Integrated analysis of dermal blister fluid proteomics and genome-wide skin gene expression in systemic sclerosis: an observational study.

Background: Skin fibrosis is a hallmark feature of systemic sclerosis. Skin biopsy transcriptomics and blister fluid proteomics give insight into the local environment of the skin. We have integrated these modalities with the aim of developing a surrogate for the modified Rodnan skin score (mRSS), using candidate genes and proteins from the skin and blister fluid as anchors to identify key analytes in the plasma. Methods: In this single-centre, prospective observational study at the Royal Free Campus, University College London, London, UK, transcriptional and proteomic analyses of blood and skin were performed in a cohort of patients with systemic sclerosis (n=52) and healthy controls (n=16). Weighted gene co-expression network analysis was used to explore the association of skin transcriptomics data, clinical traits, and blister fluid proteomic results. Candidate hub analytes were identified as those present in both blister and skin gene sets (modules), and which correlated with plasma (module membership >0·7 and gene significance >0·6). Hub analytes were confirmed using RNA transcript data obtained from skin biopsy samples from patients with early diffuse cutaneous systemic sclerosis at 12 months. Findings: We identified three modules in the skin, and two in blister fluid, which correlated with a diagnosis of early diffuse cutaneous systemic sclerosis. From these modules, 11 key hub analytes were identified, present in both skin and blister fluid modules, whose transcript and protein levels correlated with plasma protein concentrations, mRSS, and showed statistically significant correlation on repeat transcriptomic samples taken at 12 months. Multivariate analysis identified four plasma analytes as correlates of mRSS (COL4A1, COMP, SPON1, and TNC), which can be used to differentiate disease subtype. Interpretation: This unbiased approach has identified potential biological candidates that might be drivers of local skin pathogenesis in systemic sclerosis. By focusing on measurable analytes in the plasma, we generated a promising composite plasma protein biomarker that could be used for assessment of skin severity, case stratification, and as a potential outcome measure for clinical trials and practice. Once fully validated, the biomarker score could replace a clinical score such as the mRSS, which carries substantial variability. Funding: GlaxoSmithKline and UK Medical Research Council.

Molecular basis for clinical diversity between autoantibody subsets in diffuse cutaneous systemic sclerosis.

OBJECTIVES: Clinical heterogeneity is a cardinal feature of systemic sclerosis (SSc). Hallmark SSc autoantibodies are central to diagnosis and associate with distinct patterns of skin-based and organ-based complications. Understanding molecular differences between patients will benefit clinical practice and research and give insight into pathogenesis of the disease. We aimed to improve understanding of the molecular differences between key diffuse cutaneous SSc subgroups as defined by their SSc-specific autoantibodies METHODS: We have used high-dimensional transcriptional and proteomic analysis of blood and the skin in a well-characterised cohort of SSc (n=52) and healthy controls (n=16) to understand the molecular basis of clinical diversity in SSc and explore differences between the hallmark antinuclear autoantibody (ANA) reactivities. RESULTS: Our data define a molecular spectrum of SSc based on skin gene expression and serum protein analysis, reflecting recognised clinical subgroups. Moreover, we show that antitopoisomerase-1 antibodies and anti-RNA polymerase III antibodies specificities associate with remarkably different longitudinal change in serum protein markers of fibrosis and divergent gene expression profiles. Overlapping and distinct disease processes are defined using individual patient pathway analysis. CONCLUSIONS: Our findings provide insight into clinical diversity and imply pathogenetic differences between ANA-based subgroups. This supports stratification of SSc cases by ANA antibody subtype in clinical trials and may explain different outcomes across ANA subgroups in trials targeting specific pathogenic mechanisms.

Safety, pharmacokinetics and pharmacodynamics of a topical SYK inhibitor in cutaneous lupus erythematosus: A double-blind Phase Ib study.

The immunoregulator spleen tyrosine kinase (SYK) is upregulated in cutaneous lupus erythematosus (CLE). This double-blind, multicentre, Phase Ib study evaluated the safety, tolerability, pharmacokinetics, pharmacodynamics and clinical efficacy of the selective SYK inhibitor GSK2646264 in active CLE lesions. Two lesions from each participant (n = 11) were each randomized to topical application of 1% (w/w) GSK2646264 or placebo for 28 days; all participants received GSK2646264 and placebo. The primary endpoint was safety and tolerability of GSK2646264, assessed by adverse event incidence and a skin tolerability test. Secondary endpoints included change from baseline in clinical activity and mRNA expression of interferon-related genes in skin biopsies. Levels of several immune cell markers were evaluated over time. Eight (73%) participants experienced ≥ 1 adverse event (all mild in intensity), and maximal dermal response was similar for GSK2646264 and placebo. The expression of several interferon-related genes, including CXCL10 and OAS1, showed modest decreases from baseline after 28 days of treatment with GSK2646264 compared with placebo. Similar findings were observed for CD3 + T cell and CD11c + dendritic cell levels; however, overall clinical activity remained unchanged with GSK2646264 vs. placebo. Further studies are warranted to assess SYK inhibitors as potential treatment for CLE.

Depletion of LAG-3+ T Cells Translated to Pharmacology and Improvement in Psoriasis Disease Activity: A Phase I Randomized Study of mAb GSK2831781.

