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Supplementary Data. Supplemental Table S1: Summary of MEDI-573 IC50 on the proliferation of sarcoma cell lines with or without exogenously added IGF-1 or IGF-2. Supplemental Figure S1: Effect of MEDI-573 in combination with AZD2014 on SJSA-1 tumor growth in vivo.

MEDI-573 is a human antibody that neutralizes insulin-like growth factor (IGF) I and IGFII. IGFs are overexpressed in multiple types of cancer; their overexpression is a potential mechanism for resistance to IGFI receptor (IGFIR)-targeting therapy. Effects of IGF on cell proliferation, differentiation, and survival are mediated through its binding to and activation of IGFIR or insulin receptor A (IR-A). In this study, we measured the mRNA levels of IGFI, IGFII, and IGFIR in human pediatric sarcoma xenografts, and protein levels in sarcoma cell lines. MEDI-573 potently inhibited in vitro proliferation of sarcoma cell lines, with Ewing sarcoma cell lines being the most sensitive. In addition, MEDI-573 inhibited IGFI- and IGFII-induced sarcoma cell proliferation in vitro. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGFIR, pIR-A, and pAKT and significantly blocked IGFI- and IGFII-induced activation of the IGFIR and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo and inhibition correlated with neutralization of IGFI and IGFII. Combination of MEDI-573 with either rapamycin or AZD2014, another mTOR inhibitor (mTORi), significantly enhanced the antitumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by IGFI and IGFII, and inhibition of IGFI and IGFII by MEDI-573 results in significant slowing of tumor growth rate in sarcoma models, particularly in Ewing sarcoma. These data provide evidence for the potential benefits of MEDI-573 and mTORi combinations in patients with Ewing sarcoma. Mol Cancer Ther; 13(11); 2662–73. ©2014 AACR.

The gut microbiota has emerged as a key mediator of human physiology, and germ-free mice have been essential in demonstrating a role for the microbiome in disease. Preclinical models using conventional mice offer the advantage of working with a mature immune system. However, optimal protocols for fecal microbiota transplant (FMT) engraftment in conventional mice are yet to be established. Conventional BALB/c mice were randomized to receive 3-day (3d) or 3-week (3w) antibiotic (ABX) regimen in their drinking water followed by 1 or 5-daily FMTs from a human donor. Fecal samples were collected longitudinally and characterized using 16S ribosomal RNA (rRNA) sequencing. Semi-targeted metabolomic profiling of fecal samples was also done with liquid chromatography–mass spectrometry (LC-MS). Lastly, we sought to confirm our findings in BKS mice. Recovery of baseline diversity scores were greatest in the 3d groups, driven by re-emergence of mouse commensal microbiota, whereas the most resemblance to donor microbiota was seen in the 3w + 5-FMT group. Amplicon sequence variants (ASVs) that were linked to the input material (human ASVs) engrafted to a significantly greater extent when compared to mouse ASVs in the 3-week groups but not the 3-day groups. Lastly, comparison of metabolomic profiles revealed distinct functional profiles by ABX regimen. These results indicate successful model optimization and emphasize the importance of ABX duration and frequency of FMT dosing, the most stable and reliable colonization by donor ASVs was seen in the 3wk + 5-FMT group.

The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti-PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.

BackgroundMEDI9197 is an intratumorally administered toll-like receptor 7 and 8 agonist. In mice, MEDI9197 modulated antitumor immune responses, inhibited tumor growth and increased survival. This first-time-in-human, phase 1 study evaluated MEDI9197 with or without the programmed cell death ligand-1 (PD-L1) inhibitor durvalumab and/or palliative radiation therapy (RT) for advanced solid tumors.Patients and methodsEligible patients had at least one cutaneous, subcutaneous, or deep-seated lesion suitable for intratumoral (IT) injection. Dose escalation used a standard 3+3 design. Patients received IT MEDI9197 0.005–0.055 mg with or without RT (part 1), or IT MEDI9197 0.005 or 0.012 mg plus durvalumab 1500 mg intravenous with or without RT (part 3), in 4-week cycles. Primary endpoints were safety and tolerability. Secondary endpoints included pharmacokinetics, pharmacodynamics, and objective response based on Response Evaluation Criteria for Solid Tumors version 1.1. Exploratory endpoints included tumor and peripheral biomarkers that correlate with biological activity or predict response.ResultsFrom November 2015 to March 2018, part 1 enrolled 35 patients and part 3 enrolled 17 patients; five in part 1 and 2 in part 3 received RT. The maximum tolerated dose of MEDI9197 monotherapy was 0.037 mg, with dose-limiting toxicity (DLT) of cytokine release syndrome in two patients (one grade 3, one grade 4) and 0.012 mg in combination with durvalumab 1500 mg with DLT of MEDI9197-related hemorrhagic shock in one patient (grade 5) following liver metastasis rupture after two cycles of MEDI9197. Across parts 1 and 3, the most frequent MEDI9197-related adverse events (AEs) of any grade were fever (56%), fatigue (31%), and nausea (21%). The most frequent MEDI9197-related grade ≥3 events were decreased lymphocytes (15%), neutrophils (10%), and white cell counts (10%). MEDI9197 increased tumoral CD8+ and PD-L1+ cells, inducing type 1 and 2 interferons and Th1 response. There were no objective clinical responses; 10 patients in part 1 and 3 patients in part 3 had stable disease ≥8 weeks.ConclusionIT MEDI9197 was feasible for subcutaneous/cutaneous lesions but AEs precluded its use in deep-seated lesions. Although no patients responded, MEDI9197 induced systemic and intratumoral immune activation, indicating potential value in combination regimens in other patient populations.Trial registration numberNCT02556463.

