Guilherme V. Rocha

Pharmacological Characterization of a Potent Inhibitor of Autotaxin in Animal Models of Inflammatory Bowel Disease and Multiple Sclerosis.

Autotaxin is a secreted enzyme that catalyzes the conversion of lysophosphatidyl choline into the bioactive lipid mediator lysophosphatidic acid (LPA). It is the primary enzyme responsible for LPA production in plasma. It is upregulated in inflammatory conditions and inhibition of autotaxin may have anti-inflammatory activity in a variety of inflammatory diseases. To determine the role of autotaxin and LPA in the pathophysiology of inflammatory disease states, we used a potent and orally bioavailable inhibitor of autotaxin that we have recently identified, and characterized it in mouse models of inflammation, inflammatory bowel disease (IBD), multiple sclerosis (MS), and visceral pain. Compound-1, a potent inhibitor of autotaxin with an IC50 of ∼2 nM, has good oral pharmacokinetic properties in mice and results in a substantial inhibition of plasma LPA that correlates with drug exposure levels. Treatment with the inhibitor resulted in significant anti-inflammatory and analgesic effects in the carrageenan-induced paw inflammation and acetic acid-induced visceral pain tests, respectively. Compound-1 also significantly inhibited disease activity score in the dextran sodium sulfate–induced model of IBD, and in the experimental autoimmune encephalomyelitis model of MS. In conclusion, the present study demonstrates the anti-inflammatory and analgesic properties of a novel inhibitor of autotaxin that may serve as a therapeutic option for IBD, MS, and pain associated with inflammatory states.

Gene Expression and Pharmacodynamic Changes in 1,760 Systemic Lupus Erythematosus Patients From Two Phase III Trials of BAFF Blockade With Tabalumab

To characterize baseline gene expression and pharmacodynamically induced changes in whole blood gene expression in 1,760 systemic lupus erythematosus (SLE) patients from 2 phase III, 52‐week, randomized, placebo‐controlled, double‐blind studies in which patients were treated with the BAFF‐blocking IgG4 monoclonal antibody tabalumab.

Vitamin D and/or calcium deficient diets may differentially affect muscle fiber neuromuscular junction innervation

Introduction: There is evidence that supports a role for Vitamin D (Vit. D) in muscle. The exact mechanism by which Vit. D deficiency impairs muscle strength and function is not clear. Methods: Three‐week‐old mice were fed diets with varied combinations of Vit. D and Ca2+ deficiency. Behavioral testing, genomic and protein analysis, and muscle histology were performed with a focus on neuromuscular junction (NMJ) ‐related genes. Results: Vit. D and Ca2+ deficient mice performed more poorly on given behavioral tasks than animals with Vit. D deficiency alone. Genomic and protein analysis of the soleus and tibialis anterior muscles revealed changes in several Vit. D metabolic, NMJ‐related, and protein chaperoning and refolding genes. Conclusions: These data suggest that detrimental effects of a Vit. D deficient or a Vit. D and Ca2+ deficient diet may be a result of differential alterations in the structure and function of the NMJ and a lack of a sustained stress response in muscles. Muscle Nerve 54: 1120–1132, 2016

FcγRIIIa-dependent IFN-γ release in whole blood assay is predictive of therapeutic IgG1 antibodies safety

ABSTRACT Immunomodulatory monoclonal IgG1 antibodies developed for cancer and autoimmune disease have an inherent risk of systemic release of pro-inflammatory cytokines. In vitro cytokine release assays are currently used to predict cytokine release syndrome (CRS) risk, but the validation of these preclinical tools suffers from the limited number of characterized CRS-inducing IgG1 antibodies and the poor understanding of the mechanisms regulating cytokine release. Here, we incubated human whole blood from naïve healthy volunteers with four monoclonal IgG1 antibodies with different proven or predicted capacity to elicit CRS in clinic and measured cytokine release using a multiplex assay. We found that, in contrast to anti-CD52 antibodies (Campath-1H homolog) that elicited high level of multiple inflammatory cytokines from human blood cells in vitro, other IgG1 antibodies with CRS-inducing potential consistently induced release of a single tested cytokine, interferon (IFN)-γ, with a smaller magnitude than Campath. IFN-γ expression was observed as early as 2–4 h after incubation, mediated by natural killer cells, and dependent upon tumor necrosis factor and FcγRIII. Importantly, the magnitude of the IFN-γ response elicited by IgG1 antibodies with CRS-inducing potential was determined by donor FcγRIIIa-V158F polymorphism. Overall, our results highlight the importance of FcγRIIIa-dependent IFN-γ release in preclinical cytokine release assay for the prediction of CRS risk associated with therapeutic IgG1 antibodies.

