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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:Rheumatoid arthritis (RA) is characterized by chronic, uncontrolled joint inflammation and tissue destruction. Macrophages are thought to be key mediators in both the initiation and perpetuation of this pathology.1,2The RA synovium contains a complex inflammatory milieu that can stimulate macrophage-dependent production of proinflammatory cytokines through multiple signaling pathways.1,2Existing evidence indicates that toll-like receptors (TLRs) and interleukin-1 receptors (IL-1R) along with their agonists, damage-associated molecular patterns (DAMPs) and IL-1β, are highly expressed in RA joints and are important mediators of synovial macrophage activation and proinflammatory cytokine production.1-9IRAK4 (interleukin-1 receptor-associated kinase 4) is a serine/threonine kinase that facilitates TLR and IL-1R signaling in many cell types, including macrophages.10IRAK4 inhibition represents an opportunity to reduce proinflammatory cytokine production in the joints of patients with RA.Objectives:To investigate the effect of a highly selective IRAK4 inhibitor on proinflammatory cytokine production from human macrophages stimulated with synovial fluid from patients with RA.Methods:Primary human monocytes from 2 independent donors were differentiated for 6 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate human monocyte-derived macrophages (hMDMs). hMDMs were then pretreated with an IRAK4 inhibitor for 1 hour and subsequently stimulated for 24 hours with RA synovial fluid from 5 patients. Culture supernatants were then assessed for secretion of proinflammatory cytokines by MesoScale Discovery.Results:RA synovial fluid stimulation of hMDMs resulted in the production of several proinflammatory cytokines, including IL-6, IL-8, and TNFα. Pretreatment of hMDMs with an IRAK4 inhibitor resulted in the dose-dependent inhibition of IL-6, IL-8, and TNFα production, with an average EC50± SD of 27 ± 31, 26 ± 41, and 28 ± 22 nM, respectively. Maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 76 ± 8.8, 73 ± 15, and 77 ± 13, respectively. To evaluate the specific IRAK4-dependent signaling pathways mediating this response, hMDMs were pretreated with inhibitors of TLR4 (TAK242) and IL-1R (IL-1RA) prior to stimulation with RA synovial fluid. Both TAK242 and IL-1RA inhibited proinflammatory cytokine production. For TAK242, maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 39 ± 25, 48 ± 24, and 50 ± 21, respectively. For IL-1RA maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 18 ± 18, 20 ± 23, and 16 ± 18, respectively. The broad range of inhibition across each stimulation highlights the complexity and variability in the signaling pathways mediating proinflammatory cytokine production from hMDMs stimulated with RA synovial fluid, but demonstrates that RA synovial fluid can stimulate proinflammatory cytokine production in hMDMs, at least partly, through IRAK4-dependent pathways.Conclusion:This work demonstrates that IRAK4 inhibition can suppress proinflammatory cytokine production from macrophages stimulated with synovial fluid from patients with RA and supports a potential pathophysiological role for IRAK4 in perpetuating chronic inflammation in RA.References:[1]Smolen JS, et al.Nat Rev Dis Primers.2018;4:18001.[2]Udalova IA, et al.Nat Rev Rheumatol.2016;12(8):472-485.[3]Joosten LAB, et al.Nat Rev Rheumatol.2016;12(6):344-357.[4]Huang QQ, Pope RM.Curr Rheumatol Rep.2009;11(5):357-364.[5]Roh JS, Sohn DH.Immune Netw.2018;18(4):e27.[6]Sacre SM, et al.Am J Pathol.2007;170(2):518-525.[7]Ultaigh SNA, et al.Arthritis Res Ther.2011;13(1):R33.[8]Bottini N, Firestein GS.Nat Rev Rheumatol.2013;9(1):24-33.[9]Firestein GS, McInnes IB.Immunity.2017;46(2):183-196.[10]Janssens S, Beyaert R.Mol Cell.2003;11(2):293-302.Disclosure of Interests:Adam Yadon Employee of: Gilead, Debbie Ruelas Employee of: Gilead, Gundula Min-Oo Employee of: Gilead, James Taylor Employee of: Gilead, Matthew R. Warr Employee of: Gilead

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