Neutrophils abandon infection control during wound repair., The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti–PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.

ObjectiveImmune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development.MethodsVIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates.ResultsWe generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies.ConclusionsVIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.

Summary During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

Tissue residency is considered a defining feature of the innate lymphoid cell (ILC) populations located within mucosal and adipose tissues. ILCs are also present within all lymphoid tissues, but whether ILCs migrate between lymphoid and nonlymphoid sites and in what context is poorly understood. To determine whether migratory ILCs exist within peripheral lymph nodes (LNs), we labeled all cells within the brachial LN (bLN) of transgenic mice expressing a photoconvertible fluorescent protein by direct exposure to light. Tracking of cellular changes in the labeled LN revealed the gradual migration of new ILCs into the tissue, balanced by egress of ILCs dependent on sphingosine-1-phosphate receptors. Most of the migratory ILCs were ILC1s, entering LNs directly from the circulation in a CD62L- and CCR7-dependent manner and thus behaving like conventional natural killer (cNK) cells. Upon egress, both ILC1s and cNK cells were found to recirculate through peripheral LNs. A distinct population of migratory ILC2s were detected in the LN, but most of the ILC3s were tissue resident. Functionally, both migratory and resident ILC1s within LNs were able to rapidly produce IFN-γ to support the generation of robust TH1 T cell responses after immunization. Thus, migratory and resident ILC populations exist within peripheral LNs, with ILC1s, akin to cNK cells, able to traffic into these tissues where they can contribute to the initiation of adaptive immunity.

Introduction: Tertiary lymphoid structures are ectopic formations arising in areas of chronic inflammation, including cancers. TLS formation detected in tumors is thought to be associated with favorable prognosis, and markers of TLS formation and activity are currently being elucidated. The objective of this study was to determine the association between genes associated with TLS formation and outcomes in lung and bladder cancer patients treated with durvalumab (D). Methods: Previously published TLS-associated genes and gene signatures (Fridman et al., 2019) were assessed in baseline tumor biopsy RNA sequencing data from D-treated (monotherapy) non-small cell lung (NSCLC, n = 97) and bladder (BLCA, n = 62) cancer patients from a nonrandomized phase Ib/II clinical trial (1108/NCT01693562), as well as in lung adenocarcinoma (LUAD, n = 594), squamous cell carcinoma (LUSC, n = 551) and bladder cancer (BLCA, n = 433) cohorts from TCGA. Kaplan-Meier analysis was performed to determine associations between TLS-related signature expression at baseline and overall survival (OS) in D-treated patients, as well as in LUAD, LUSC, and BLCA patients from TCGA to determine prognostic significance of these signatures. TLS-related signatures were also compared in D-treated responders and non-responders via ANOVA. Results: Higher levels of CXCL13 transcript in D-treated patients was associated with better median OS (20.2 vs. 6.5 months in CXCL13-high vs. low patients respectively, p = 0.01). CXCL13 expression was not significantly associated with survival in either LUAD or LUSC TCGA patients. Elevated expression of TLS-related chemokine and T-follicular helper cell (Tfh) signatures were also linked to better OS in D-treated patients (p < 0.05) compared to lung cancer patients in TCGA. These signatures were significantly elevated in D-treated responders compared to patients with progressive disease (fold-change > 1.5, p < 0.05). In D-treated BLCA patients, elevated chemokine and Tfh signatures were associated with better OS (median OS not reached in signature-high patients, p < 0.05). Both signatures were not associated with OS in BLCA patients from TCGA. In BLCA D-treated complete responders (n = 4), all TLS-related markers were significantly elevated compared to patients with progressive disease. Elevated levels of CXCL13 transcript and either PDL1 protein or interferon-γ gene signature levels tracked with better OS in D-treated patients. Assessment of TLS structures via immunohistochemistry in the same patients is currently being explored. Conclusions: TLS-related genes were elevated at baseline in D-treated NSCLC and BLCA patients with better response and OS. Modulation of these signatures was predictive of IO response, as they were not associated with survival in treatment naïve patients in TCGA. These results enhance understanding of mechanisms driving increased immunogenicity in IO-responsive patients. Citation Format: Sriram Sridhar, Christopher M. Morehouse, Yashaswi Shreshta, Marlon Rebelatto, Ikbel Achour, Ioannis Kagiampakis, Ezogelin Oflazoglu, Katie Streicher. Biomarkers associated with tertiary lymphoid structures are elevated in lung and bladder cancer patients who respond to durvalumab treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5667.

