Laura Richman

Neutralization of interferon‐α/β–inducible genes and downstream effect in a phase I trial of an anti–interferon‐α monoclonal antibody in systemic lupus erythematosus

OBJECTIVE Type I interferons (IFNs) play an important role in the pathogenesis of systemic lupus erythematosus (SLE). This phase Ia trial was undertaken to evaluate the safety, pharmacokinetics, and immunogenicity of anti-IFNalpha monoclonal antibody (mAb) therapy in SLE. During the trial, we also examined whether overexpression of an IFNalpha/beta-inducible gene signature in whole blood could serve as a pharmacodynamic biomarker to evaluate IFNalpha neutralization and investigated downstream effects of neutralizing IFNalpha on BAFF and other key signaling pathways, i.e., granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), tumor necrosis factor alpha (TNFalpha), and IL-1beta, in SLE. METHODS Affymetrix Human Genome U133 Plus 2.0 microarrays were used to profile whole blood and lesional skin of patients receiving standard therapy for mild to moderate SLE. Selected IFNalpha/beta-inducible proteins were analyzed by immunohistochemistry. RESULTS With the study treatment, we observed anti-IFNalpha mAb-specific and dose-dependent inhibition of overexpression of IFNalpha/beta-inducible genes in whole blood and skin lesions from SLE patients, at both the transcript and the protein levels. In SLE patients with overexpression of messenger RNA for BAFF, TNFalpha, IL-10, IL-1beta, GM-CSF, and their respective inducible gene signatures in whole blood and/or skin lesions, we observed a general trend toward suppression of the expression of these genes and/or gene signatures upon treatment with anti-IFNalpha mAb. CONCLUSION IFNalpha/beta-inducible gene signatures in whole blood are effective pharmacodynamic biomarkers to evaluate anti-IFNalpha mAb therapy in SLE. Anti-IFNalpha mAb can neutralize overexpression of IFNalpha/beta-inducible genes in whole blood and lesional skin from SLE patients and has profound effects on signaling pathways that may be downstream of IFNalpha in SLE.

Safety profile and clinical activity of sifalimumab, a fully human anti-interferon α monoclonal antibody, in systemic lupus erythematosus: a phase I, multicentre, double-blind randomised study

Background Type I interferons (IFNs) appear to play a central role in disease pathogenesis in systemic lupus erythematosus (SLE), making them potential therapeutic targets. Methods Safety profile, pharmacokinetics, immunogenicity, pharmacodynamics and clinical activity of sifalimumab, an anti-IFNα monoclonal antibody, were assessed in a phase I, multicentre, randomised, double-blind, dose-escalation study with an open-label extension in adults with moderately active SLE. Subjects received one intravenous dose of sifalimumab (n=33 blinded phase, 0.3, 1, 3, 10 or 30 mg/kg; n=17 open-label, 1, 3, 10 or 30 mg/kg) or placebo (n=17). Each phase lasted 84 days. Results Adverse events (AEs) were similar between groups; about 97% of AEs were grade 1 or 2. All grade 3 and 4 AEs and all serious AEs (2 placebo, 1 sifalimumab) were deemed unrelated to the study drug. No increase in viral infections or reactivation was observed. Sifalimumab caused dose-dependent inhibition of type I IFN-induced mRNAs (type I IFN signature) in whole blood and corresponding changes in related proteins in affected skin. Exploratory analyses showed consistent trends toward improvement in disease activity in sifalimumab-treated versus placebo-treated subjects. A lower proportion of sifalimumab-treated subjects required new or increased immunosuppressive treatments (12% vs 41%; p=0.03) and had fewer Systemic Lupus Erythematosus Disease Activity Index flares (3% vs 29%; p=0.014). Conclusions Sifalimumab had a safety profile that supports further clinical development. This trial demonstrated that overexpression of type I IFN signature in SLE is at least partly driven by IFNα, and exploratory analyses suggest that IFNα inhibition may be associated with clinical benefit in SLE. Trial registration number NCT00299819.

Type I Interferon: Potential Therapeutic Target for Psoriasis?

Background Psoriasis is an immune-mediated disease characterized by aberrant epidermal differentiation, surface scale formation, and marked cutaneous inflammation. To better understand the pathogenesis of this disease and identify potential mediators, we used whole genome array analysis to profile paired lesional and nonlesional psoriatic skin and skin from healthy donors. Methodology/Principal Findings We observed robust overexpression of type I interferon (IFN)–inducible genes and genomic signatures that indicate T cell and dendritic cell infiltration in lesional skin. Up-regulation of mRNAs for IFN-α subtypes was observed in lesional skin compared with nonlesional skin. Enrichment of mature dendritic cells and 2 type I IFN–inducible proteins, STAT1 and ISG15, were observed in the majority of lesional skin biopsies. Concordant overexpression of IFN-γ and TNF-α–inducible gene signatures occurred at the same disease sites. Conclusions/Significance Up-regulation of TNF-α and elevation of the TNF-α–inducible gene signature in lesional skin underscore the importance of this cytokine in psoriasis; these data describe a molecular basis for the therapeutic activity of anti–TNF-α agents. Furthermore, these findings implicate type I IFNs in the pathogenesis of psoriasis. Consistent and significant up-regulation of type I IFNs and their associated gene signatures in psoriatic skin suggest that type I IFNs may be potential therapeutic targets in psoriasis treatment.