Activated T cells drive a range of immune-mediated inflammatory diseases. LAG-3 is transiently expressed on recently activated CD4+ and CD8+ T cells. We describe the engineering and first-in-human clinical study (NCT02195349) of GSK2831781 (an afucosylated humanized IgG1 monoclonal antibody enhanced with high affinity for Fc receptors and LAG-3 and antibody-dependent cellular cytotoxicity capabilities), which depletes LAG-3 expressing cells. GSK2831781 was tested in a phase I/Ib, double-blind, placebo-controlled clinical study, which randomized 40 healthy participants (part A) and 27 patients with psoriasis (part B) to single doses of GSK2831781 (up to 0.15 and 5 mg/kg, respectively) or placebo. Adverse events were generally balanced across groups, with no safety or tolerability concern identified. LAG-3+ cell depletion in peripheral blood was observed at doses ≥ 0.15 mg/kg and was dose-dependent. In biopsies of psoriasis plaques, a reduction in mean group LAG-3+ and CD3+ T-cell counts was observed following treatment. Downregulation of proinflammatory genes (IL-17A, IL-17F, IFNγ, and S100A12) and upregulation of the epithelial barrier integrity gene, CDHR1, was observed with the 5 mg/kg dose of GSK2831781. Psoriasis disease activity improved up to day 43 at all GSK2831781 doses (0.5, 1.5, and 5 mg/kg) compared with placebo. Depletion of LAG-3-expressing activated T cells is a novel approach, and this first clinical study shows that GSK2831781 is pharmacologically active and provides encouraging early evidence of clinical effects in psoriasis, which warrants further investigation in T-cell-mediated inflammatory diseases.

Lymphocyte Activation Gene (LAG)-3 Is Associated With Mucosal Inflammation and Disease Activity in Ulcerative Colitis.

BACKGROUND AND AIMS: Lymphocyte activation gene [LAG]-3 is an immune checkpoint and its expression identifies recently activated lymphocytes that may contribute to inflammation. We investigated the role of LAG-3 by analysing its expression and function in immune cells from blood and tissue of patients with ulcerative colitis [UC]. METHODS: The phenotypic properties of LAG-3+ T cells were determined by flow cytometry, qRT-PCR and single-cell RNA-sequencing. LAG-3+ cells were quantified and correlated with disease activity. The functional effects of LAG-3+ cells were tested using a depleting anti-LAG-3 monoclonal antibody [mAb] in a mixed lymphocyte reaction [MLR]. RESULTS: LAG-3+ cells in the blood were negligible. LAG-3+ lymphocytes were markedly increased in inflamed mucosal tissue and both frequencies of LAG-3+ T cells and transcript levels of LAG3 correlated with endoscopic severity. LAG-3 expression was predominantly on effector memory T cells, and single-cell RNA-sequencing revealed LAG3 expression in activated and cytokine-producing T cell subsets. Foxp3+CD25hi Tregs also expressed LAG-3, although most mucosal Tregs were LAG-3-. Mucosal LAG-3+ cells produced mainly interferon γ [IFNγ] and interleukin-17A. LAG-3+ cell numbers decreased in patients who responded to biologics, and remained elevated in non-responders. Treatment with a depleting anti-LAG-3 mAb led to a reduction in proliferation and IFNγ production in an MLR. CONCLUSIONS: LAG-3+ cells are increased in the inflamed mucosa, predominantly on effector memory T cells with an activated phenotype and their cell numbers positively correlate with disease activity. Depleting LAG-3 eliminates activated proliferating T cells, and hence LAG-3 could be a therapeutic target in UC.

In vivo affinity and target engagement in skin and blood in a first-time-in-human study of an anti-oncostatin M monoclonal antibody.

AIMS: The oncostatin M (OSM) pathway drives fibrosis, inflammation and vasculopathy, and is a potential therapeutic target for inflammatory and fibrotic diseases. The aim of this first-time-in-human experimental medicine study was to assess the safety, tolerability, pharmacokinetics and target engagement of single subcutaneous doses of GSK2330811, an anti-OSM monoclonal antibody, in healthy subjects. METHODS: This was a phase I, randomized, double-blind, placebo-controlled, single-dose escalation, first-time-in-human study of subcutaneously administered GSK2330811 in healthy adults (NCT02386436). Safety and tolerability, GSK2330811 pharmacokinetic profile, OSM levels in blood and skin, and the potential for antidrug antibody formation were assessed. The in vivo affinity of GSK2330811 for OSM and target engagement in serum and skin blister fluid (obtained via a skin suction blister model) were estimated using target-mediated drug disposition (TMDD) models in combination with compartmental and physiology-based pharmacokinetic (PBPK) models. RESULTS: Thirty subjects were randomized to receive GSK2330811 and 10 to placebo in this completed study. GSK2330811 demonstrated a favourable safety profile in healthy subjects; no adverse events were serious or led to withdrawal. There were no clinically relevant trends in change from baseline in laboratory values, with the exception of a reversible dose-dependent reduction in platelet count. GSK2330811 exhibited linear pharmacokinetics over the dose range 0.1-6 mg kg-1 . The estimated in vivo affinity (nM) of GSK2330811 for OSM was 0.568 [95% confidence interval (CI) 0.455, 0.710] in the compartmental with TMDD model and 0.629 (95% CI 0.494, 0.802) using the minimal PBPK with TMDD model. CONCLUSIONS: Single subcutaneous doses of GSK2330811 were well tolerated in healthy subjects. GSK2330811 demonstrated sufficient affinity to achieve target engagement in systemic circulation and target skin tissue, supporting the progression of GSK2330811 clinical development.