Transitional type-1 (T1) cells are peripheral immature B cells known to populate the spleen (spl) after completing their BCR assembly in the bone marrow (BM). To advance the understanding of splenic T1 (CD19posCD24hiCD21neg) B cells, we addressed the heterogeneity and biology of these cells using flow cytometry combined with genetically modified mice. Most recent emigrant T1 cells were selected by excluding CD23pos and including CD93high (AA4.1) B cells termed T12123DN. Transcriptomic analysis identified RAG1 and 2 as signature genes for this B cell population. Further separation of T12123DN cells based on surface IgM expression revealed a previously undescribed cell subset with undetectable cell surface IgM (-IgMneg). The spl-IgMneg subsets expresses RAG1/2 and actively undergoes Igk gene rearrangement at levels comparable to BM pre-B cells. Upon in vitro exposure to BAFF or transplantation into immunodeficient hosts, spl-IgMneg cells can give rise to fully mature IgMposIgDpos B cells. Furthermore, BAFF-R and NF-kB pathways are required for their efficient maturation. Our findings suggest that the spl-T1 population encompasses a subset of B cells that resemble but are distinct from the developing B cells in the BM. These spl-IgMneg B cells may represent receptor editing B cells, and/or precursor B cells undergoing BCR assembly and selection in the periphery, possibly providing an opportunity for tolerance induction to tissue restricted self-antigens and microbiota-derived antigens.

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.

Long-lived antibody-secreting cells (ASCs) are critical for the maintenance of humoral immunity through the continued production of antibodies specific for previously encountered pathogen or vaccine antigens. Recent reports describing humoral immune memory have suggested the importance of long-lived CD19− bone marrow (BM) ASCs, which secrete antibodies recognizing previously encountered vaccine antigens. However, these reports do not agree upon the unique contribution of the CD19+ BM ASC subset toward humoral immunity. Here, we found both CD19+ and negative ASCs from human BM were similar in functional capacity to react to a number of vaccine antigens via ELISpot assays. The CD19+ cells were the predominant ASC population found in lymphoid tissues, and unlike the CD19− ASCs, which were found only in spleen and BM, the CD19+ ASCs were found in tonsil and blood. CD19+ ASCs from the BM, spleen, and tonsil were capable of recognizing polio vaccine antigens, indicating the CD19+ ASC cells play a novel role in long-lasting immune defense. Comparative gene expression analysis indicated CD19+ and negative BM ASCs differed significantly by only 14 distinct messenger RNAs and exhibited similar gene expression for cell cycle, autophagy, and apoptosis control necessary for long life. In addition, we show identical CDR-H3 sequences found on both BM ASC subsets, indicating a shared developmental path. Together, these results provide novel insight for the distribution, function, genetic regulation, and development of long-lived ASCs and may not only impact improved cell therapies but also enhance strategies for vaccine development.