Patients with systemic lupus erythematosus, myositis, rheumatoid arthritis and scleroderma share activation of a common type I interferon pathway

Objective To characterise activation of the type I interferon (IFN) pathway in patients with systemic lupus erythematosus (SLE), dermatomyositis (DM), polymyositis (PM), rheumatoid arthritis (RA) and systemic scleroderma (SSc) and to evaluate the potential to develop a molecular diagnostic tool from the peripheral blood that reflects this activation in disease-affected tissues. Methods Overexpressed transcripts were identified in the whole blood (WB) of 262 patients with SLE, 44 with DM, 33 with PM, 28 with SSc and 89 with RA and compared with 24 healthy subjects using Affymetrix microarrays. A five gene type I IFN signature was assessed in these subjects to identify subpopulations showing both activation and concordance of the type I IFN pathway in the peripheral blood and disease-affected tissues of each disease and to correlate activation of this pathway in the WB with clinical measurements. Results A common set of 36 type I IFN inducible transcripts were identified among the most overexpressed in the WB of all subjects. Significant activation of the type I IFN pathway in subgroups of each of the five diseases studied was observed. Baseline disease activity measurements correlated with a type I IFN gene signature in the WB of subjects with SLE, PM and SSc, as did various serum autoantibody levels in subjects with SLE and DM. This signature was also well correlated between disease-affected tissue and WB in subjects with SLE, DM, PM and SSc. Conclusions The results indicate that the type I IFN pathway is activated in patient subsets of five rheumatic diseases and suggest that these subsets may benefit from anti-IFN therapy.

A phase 1b clinical trial evaluating sifalimumab, an anti-IFN-α monoclonal antibody, shows target neutralisation of a type I IFN signature in blood of dermatomyositis and polymyositis patients

Objective To assess the pharmacodynamic effects of sifalimumab, an investigational anti-IFN-α monoclonal antibody, in the blood and muscle of adult dermatomyositis and polymyositis patients by measuring neutralisation of a type I IFN gene signature (IFNGS) following drug exposure. Methods A phase 1b randomised, double-blinded, placebo controlled, dose-escalation, multicentre clinical trial was conducted to evaluate sifalimumab in dermatomyositis or polymyositis patients. Blood and muscle biopsies were procured before and after sifalimumab administration. Selected proteins were measured in patient serum with a multiplex assay, in the muscle using immunohistochemistry, and transcripts were profiled with microarray and quantitative reverse transcriptase PCR assays. A 13-gene IFNGS was used to measure the pharmacological effect of sifalimumab. Results The IFNGS was suppressed by a median of 53–66% across three time points (days 28, 56 and 98) in blood (p=0.019) and 47% at day 98 in muscle specimens post-sifalimumab administration. Both IFN-inducible transcripts and proteins were prevalently suppressed following sifalimumab administration. Patients with 15% or greater improvement from baseline manual muscle testing scores showed greater neutralisation of the IFNGS than patients with less than 15% improvement in both blood and muscle. Pathway/functional analysis of transcripts suppressed by sifalimumab showed that leucocyte infiltration, antigen presentation and immunoglobulin categories were most suppressed by sifalimumab and highly correlated with IFNGS neutralisation in muscle. Conclusions Sifalimumab suppressed the IFNGS in blood and muscle tissue in myositis patients, consistent with this molecules mechanism of action with a positive correlative trend between target neutralisation and clinical improvement. These observations will require confirmation in a larger trial powered to evaluate efficacy.

A novel oncogenic role for the miRNA-506-514 cluster in initiating melanocyte transformation and promoting melanoma growth.

Malignant melanoma is the most aggressive form of skin cancer and its incidence has doubled in the last two decades. It represents only 4% of skin cancer cases per year, but causes as many as 74% of skin cancer deaths. Early detection of malignant melanoma is associated with survival rates of up to 90%, but later detection (stage III to stage IV) is associated with survival rates of only 10%. Dysregulation of microRNA (miRNA) expression has been linked to tumor development and progression by functioning either as a tumor suppressor, an oncogene or a metastasis regulator in multiple cancer types. To understand the role of miRNA in the pathogenesis of malignant melanoma and identify biomarkers of metastasis, miRNA expression profiles in skin punches from 33 metastatic melanoma patients and 14 normal healthy donors were compared. We identified a cluster of 14 miRNAs on the X chromosome, termed the miR-506-514 cluster, which was consistently overexpressed in nearly all melanomas tested (30–60 fold, P<0.001), regardless of mutations in N-ras or B-raf. Inhibition of the expression of this cluster as a whole, or one of its sub-clusters (Sub-cluster A) consisting of six mature miRNAs, led to significant inhibition of cell growth, induction of apoptosis, decreased invasiveness and decreased colony formation in soft agar across multiple melanoma cell lines. Sub-cluster A of the miR-506-514 cluster was critical for maintaining the cancer phenotype, but the overexpression of the full cluster was necessary for melanocyte transformation. Our results provide new insights into the functional role of this miRNA cluster in melanoma, and suggest new approaches to treat or diagnose this disease.

