Background Interleukin-6 (IL-6) has been shown to be elevated in patients (pts) with AS and correlates with disease activity.1 Case reports concerning the use of TCZ in pts with AS have been published.2-5 The efficacy and safety of TCZ, an inhibitor of IL-6 receptor signalling, as a treatment for AS have not been evaluated in a placebo-controlled trial. Objectives To examine the efficacy and safety of TCZ for the treatment of AS. Methods Study Design: Phase 2, multicentre, randomised, double-blind, placebo-controlled study, 12-week (wk) duration. Patients: Definite diagnoses of AS by the modified New York criteria and Bath AS Disease Activity Index (BASDAI) score ≥4.0; adults who were NSAID inadequate responders (IR) and anti-TNF naive. Study Drugs: TCZ 8 mg/kg or placebo (PBO) IV every 4 wks. Primary Endpoints: 20% improvement in assessment of AS (ASAS20) response and safety at wk 12. Results Of 102 pts enrolled, 99 (48 TCZ, 51 PBO) completed 12 wks. Pt demographics and disease status were in keeping with the target population (Table). The proportion of pts achieving ASAS20 and ASAS40 at wk 12 was not significantly different for TCZ vs PBO (37% vs 28%, p=0.2823; 12% vs 20%, p=0.2694) (Table). No differences between TCZ and PBO were observed for (1) higher percentage ASAS responses, (2) change in individual ASAS components, (3) BASDAI endpoints. These findings were independent of baseline CRP level. There was a reduction in CRP and in AS Disease Activity Score (ASDAS)–CRP with TCZ but not PBO (difference in median CRP change between arms, –1.2 mg/dl, p<0.0001; difference in mean ASDAS-CRP change between arms, –0.9, p<0.0001). Rates/100 patient-years of adverse events (AEs) for TCZ and PBO were 262.4 and 226.9; rates of serious AEs were 17.5 and 0. No AEs led to withdrawal during this 12-wk study. TCZ 8 mg/kg (N=51)PBO (N=51) Demographics, n (%)  Age, mean (range)41.6 (23-69) y42.7 (19-84) y  SexM 36 (71)/F 15 (29)M 40 (78)/F 11 (22)  HLA-B27 statusPositive 43 (84.3)Positive 45 (88.2) Negative 8 (15.7)Negative 6 (11.8)  Disease duration≤10 y 39 (76.5)≤10 y 34 (66.7) >10 y 12 (23.5)>10 y 17 (33.3)  Baseline BASDAI, mean (range)6.62 (2.9-9.5)6.77 (4.2-9.7) Efficacy Endpoint, n (%)  ASAS2019 (37.3)14 (27.5)  ASAS20 (baseline CRP <3× ULN)6 (23.1)6 (27.3)  ASAS20 (baseline CRP ≥3× ULN)13 (52.0)(27.6)  ASAS406 (11.8)10 (19.6)  BASDAI (<4)1 (2.0)0 (0.0)  BASDAI503 (5.9)5 (9.8)  ASDAS-CRP (mean change)–1.40–0.44 Conclusions The study failed to demonstrate the efficacy of TCZ over placebo for the treatment of symptoms of AS, irrespective of baseline CRP level. CRP levels declined with TCZ, suggesting adequate IL-6R blockade. The change in ASDAS-CRP was driven by the decrease in CRP. The safety profile of TCZ in this study was consistent with that seen in RA pts. References Gratacόs J et al. Br J Rheumatol 1994;33:927-31. Brulhart L et al. Joint Bone Spine 2010;77;625-6. Henes JC et al. Ann Rheum Dis 2010;69:2217-8. Shima Y et al. Mod Rheumatol 2011;21:436-9. Cohen JD et al. J Rheumatol 2011;38:1527. Disclosure of...