OBJECTIVE B cells impact the progression of systemic sclerosis (SSc; scleroderma) through multiple pathogenic mechanisms. CD19 inhibition in mice reduced skin thickness, collagen production, and autoantibody levels, consistent with CD19 expression on plasma cells (PCs), the source of antibody production. PC depletion could effectively reduce collagen deposition and inflammation in SSc; therefore, we investigated the effects of PC depletion on SSc disease activity. METHODS A PC gene signature was evaluated in SSc skin biopsy samples in 2 phase I clinical trials. We assessed microarray data from tissue from public studies of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), dermatomyositis (DM), systemic lupus erythematosus (SLE), and atopic dermatitis, as well as blood from a phase IIb clinical trial in SLE. RESULTS The PC signature was elevated in SSc skin specimens compared to healthy donor skin (P = 2.28 × 10-6 ) and correlated with the baseline modified Rodnan skin thickness score (MRSS) (r = 0.64, P = 0.0004). Patients with a high PC signature at baseline showed greater improvement in the MRSS (mean ± SD change 35 ± 16%; P = 6.30 × 10-4 ) following anti-CD19 treatment with inebilizumab (MEDI-551) than did patients with a low PC signature at baseline (mean ± SD change 8 ± 12%; P = 0.104). The PC signature was overexpressed in tissue from patients with SLE, DM, COPD, interstitial lung disease, and IPF relative to controls (all fold change >2; P < 0.001). The PC signature also differed significantly between SLE patients with mild-to-moderate disease and those with severe disease (SLE Disease Activity Index cutoff at 10) (fold change 1.44; P = 3.90 × 10-3 ) and correlated significantly with the degree of emphysema in COPD (r = 0.53, P = 7.55 × 10-8 ). CONCLUSION Our results support the notion that PCs have a role in the pathogenesis of SSc and other autoimmune or pulmonary indications. An elevated pretreatment PC signature was associated with increased benefit from MEDI-551 in SSc.

Purpose: To identify a predictive biomarker for durvalumab, an anti–programmed death ligand 1 (PD-L1) mAb. Experimental Design: RNA sequencing of 97 advanced-stage non–small cell lung carcinoma (NSCLC) biopsies from a nonrandomized phase Ib/II clinical trial (1108/NCT01693562) were profiled to identify a predictive signature; 62 locally advanced or metastatic urothelial cancer tumors from the same study were profiled to confirm predictive utility of the signature. Thirty NSCLC patients provided pre- and posttreatment tumors for messenger RNA (mRNA) analysis. NSCLC with ≥25% tumor cells and urothelial cancer with ≥25% tumor or immune cells stained for PD-L1 at any intensity were scored PD-L1 positive (PD-L1+). Kaplan–Meier and Cox proportional hazards analyses were used to adjust for gender, age, prior therapies, histology, ECOG status, liver metastasis, and smoking. Tumor mutation burden (TMB) was calculated using data from The Cancer Genome Atlas (TCGA). Results: In the NSCLC discovery set, a four-gene IFNγ-positive (IFNγ+) signature comprising IFNγ, CD274, LAG3, and CXCL9 was associated with higher overall response rates, longer median progression-free survival, and overall survival compared with signature-low patients. IFNγ+-signature NSCLC patients had improved survival regardless of IHC PD-L1 status. These associations were replicated in a urothelial cancer cohort. The IFNγ+ signature was induced 2-fold (P = 0.003) by durvalumab after 8 weeks of therapy in patients with NSCLC, and baseline signature was associated with TMB but not survival in TCGA data. Conclusions: The IFNγ+ mRNA signature may assist in identifying patients with improved outcomes with durvalumab, independent of PD-L1 assessed by IHC. Clin Cancer Res; 24(16); 3857–66. ©2018 AACR.

