potential for an improved safety profile. Disclosure of Interest J. Di Paolo Shareholder of: Gilead Sciences, P. Blomgren Shareholder of: Gilead Sciences, M. Dolton Shareholder of: Gilead Sciences, R. Jones Shareholder of: Gilead Sciences, J. Kropf Shareholder of: Gilead Sciences, T. Lee Shareholder of: Gilead Sciences, S. Mitchell Shareholder of: Gilead Sciences, B. Murray Shareholder of: Gilead Sciences, K. Suekawa-Pirrone Shareholder of: Gilead Sciences, S. Wise Shareholder of: Gilead Sciences, J. Xu Shareholder of: Gilead Sciences, Z. Zhao Shareholder of: Gilead Sciences, K. Currie Shareholder of: Gilead Sciences