INTRODUCTION: Blockade of complement factor C5 has demonstrated benefit in Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Hemolytic Uremic Syndrome and Generalized Myasthenia Gravis. We developed a human IgG4P antibody, REGN3918, that binds with high affinity to wild-type and variant (R885H/C) human C5. REGN3918 was well-tolerated in monkey toxicology studies with up to 26 weeks of dosing at up to 100 mg/kg/week. This finding was supportive of conducting this first-in-human study of REGN3918 in healthy volunteers. OBJECTIVE: The primary objective of this ongoing study is to evaluate the safety and tolerability of single ascending intravenous (IV) and subcutaneous (SC) doses and a multiple dose regimen consisting of an IV loading dose plus multiple weekly SC doses of REGN3918 administered in healthy volunteers. The secondary objectives of the study are to assess the pharmacokinetic (PK) and pharmacodynamic (PD) profile of REGN3918. METHODS: 56 subjects were randomized to 4 sequential ascending IV dose cohorts plus 2 sequential ascending SC cohorts followed by 1 multiple dose cohort (consisting of an IV loading dose and weekly SC doses). Each cohort consisted of 8 subjects randomized to receive REGN3918 or placebo (6 active: 2 placebo). An adaptive design was implemented to allow for dose level and dosing interval adjustment utilizing in-study PK and PD measures. The PD profile of REGN3918 was assessed utilizing a sheep red blood cell complement activity assay (CH50 assay) as well as serum concentrations of total C5. REGN3918 was administered as follows:Cohort 1: 1 mg/kg IV, single doseCohort 2a: 3 mg/kg IV, single doseCohort 2b: 300 mg SC, single doseCohort 3a: 10 mg/kg IV, single doseCohort 3b: 600 mg SC, single doseCohort 4: 30 mg/kg IV, single doseCohort 5: Loading dose of 15 mg/kg IV followed by 4 repeat SC doses of 400 mg administered once weekly for four weeks. RESULTS: REGN3918 was found to be well tolerated in single doses of up to 30 mg/kg IV and 600 mg SC. The multiple dose Cohort 5 has completed dosing in all subjects and is currently in safety follow-up. Thus far, there has been one SAE, salpingitis in a subject with an intra-uterine contraceptive device. The SAE occurred in a Cohort 5 subject after completion of dosing and has since resolved. REGN3918 exhibited dose-dependent increases in exposure in serum, with a trend toward prolonged serum concentrations at IV doses ≥10 mg/kg. Following SC administration, concentrations of REGN3918 in serum peaked at 4 to 8 days post dose and bioavailability was estimated as approximately 70%. REGN3918 exposure led to dose-dependent inhibition of CH50. In all 4 IV dosing cohorts, suppression of hemolysis was observed at 15 min post-injection. Complete suppression of hemolysis was achieved with ≥ 3mg/kg dosing. At 30 mg/kg, complete suppression of hemolysis was maintained for >6 weeks, consistent with observed prolonged REGN3918 concentrations following this dose. In the 2 SC cohorts, peak suppression of hemolysis was observed 3-7 days post dosing, again consistent with observed peak concentrations of REGN3918 in serum. In the multiple dose cohort 5, complete suppression of CH50 was observed over the four-week dosing period. CONCLUSIONS: REGN3918 was well tolerated and resulted in dose-dependent inhibition of hemolytic activity in normal healthy volunteers. A single IV infusion of R3918, at 30 mg/kg, blocked hemolytic activity completely for >6 weeks. Complete inhibition of hemolytic activity was maintained over a 4-week dosing period by a weekly SC regimen following an IV loading dose. Disclosures Weyne: Regeneron Pharmaceuticals: Employment, Equity Ownership. Ni:Regeneron Pharmaceuticals: Employment, Equity Ownership. DelGizzi:Regeneron Pharmaceuticals: Employment, Equity Ownership. Godin:Regeneron Pharmaceuticals: Employment. Morton:Regeneron Pharmaceuticals: Employment, Equity Ownership. Prasad:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rankin:Regeneron Pharmaceuticals: Employment, Equity Ownership. Simek-Lemos:Regeneron Pharmaceuticals: Employment, Equity Ownership. Wang:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rippley:Regeneron Pharmaceuticals: Employment, Equity Ownership. Harari:Regeneron Pharmaceuticals, Inc.: Employment.