Background Type I interferon (IFN) has been implicated in systemic lupus erythematosus (SLE) pathogenesis, and the majority of patients with SLE have elevated expression of type I IFN-inducible genes in their blood. Anifrolumab, a fully human, IgG1κ monoclonal antibody against the type I IFN receptor, is in Phase III development for the treatment of moderate to severe SLE (NCT02446912 and NCT02446899). Objectives We sought to understand other molecular pathways (either dependent on or independent of type I IFN signaling), to elucidate heterogeneous mechanisms in SLE, and to identify patient subsets for personalized disease management. Methods Baseline blood samples from adult patients with moderate to severe SLE from two Phase IIb clinical studies (NCT01438489, N=265; NCT01283139, N=416) were profiled with whole genome array analyses. Type I IFN gene signature (IFNGS) test status was determined by a central laboratory utilizing an analytically validated four gene (IFI27, IFI44, IFI44L, RSAD2) quantitative polymerase chain reaction-based test from patients9 whole blood. A predetermined, delta Ct-based cut-off point, in the trough of the bimodal distribution, was utilized to segregate type I IFNGS test–high from –low patients at baseline. Blood from healthy controls was stimulated ex vivo with IFN-β, IFN-γ, IFN-λ, IFN-ω, or a pool of all IFN-α subtypes, with or without blocking antibodies for each IFN type, to develop IFN-type-specific signatures. Cell type- and cytokine pathway-specific gene signatures derived from the literature were also evaluated with the Phase IIb sample data. A Fisher9s exact test was used for enrichment calculations (signatures cut at median), and comparisons were adjusted for multiplicity through false discovery rate. Results A total of 79% of SLE patients in the combined study population had a type I IFNGS test–high status. From the type I IFNGS test–high patients, 29/95 signatures evaluated had significant enrichment, including those for B cells (q=1.17E-17, odds ratio [OR]=6.4), plasma cells (q=6.96E-11, OR=3.9), and CD40L signaling (q=1.07E-08, OR=3.3), relative to type I IFNGS test–low patients. In contrast, type I IFNGS test–low patients had enrichment for eosinophils (q=5.4E-6, OR=0.39) and type II IFN (IFN-γ) specifically inducible gene signatures (q=4.6E-3, OR=0.47). These findings were significant for the combined study population, as well as for the NCT01438489 study population, and were either significant or trending for the NCT01283139 population (q Conclusions SLE patients who are type I IFNGS test–high had elevated concentrations of B cells, plasma cells, and other inflammatory cytokine pathways. Type I IFNGS test–low patients, by contrast, were enriched for eosinophil and type II IFN pathways. These observations provide new insights into the molecular heterogeneity underlying SLE and suggest new therapeutic approaches, particularly for type I IFNGS test–low patients. Acknowledgements Funded by MedImmune. Medical writing support was provided by R. Plant, QXV Comms, an Ashfield business, UK. Disclosure of Interest H. Liu Employee of: MedImmune LLC, B. Higgs Shareholder of: AstraZeneca, Employee of: MedImmune LLC, W. Rees Employee of: MedImmune LLC, C. Morehouse Shareholder of: AstraZeneca, Employee of: MedImmune LLC, K. Streicher Employee of: MedImmune LLC, P. Brohawn Shareholder of: AstraZeneca, Employee of: MedImmune, G. Illei Shareholder of: AstraZeneca, Employee of: MedImmune LLC, K. Ranade Employee of: MedImmune LLC

MEDI-573 is a human antibody that neutralizes insulin-like growth factor (IGF) I and IGFII. IGFs are overexpressed in multiple types of cancer; their overexpression is a potential mechanism for resistance to IGFI receptor (IGFIR)-targeting therapy. Effects of IGF on cell proliferation, differentiation, and survival are mediated through its binding to and activation of IGFIR or insulin receptor A (IR-A). In this study, we measured the mRNA levels of IGFI, IGFII, and IGFIR in human pediatric sarcoma xenografts, and protein levels in sarcoma cell lines. MEDI-573 potently inhibited in vitro proliferation of sarcoma cell lines, with Ewing sarcoma cell lines being the most sensitive. In addition, MEDI-573 inhibited IGFI- and IGFII-induced sarcoma cell proliferation in vitro. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGFIR, pIR-A, and pAKT and significantly blocked IGFI- and IGFII-induced activation of the IGFIR and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo and inhibition correlated with neutralization of IGFI and IGFII. Combination of MEDI-573 with either rapamycin or AZD2014, another mTOR inhibitor (mTORi), significantly enhanced the antitumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by IGFI and IGFII, and inhibition of IGFI and IGFII by MEDI-573 results in significant slowing of tumor growth rate in sarcoma models, particularly in Ewing sarcoma. These data provide evidence for the potential benefits of MEDI-573 and mTORi combinations in patients with Ewing sarcoma. Mol Cancer Ther; 13(11); 2662–73. ©2014 AACR.