Inhibition of Myogenic MicroRNAs 1, 133, and 206 by Inflammatory Cytokines Links Inflammation and Muscle Degeneration in Adult Inflammatory Myopathies

The molecular basis of inflammatory myopathies such as dermatomyositis (DM), polymyositis, and inclusion body myositis, which share the characteristics of chronic muscle inflammation and skeletal muscle wasting, are poorly understood. As such, effective targeted treatments for these diseases are lacking, resulting in critical unmet medical needs for these devastating diseases. The purpose of this study was to identify possible new targets for drug development by exploring the mechanism by which inflammation may play a role in the pathology of the inflammatory myopathies.

Use of type I interferon-inducible mRNAs as pharmacodynamic markers and potential diagnostic markers in trials with sifalimumab, an anti-IFNα antibody, in systemic lupus erythematosus

Type I interferons are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Type I interferon-inducible mRNAs are widely and concordantly overexpressed in the periphery and involved tissues of a subset of SLE patients, and provide utility as pharmacodynamic biomarkers to aid dose selection, as well as potential indicators of patients who might respond favorably to anti-IFNα therapy in SLE. We implemented a three-tiered approach to identify a panel of type I interferon-inducible mRNAs to be used as potential pharmacodynamic biomarkers to aid dose selection in clinical trials of sifalimumab, an anti-IFNα monoclonal antibody under development for the treatment of SLE. In a single-dose escalation phase 1 trial, we observed a sifalimumab-specific and dose-dependent inhibition of the overexpression of type I interferon-inducible mRNAs in the blood of treated subjects. Inhibition of expression of type I interferon-inducible mRNAs and proteins was also observed in skin lesions of SLE subjects from the same trial. Inhibiting IFNα resulted in a profound downstream effect in these SLE subjects that included suppression of mRNAs of B-cell activating factor belonging to the TNF family and the signaling pathways of TNFα, IL-10, IL-1β, and granulocyte-macrophage colony-stimulating factor in both the periphery and skin lesions. A scoring method based on the expression of type I interferon-inducible mRNAs partitioned SLE patients into two distinct subpopulations, which suggests the possibility of using these type I interferon-inducible genes as predictive biomarkers to identify SLE patients who might respond more favorably to anti-type I interferon therapy.

MicroRNA‐206 induces G1 arrest in melanoma by inhibition of CDK4 and Cyclin D

Expression profiling of microRNAs in melanoma lesional skin biopsies compared with normal donor skin biopsies, as well as melanoma cell lines compared with normal melanocytes, revealed that hsa‐miR‐206 was down‐regulated in melanoma (−75.4‐fold, P = 1.7 × 10−4). MiR‐206 has been implicated in a large number of cancers, including breast, lung, colorectal, ovarian, and prostate cancers; however, its role in tumor development remains largely unknown, its biologic function is poorly characterized, and its targets affecting cancer cells are largely unknown. MiR‐206 reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatics identified cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate targets. Western blots and 3′UTR reporter gene assays revealed that miR‐206 inhibited translation of CDK4, Cyclin D1, and Cyclin C. Additionally, hsa‐miR‐206 transfection induced G1 arrest in multiple melanoma cell lines. These observations support hsa‐miR‐206 as a tumor suppressor in melanoma and identify Cyclin C, Cyclin D1, and CDK4 as miR‐206 targets.

Pharmacogenomics and Translational Simulations to Bridge Indications for an Anti-Interferon-α Receptor Antibody

A type I interferon (IFN) gene signature shared by systemic lupus erythematous (SLE) and systemic sclerosis (SSc) was used to evaluate an anti‐type I IFN‐α receptor (IFN‐αR) monoclonal antibody, MEDI‐546, in a phase I trial in SSc. MEDI‐546 suppressed IFN signature in blood and skin of SSc patients in a dose‐dependent manner. To bridge clinical indications to SLE, we developed a model incorporating (i) pharmacokinetics (PK) and pharmacodynamics (PD) in SSc patients, (ii) internalization kinetics of MEDI‐546/IFN‐αR complex, and (iii) the different IFN signatures between SSc and SLE. Simulations predicted that i.v. administration of MEDI‐546 at 300‐ or 1,000‐mg monthly doses could suppress IFN signature in blood to levels of healthy subjects in 53 and 68% of SLE patients, respectively. An innovative approach utilizing a novel biomarker characterized the PD of MEDI‐546 by modeling and simulation and allowed rapid progression of MEDI‐546 from a phase I study in SSc to a randomized, multiple‐dose phase II trial.