INTRODUCTION: Blockade of complement factor C5 has demonstrated benefit in paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, generalized myasthenia gravis and neuromyelitis optica. We have completed a Phase I study of pozelimab, a fully human anti-C5 IgG4, in healthy volunteers. Pozelimab was well tolerated and resulted in dose-dependent inhibition of hemolytic activity through the classical complement pathway in normal healthy volunteers. Complete inhibition of hemolytic activity was maintained over a 4-week dosing period by a weekly subcutaneous (SC) regimen following an intravenous (IV) loading dose (ASH2018 abstract). OBJECTIVE: To further characterize the impact of pozelimab on the alternative complement pathway activity, we investigated the effect of pozelimab on alternative pathway-mediated hemolysis using an AH50 assay in the completed first-in-human (FIH) study. In addition, we compared the effect of pozelimab in both alternative and classical pathway hemolysis assays with those of in-house eculizumab and in-house ravulizumab in pooled normal human serum (NHS) samples, ex vivo. METHODS: In total, 56 subjects were randomized to 4 sequential ascending IV single dose cohorts plus 2 sequential ascending SC single dose cohorts followed by 1 multiple dose cohort (consisting of an IV loading dose and weekly SC doses). Each cohort consisted of 8 subjects randomized to receive pozelimab or placebo (6 active: 2 placebo). Serum collected at multiple time-points was utilized to assess the effect of pozelimab on alternative pathway activity. For ex vivo spike experiments, pooled NHS was used to compare the hemolytic function of pozelimab, in-house produced eculizumab and in-house produced ravulizumab. Comparator antibodies were synthesized from published sequence. The alternative pathway (AP) and classical pathway (CP) hemolysis assays were performed based on lysis of rabbit red blood cells (RBCs) and sensitized sheep RBCs, respectively. Both assays measure the amount of hemoglobin released from red blood cells at 412 nm. RESULTS: In the FIH study, baseline AH50 was comparable across treatment groups with a mean of 110 U/mL (standard deviation = 19, n = 56). Pozelimab exposure led to dose-dependent inhibition of AH50. In all 4 IV dosing cohorts, peak suppression of hemolysis was observed at end of infusion (EOI). Maximal suppression of hemolysis was approximately −85% change from baseline. This was achieved with the 30 mg/kg IV group and the repeat dose 15 mg/kg IV + 400 mg SC QW group. In the 2 SC cohorts, peak suppression of hemolysis was observed 3-7 days post dosing, which was consistent with observed peak concentrations of pozelimab in serum. In an ex vivo spike study, pozelimab, in-house eculizumab and in-house ravulizumab were spiked into 10, 25 or 48% pooled NHS for AP, and 5, 10 or 25% for CP. The results from AP hemolysis assays showed that, for a given concentration of spiked antibody, the maximal suppression of hemolysis for all the antibodies decreased with increased percentage of serum (Figure). The maximal suppression of hemolysis was consistently higher (32-169%) for pozelimab compared with in-house eculizumab, and lower for in-house ravulizumab compared with pozelimab and in-house eculizumab at all serum percentages tested. The results from CP hemolysis assays showed that, although the maximal suppression of hemolysis was similar for all antibodies tested, in-house ravulizumab was required to be at least a log higher in concentration to achieve a similar effect as the other two anti-C5 antibodies. CONCLUSIONS: Ex vivo studies with pooled NHS demonstrate that pozelimab robustly blocks both CP and AP hemolysis. In-house ravulizumab appeared to be less potent compared with in-house eculizumab in both CP and AP hemolysis assays. The Phase I healthy volunteer study of pozelimab demonstrated dose-dependent and significant inhibition of alternative pathway hemolysis, with the maximal suppression of hemolysis approximately −85% change from baseline. Figure Disclosures Devalaraja-Narashimha: Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Ni:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Huang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Wang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Chaudhari:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Prasad:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Harari:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Rankin:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Morton:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Weyne:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. OffLabel Disclosure: The drug is pozelimab and it is a fully human anti-C5 IgG4 being tested a Phase 1 study in healthy volunteers.