Patients who respond to PD-L1 / PD-1 immune checkpoint blockade tend to have an inflamed, TH1 polarised tumor microenvironment (TME), characterised by expression of interferon-γ (IFNγ) and PD-L1. Novel therapies that induce TH1 transformation of the patient TME therefore have the potential to enhance anti-tumor immunity. As a central mediator of TH1 immune responses, interleukin 12 (IL-12) directly induces IFNγ release from activated NK, NKT and T cells, and is known to play a key role in driving anti-tumor responses. However systemic recombinant IL-12 was poorly tolerated in early clinical trials. We therefore designed MEDI1191 as a novel IL-12-based therapy designed for injection directly into tumors, composed of a lipid nanoparticle (LNP)-formulated mRNA encoding human IL-12. We previously reported that intratumoral (IT) mouse (m) IL-12 mRNA, the surrogate for MEDI1191, promotes cytotoxic T cell-dependent anti-tumor immunity and enhances responses to PD-L1 blockade in pre-clinical models. Here, we demonstrate that IFNγ is also required for the anti-tumor activity of mIL-12 mRNA. A single dose of mIL-12 mRNA significantly increased expression of IFNγ and TH1 genes in MC38 tumor-bearing mice. Treatment with an IFNγ neutralising antibody blocked mIL-12 mRNA anti-tumor activity in this model. In addition, we report here that MC38 tumor rejection in response mIL-12 mRNA / anti-PD-L1 combination therapy correlates with increased cytotoxic T cell infiltration into tumors, and expansion of tumor-reactive T cells in the periphery. We next investigated the pharmocodynamic activity of MEDI1191 in patient tumor-derived models. A single IT dose of MEDI1191 induced human IL-12p70 expression in mice bearing four different patient-derived xenograft tumors. Furthermore, in an ex vivo patient tumor slice culture assay, MEDI1191 induced dose-dependent IL-12 release, IFNγ expression and upregulation of TH1-signature gene expression. IL-12 protein secretion was induced in slices of all patient tumors tested. However, the magnitude of the IFNγ response to MEDI1191 varied between patient tumors. Quantification of the tumoral T cell and NK cell numbers within the patient tumor samples revealed a positive correlation between MEDI1191-induced IFNγ release and baseline tumor NK infiltrate. These preclinical data demonstrate the potential for MEDI1191 to induce IFNγ-dependent TH1 transformation of the TME, and support the development of MEDI1191 as a potential treatment for patients with solid tumors, alone and in combination with inhibitors of the PD-1/PD-L1 T cell checkpoint. Citation Format: Nadia Luheshi, Susannah Hewitt, Fabien Garcon, Shannon Burke, Amanda Watkins, Kristen Arnold, John Zielinski, Philip Martin, Michael Sulikowski, Christopher Bagnall, Jean-Martin Lapointe, Gordon Moody, Han Si, Christopher Morehouse, Robert W. Wilkinson, Ronald Herbst, Joshua Frederick. MEDI1191, a novel IL-12 mRNA therapy for intratumoral injection to promote TH1 transformation of the patient tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5017.

Objective 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. Methods We compared expression levels of inflammatory cytokines and microRNAs (miRNAs) between muscle biopsy samples from patients with inflammatory myopathies and those from donors without myositis. In vitro human and mouse model systems were then used to characterize the role of these cytokines and microRNAs on myoblast-to-myocyte differentiation. Results We observed increased expression of inflammatory cytokines, including tumor necrosis factor α (TNFα), interferon-α (IFNα), IFNβ, and interleukin-1β, in different subtypes of inflammatory myopathies. We observed decreased expression of microRNA-1 (miR-1), miR-133a, and miR-133b in all of the inflammatory myopathy subtypes we evaluated, as well as decreased expression of miR-206 in DM; these miRNAs are essential for adult skeletal muscle differentiation and maintenance. TNFα was significantly inversely correlated with decreased myogenic miRNA expression in the inflammatory myopathy subtypes. In mechanistic studies, TNFα inhibited the expression of myogenic miRNAs and suppressed the differentiation of C2C12 myoblasts to myocytes/myotubes in an NF-κB–dependent manner. This block in differentiation by TNFα was relieved by overexpression of miR-1, miR-206, or miR-133a/b. Conclusion Taken together, these results provide a new mechanistic link between the action of proinflammatory cytokines and the degenerative pathology of inflammatory myopathies, and suggest therapeutic approaches for these diseases.

Objective Production of pathogenic autoantibodies by self-reactive plasma cells (PCs) is a hallmark of autoimmune diseases. We undertook this study to investigate the prevalence of PCs and their relationship to known pathogenic pathways to increase our understanding of the role of PCs in disease progression and treatment response. Methods We developed a sensitive gene expression–based method to overcome the challenges of measuring PCs using flow cytometry. Whole-genome microarray analysis of sorted cellular fractions identified a panel of genes, IGHA1, IGJ, IGKC, IGKV4-1, and TNFRSF17, expressed predominantly in PCs. The sensitivity of the PC signature score created from the combined expression levels of these genes was assessed through ex vivo experiments with sorted cells. This PC gene expression signature was used for monitoring changes in PC levels following anti-CD19 therapy, for evaluating the relationship between PCs and other autoimmune disease–related genes, and for estimating PC levels in affected blood and tissue from patients with multiple autoimmune diseases. Results The PC signature was highly sensitive and capable of detecting a change in as few as 360 PCs. The PC signature was reduced more than 90% in scleroderma patients following anti-CD19 treatment, and this reduction was highly correlated (r = 0.80) with inhibition of collagen gene expression. Evaluation of multiple autoimmune diseases revealed that 30–35% of lupus and rheumatoid arthritis patients had increased levels of PCs. Conclusion This newly developed PC signature provides a robust and accurate method of measuring PC levels in the clinic. Our results highlight subsets of patients across multiple autoimmune diseases who may benefit from PC-depleting therapy.