P059 Ex vivo comparison of baricitinib, upadacitinib, filgotinib and tofacitinib for cytokine signalling in human leukocyte subpopulations

Introduction Baricitinib (BARI), an oral selective Janus kinase (JAK) 1/2 inhibitor, approved in the EU for moderate to severe active RA. Objectives To compare in vitro cellular pharmacology of BARI to upadacitinib (ABT), filgotinib (FILGO), and tofacitinib (TOFA), three JAK inhibitors (JAKis) currently approved or in clinical development. Methods Peripheral blood mononuclear cells from healthy donors (n=6–12) were incubated with different JAKis. After cytokine stimulation, phosphorylated signal transducer and activator of transcription (pSTAT) levels were measured and IC50 calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of in vitro analysis was assessed using calculated mean concentration-time (CT) profiles over 24 hour JAKi-treated subjects (BARI 4 mg QD; ABT 15 and 30 mg QD; FILGO 100 and 200 mg QD; TOFA 5 and 10 mg BID). Time above IC50 (T>IC; h/day) and average daily% inhibition of pSTAT formation (%SI) were calculated for each JAKi, cytokine, and cell type. Results Tested cytokines did not signal in all cell types. When signalling was detected, IC50,%SI, and T>IC for a particular JAKi exhibited similar dose dependent inhibition across cell types. For JAK1/3 dependent signalling across 4 cytokines (IL-2, 4, 15, 21), IC50 for ABT and TOFA were more potent than BARI; FILGO was the least potent. Overlaid on CT profiles, this indicated generally higher%SI and longer T>IC for ABT and TOFA compared to BARI and FILGO. For IL-6 (JAK1/2),%SI and T>IC was TOFA>BARI/ABT>FILGO and for IL-10 (JAK1/TYK2),%SI was TOFA>BARI/ABT>FILGO. IFN-γ (JAK1/2) was modulated only by BARI, ABT, and TOFA. IFN-α (JAK1/TYK2) signalling was most potently inhibited by BARI and ABT. FILGO did not appear to modulate GM-CSF signalling (JAK2/2), while%SI and T>IC were similar between BARI and ABT. Conclusions JAKis modulate distinct cytokine pathways to differing degrees and durations over 24 hour. BARI and FILGO inhibited JAK1/3 signalling less than ABT and TOFA. No JAKis agent potently or continuously inhibited an individual cytokine signalling pathway throughout dosing interval, implying the varying efficacy and safety profiles of JAKis across disease states. Acknowledgements Study support Eli Lilly and Company and Incyte Corporation. Encore of ACR/ARHP-2017 Annual Scientific Meeting, Nov 4–8, 2016; San Diego, CA, USA. Disclosure of interest None declared

Vitamin D receptor activation reduces VCaP xenograft tumor growth and counteracts ERG activity despite induction of TMPRSS2:ERG

Whether vitamin D is chemopreventive and/or has potential therapeutically in prostate cancer is unresolved. One confounding factor is that many prostate cancers express a TMPRSS2:ERG fusion gene whose expression is increased both by androgens and by vitamin D receptor (VDR) activation. Two challenges that limit VDR agonist use clinically are hypercalcemia and the cooperation of VDR with ERG to hyper-induce the 1α,25-dihydroxyvitamin D3 metabolizing enzyme, CYP24A1, thus reducing VDR activity. Using the VCaP TMPRSS2:ERG positive cell line as a model, we found that a nonsecosteroidal CYP24A1 resistant VDR agonist, VDRM2, substantially reduces growth of xenograft tumors without inducing hypercalcemia. Utilizing next generation RNA sequencing, we found a very high overlap of 1,25D(OH)2D3 and VDRM2 regulated genes and by drawing upon previously published datasets to create an ERG signature, we found activation of VDR does not induce ERG activity above the already high basal levels present in VCaP cells. Moreover, we found VDR activation opposes 8 of the 10 most significant ERG regulated Hallmark gene set collection pathways from Gene Set Enrichment Analysis (GSEA). Thus, a CYP24A1 resistant VDR agonist may be beneficial for treatment of TMPRSS2:ERG positive prostate cancer; one negative consequence of TMPRSS2:ERG expression is inactivation of VDR signaling.