Background/Purpose: In adults with inflammatory arthritis, insulin resistance (IR) is associated with diabetes and cardiovascular disease. Interleukin-6 (IL-6) is postulated to play a mechanistic role in IR. The aim of this study was to evaluate the degree of IR among children with systemic juvenile idiopathic arthritis (sJIA) and to determine whether treatment with tocilizumab (TCZ) results in attenuation of IR in sJIA.Methods: Patients from TENDER 1 were included if baseline and week 6 fasting insulin were measured. Glucocorticoid tapering was not permitted until week 6. Insulin sensitivity was quantified using the homeostatic model of insulin resistance (HOMA-IR). Patients were classified as having IR if their HOMA-IR was Ն2.2 U. Change in HOMA-IR after 6 weeks was assessed using paired t-test. Baseline associations with HOMA-IR and factors predicting change of HOMA-IR from baseline were assessed using regression analyses. Factors changing in association with HOMA-IR change were assessed.Results: Ninety-two patients with sJIA were analyzed. 62 were randomly assigned to TCZ and 30 to placebo, 12 of whom required escape therapy with TCZ by week 6. At baseline, 40 patients (43%) had IR. Baseline HOMA-IR was associated with higher standardized body mass index and higher IL-6 levels (␤-

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The Earable device is a behind-the-ear wearable originally developed to measure cognitive function. Since Earable measures electroencephalography (EEG), electromyography (EMG), and electrooculography (EOG), it may also have the potential to objectively quantify facial muscle and eye movement activities relevant in the assessment of neuromuscular disorders. As an initial step to developing a digital assessment in neuromuscular disorders, a pilot study was conducted to determine whether the Earable device could be utilized to objectively measure facial muscle and eye movements intended to be representative of Performance Outcome Assessments, (PerfOs) with tasks designed to model clinical PerfOs, referred to as mock-PerfO activities. The specific aims of this study were: To determine whether the Earable raw EMG, EOG, and EEG signals could be processed to extract features describing these waveforms; To determine Earable feature data quality, test re-test reliability, and statistical properties; To determine whether features derived from Earable could be used to determine the difference between various facial muscle and eye movement activities; and, To determine what features and feature types are important for mock-PerfO activity level classification. A total of N = 10 healthy volunteers participated in the study. Each study participant performed 16 mock-PerfOs activities, including talking, chewing, swallowing, eye closure, gazing in different directions, puffing cheeks, chewing an apple, and making various facial expressions. Each activity was repeated four times in the morning and four times at night. A total of 161 summary features were extracted from the EEG, EMG, and EOG bio-sensor data. Feature vectors were used as input to machine learning models to classify the mock-PerfO activities, and model performance was evaluated on a held-out test set. Additionally, a convolutional neural network (CNN) was used to classify low-level representations of the raw bio-sensor data for each task, and model performance was correspondingly evaluated and compared directly to feature classification performance. The model’s prediction accuracy on the Earable device’s classification ability was quantitatively assessed. Study results indicate that Earable can potentially quantify different aspects of facial and eye movements and may be used to differentiate mock-PerfO activities. Specially, Earable was found to differentiate talking, chewing, and swallowing tasks from other tasks with observed F1 scores >0.9. While EMG features contribute to classification accuracy for all tasks, EOG features are important for classifying gaze tasks. Finally, we found that analysis with summary features outperformed a CNN for activity classification. We believe Earable may be used to measure cranial muscle activity relevant for neuromuscular disorder assessment. Classification performance of mock-PerfO activities with summary features enables a strategy for detecting disease-specific signals relative to controls, as well as the monitoring of intra-subject treatment responses. Further testing is needed to evaluate the Earable device in clinical populations and clinical development settings.