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

International audience; It has recently been demonstrated that memory B cells can reenter and reengage germinal center (GC) reactions, opening the possibility that multi-hit lymphomagenesis gradually occurs throughout life during successive immunological challenges. Here, we investigated this scenario in follicular lymphoma (FL), an indolent GC-derived malignancy. We developed a mouse model that recapitulates the FL hallmark t(14;18) translocation, which results in constitutive activation of antiapoptotic protein B cell lymphoma 2 (BCL2) in a subset of B cells, and applied a combination of molecular and immunofluorescence approaches to track normal and t(14;18)(+) memory B cells in human and BCL2-overexpressing B cells in murine lymphoid tissues. BCL2-overexpressing B cells required multiple GC transits before acquiring FL-associated developmental arrest and presenting as GC B cells with constitutive activation-induced cytidine deaminase (AID) mutator activity. Moreover, multiple reentries into the GC were necessary for the progression to advanced precursor stages of FL. Together, our results demonstrate that protracted subversion of immune dynamics contributes to early dissemination and progression of t(14;18)(+) precursors and shapes the systemic presentation of FL patients.

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.

The objective of this study was to evaluate the relationship between blood mRNA, disease activity and treatment effects in a longitudinal study of patients with dermatomyositis (DM) or polymyositis (PM). In all, 24 patients with DM or PM were followed for up to 6 years (mean of 1.9 years) at 2-7 follow-up visits while receiving standard clinical care. Clinical data and blood samples collected at 80 patient visits were used for the analysis of cytokine-induced gene expression for the signaling pathways of type 1 interferon (IFN), tumor necrosis factor-α, IL-1β, granulocyte-monocyte colony-stimulating factor, IL-10 and IL-13. A type 1 IFN signature score, but not other cytokine signature scores in the blood of patients with DM or PM, correlated highly with disease activity, decreased significantly with immunomodulatory therapies and showed concordant changes with major changes in disease activity. Type 1 IFN signature score in the blood correlates with disease activity in longitudinal follow-up of individual patients with DM or PM. The type 1 IFN-inducible gene transcripts in the blood have potential utility for monitoring disease activity in patients with DM or PM.

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.

Tumors are characterized by properties of genetic instability, heterogeneity, and significant oligoclonality. Elucidating this intratumoral heterogeneity is challenging but important. In this study, we propose a framework, BubbleTree, to characterize the tumor clonality using next generation sequencing (NGS) data. BubbleTree simultaneously elucidates the complexity of a tumor biopsy, estimating cancerous cell purity, tumor ploidy, allele-specific copy number, and clonality and represents this in an intuitive graph. We further developed a three-step heuristic method to automate the interpretation of the BubbleTree graph, using a divide-and-conquer strategy. In this study, we demonstrated the performance of BubbleTree with comparisons to similar commonly used tools such as THetA2, ABSOLUTE, AbsCN-seq and ASCAT, using both simulated and patient-derived data. BubbleTree outperformed these tools, particularly in identifying tumor subclonal populations and polyploidy. We further demonstrated BubbleTree's utility in tracking clonality changes from patients’ primary to metastatic tumor and dating somatic single nucleotide and copy number variants along the tumor clonal evolution. Overall, the BubbleTree graph and corresponding model is a powerful approach to provide a comprehensive spectrum of the heterogeneous tumor karyotype in human tumors. BubbleTree is R-based and freely available to the research community (https://www.bioconductor.org/packages/release/bioc/html/BubbleTree.html).