42 Gene expression profile from 1,760 sle patients reveals novel complex interferon responsive gene networks

Background and aims RNA profiling was performed on 1760 SLE patients from two, large Phase III clinical trials, ILLUMINATE-1 and −2. SLE was compared to both healthy controls and other autoimmune diseases, including rheumatoid arthritis, psoriasis and psoriatic arthritis. The goals of this study were to characterise gene expression networks in SLE using these large cohorts, and to compare gene expression phenotypes in SLE to healthy controls and other autoimmune diseases. Methods Blood was collected at baseline and RNA was interrogated on all samples using Affymetrix HTA 2.0 microarrays and on select SLE samples using NanoString nCounterTM. Complete demographics, serum IgG anti-dsDNA antibodies, and complement were measured in SLE. Analyses of gene expression and gene pathways were performed. Results Baseline elevation of interferon responsive genes (IRG) was detected in SLE and associated with younger age, elevated anti-dsDNA antibodies, elevated SLEDAI and decreased levels of C3. Significant differences in SLEDAI organ domain involvement between IRG-positive and IRG-negative groups were observed. Elevated expression of IRG, genes involved with B cell and plasma cell biology, and with cell cycling and signalling were detected in SLE. A bimodal expression pattern of IRG was unique to SLE. Substantial heterogeneity of expression of IRG and complex relationships in interferon (IFN) gene networks were observed. Conclusions There was substantial heterogeneity of gene expression in IFN gene networks when examining individual IFN genes and complex relationships were observed among IFN gene networks. Low IFI27 was identified as a novel subtype of IFN signature in SLE.

OP0079 Pharmacodynamic Changes in Gene Expression Observed in Two Phase 3 Trials of Baff Blockade with Tabalumab in SLE

Background RNA gene expression profiling was performed on 1,760 SLE patients randomly selected from Illuminate Trials 1 & 2 which studied the anti-BAFF, IgG4 monoclonal antibody, tabalumab, for efficacy in SLE (Isenberg D., et al. ACR Boston 2104). The baseline disease activity and clinical characteristics were balanced across treatment and placebo groups in both Trial 1 (N=1164) & 2 (N=1124). The primary endpoint of SRI-5 was not met in Trial 1 but was met in Trial 2 for the 120mg Q2week (W) dose (p=0.002). Objectives The present study characterized baseline and pharmacodynamic (PD)-induced changes in gene expression from these two cohorts of SLE patients. Methods Whole blood was collected in Tempus tubes at baseline, weeks 16 and 52. RNA was extracted and analyzed using Affymetrix HTA 2.0 whole genome microarrays. Serum levels of IgG anti-dsDNA antibodies (abs) were measured by ELISA. Serum complement components C3 and C4 were measured by nephelometry and B cells enumerated using flow cytometry. Statistical analyses to identify PD-induced gene changes were conducted with Q2W and Q4W cohorts using a mixed effects model with relative gene expression as the response and inclusion of baseline clinical covariates. Results Statistically significant PD changes were observed in both trials in the Q2W and Q4W 120mg dosing arms compared to placebo for the following biomarkers: anti-dsDNA abs, C3 & C4, total serum IgG, IgM & IgA, and peripheral blood B cell number (all with p<0.001). Changes in gene expression in tabalumab treated patients were compared to placebo in 422 randomly selected patients at baseline, weeks 16 and 52. Statistically significant changes were identified in 410 genes (false discovery rate qval<0.20 Trial 1 and p<0.20 Trial 2) including: plasma cell markers, B cell markers, TNF superfamily members, Fc & Fc-like receptors, complement receptors and a pain receptor, SCN3A. There were no changes in monocyte or neutrophil specific markers. A group of 34 pre-specified interferon responsive genes (IRG) were examined and baseline elevation of IRG was significantly associated with elevated anti-dsDNA abs and decreased levels of C3 & C4. B cell number as measured by flow cytometry correlated with gene expression of B cell-associated genes including CD19. PD changes in predefined B cell and plasma cell genes were observed in the treatment groups and associated with changes in anti-dsDNA abs, serum Ig and complement levels, and cell number; however, treatment with tabalumab was not associated with changes in IRG or BAFF gene expression. Conclusions Pharmacodynamic changes associated with tabalumab treatment included anti-dsDNA abs, complement components C3 & C4, serum IgG, IgA & IgM, and B cell numbers. Statistically significant changes were observed in 410 genes, broadly consistent with the mechanism of BAFF blockade. Changes in expression of a number of additional genes were unexpected and suggest that these may be of importance in anti-BAFF drug response. The data here confirm previous smaller studies that a Type I interferon signature may represent a distinctive subset of SLE patients. Disclosure of Interest R. Hoffman Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, J. Merrill: None declared, M. Alarcόn-Riquelme: None declared, M. Petri: None declared, E. Dow Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, E. Nantz Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, L. Nisenbaum Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, K. Schroeder Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, W. Komocsar Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, N. Perumal Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, G. Rocha Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, R. Higgs Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company