Dysfunctional leukocyte-endothelial interactions are thought to play a key role in systemic lupus erythematosus pathogenesis. We questioned the importance of TNF-α and IL-1 for endothelial activation in MRL/lpr lupus-prone mice. Endothelial ICAM-1 and VCAM-1 expression increased significantly with disease evolution in kidney, heart, and brain, as shown by i.v. injected radiolabeled Ab uptake. Lung endothelial VCAM-1 also increased, while lung endothelial ICAM-1 did not rise above a high basal level. Immunoassays showed a significantly raised circulating level of TNF-α by 14 wk, with levels of circulating IL-1α and IL-1β being additionally raised by 20 wk. With 14-wk-old MRL/lpr, anti-TNF-α antiserum inhibited expression of ICAM-1 and VCAM-1 by endothelial cells cultured with sera in vitro, and uptake of anti-ICAM-1 and anti-VCAM-1 mAb in lung, kidney, brain, and heart in vivo. In contrast, both anti-TNF-α and anti-IL-1 antisera were required for maximal inhibition in vitro and in vivo at 20 wk. These data indicate that TNF-α is largely responsible for the early up-regulation of endothelial ICAM-1 and VCAM-1, but that IL-1 enhances expression in late disease. Our observations provide novel insights of possible relevance to understanding endothelial activation in systemic lupus erythematosus, and highlight an approach that can be extended to dissecting other chronic inflammatory diseases.

The Notch signaling pathway is an evolutionarily conserved, cell-cell communication system with critical functions in organogenesis and tissue homeostasis, including hemato- and immuno-poiesis. Recent data have revealed important roles for Notch in the regulation of mature T cell differentiation and function. Studies in mouse models have identified Notch as a critical regulator of pathogenic T-cell responses during acute GVHD (aGVHD) (Zhang Y, 2011, Blood). However, the exact biological effects and the therapeutic potential of Notch pathway manipulation in clinical settings remains unclear. To address this question, we tested the activity of Notch pathway blockade in a non-human primate (NHP) aGVHD model, previously shown to exhibit donor T cell-intrinsic activation of the Notch pathway during aGVHD (Furlan SN, 2015, Sci Transl Med). To inhibit the Notch pathway, we used a blocking mAb to the Notch ligand DLL4, identified as the dominant ligand in a mouse aGVHD model (Tran, 2013, JCI; Chung, 2017, JCI). Prophylactic treatment regimens with either a single administration of anti-DLL4 mAb on day 0 (3 mg/kg), or with 3 doses (3 mg/kg each) on days 0, 7 and 14 significantly improved GVHD-free survival of allo-HCT recipients (median survival time (MST) = 26.5 days for the single dose regimen, and MST = 26 days for the triple dose regimen) in comparison with unprophylaxed controls (MST = 8 days, p<0.005; Fig 1A). Importantly, donor cell engraftment and hematopoietic reconstitution were normal (Fig 1B). Of note, DLL4 blockade dramatically changed the clinical phenotype of break-through GVHD, providing complete protection from gastrointestinal (GI) aGVHD, and instead showing clinical manifestations predominantly restricted to liver (Fig 1C, D). We next sought to determine the mechanism of GI aGVHD protection caused by DLL4 inhibition. Notably, anti-DLL4 GVHD prophylaxis decreased the expression of the gut-homing α4β7 integrin on CD8 T cells from the peripheral blood, spleen, mesenteric lymph nodes and colon in treated recipients (Fig 2A). Intestinal CD8 T cells also displayed a skewing towards a mature, effector-memory phenotype (Fig 2B) and reduced Ki67+ proliferation (Fig 2C). In contrast, CD8 T cells isolated from liver, the main site of break-through GVHD in anti-DLL4 mAb-treated animals, demonstrated a similar proliferation rate in anti-DLL4-treated compared to unprophylaxed cohorts (Fig 2C). Moreover, anti-DLL4 mAb prophylaxis was associated with an improved regulatory T cell/conventional T cell ratio in the colon but not in liver (Fig 2D). Collectively, our results show that the function of individual Notch ligands is preserved from mice to primates in aGVHD. We provide the first demonstration of the important role of Notch signaling in alloimmune T cell activation in a large animal model of aGVHD, and the therapeutic potential of DLL4 blockade for aGVHD prevention. Disclosures Tkachev: Regeneron Pharmaceuticals, Inc.: Research Funding. Kuhnert:Regeneron Pharmaceuticals, Inc.: Employment. Blazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Carpenter:Regeneron Pharmaceuticals, Inc.: Employment. Harari:Regeneron Pharmaceuticals, Inc.: Employment. Thurston:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Kean:Regeneron Pharmaceuticals, Inc.: Research Funding.