Background Sifalimumab is a fully human, IgG 1 κ monoclonal antibody in Phase IIb clinical development for systemic lupus erythematosus (SLE). Sifalimumab binds to and neutralizes the majority of IFN-α subtypes. Objectives We assessed the pharmacokinetic and pharmacodynamic effects of sifalimumab through analysis of the blood of adult patients with moderate to severe SLE enrolled in a Phase IIb study. Methods Adult patients who satisfied ACR classification criteria for SLE were enrolled in a Phase IIb randomized controlled trial 1 and received sifalimumab 200, 600, or 1,200 mg intravenously every 4 weeks, or placebo (following starting doses at Days 1, 15, and 29). Blood specimens were collected for PK and PD assessments at several time points from pre-dosage to 516 days after initial administration. Sifalimumab drug concentrations were measured via a validated electrochemiluminescence assay, and PK parameters were determined by noncompartmental analysis. Transcript profiling was conducted through real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) on a type I IFN-inducible gene signature (IFNGS). Results Sifalimumab serum concentrations exhibited linear and dose-proportional PK over the 200 to 1,200 mg every 4 weeks dosage range. Mean maximum concentration (C max ) and trough concentrations (C trough ), respectively, ranged from 117 to 562 ug/mL and 19.9 to 150 ug/mL, respectively, at steady state. Mean clearance and half-life ranged from 0.11 to 0.14 L/day and 24 to 26 days. A total of 349 of 431 patients (81%) were positive for the IFNGS in whole blood at baseline. Expression of IFNGS in whole blood decreased following sifalimumab administration for all dosages in patients positive for the IFNGS at baseline. Neutralization scores were calculated for each available time point (Days 15 to 365), based on patients9 Day-1 baseline expression IFNGS values. Percentage neutralization scores for each dosage arm were summarized (mean ± SE) for all available time points. In the 200-mg every 4 week dosage arm, a moderate mean signature neutralization (6.1–21.8%) was observed at Day 15 and maintained through Day 365. Maximum mean neutralization (21.8%) was observed at Day 15. In the 600-mg every 4 week dosage arm, moderate mean signature neutralization (8.3–27%) was observed at Day 15 and maintained through Day 365. Maximum mean neutralization (27%) was observed at Day 15. Finally, in the 1,200 mg every 4 week dosage arm, slightly more substantial mean signature neutralization (17.7–25.3%) was observed at Day 15 and maintained through Day 365. Maximum mean neutralization (25.3%) was observed at Day 15. IFNGS induction was noted for the placebo at each time point assessed. For all patients who received sifalimumab, maximum mean signature neutralization was observed at the first time point assessed (Day 15), while near-equivalent to sub-maximal neutralization magnitudes were noted at subsequent time points. Conclusions Sifalimumab PK was linear and dose-proportional. Sifalimumab demonstrated expected mechanism of action in SLE. Target engagement of sifalimumab was confirmed with inhibition of the IFNGS. As expected, inhibition of IFN signature was less than complete over the dosage range tested. References Khamashta M, et al. Arthritis Rheumatol. 2014;66:3530–31 (Abs L4). Acknowledgements Funded by MedImmune. Disclosure of Interest C. Morehouse Shareholder of: AstraZeneca, Employee of: MedImmune, P. Brohawn Shareholder of: AstraZeneca, Employee of: MedImmune, B. Higgs Shareholder of: AstraZeneca, Employee of: MedImmune, B. Zheng Employee of: MedImmune, Y. Yao Shareholder of: AstraZeneca, Employee of: MedImmune, L. Roskos Shareholder of: AstraZeneca, Employee of: MedImmune, G. Robbie Shareholder of: AstraZeneca, Employee of: MedImmune

Intratumor heterogeneity can confound the results of mutation analyses in oncodriver genes using traditional methods thereby challenging the application of targeted cancer therapy strategies for patients. Ultradeep sequencing can detect low frequency and expanded clonal mutations in primary tumors to better inform treatment decisions. KRAS coding exons in 61 treatment-naïve colorectal cancer (CRC) tumors and KRAS, EGFR, ALK, and MET in lung tumors from three Chinese non-small cell lung cancer (NSCLC) patients were sequenced using ultradeep sequencing methods. Forty-one percent of CRC patients (25/61) harbored mutations in the KRAS active domain, eight of which (13%) were not detected by Sanger sequencing. Three (of eight) had frequencies less than 10% and one patient harbored more than one mutation. Low frequency KRAS active (G12R) and EGFR kinase domain mutations (G719A) were identified in one NSCLC patient. A second NSCLC patient showed an EML4-ALK fusion with ALK, EGFR, and MET mutations. A third NSCLC patient harbored multiple low frequency mutations in KRAS, EGFR, and MET as well as ALK gene copy number increases. Within the same patient, multiple low frequency mutations occurred within a gene. A complex pattern of intrinsic low frequency driver mutations in well-known tumor oncogenes may exist prior to treatment, resulting in resistance to targeted therapies. Current targeted cancer therapies usually lack durability and demonstrate limited overall efficacy in patients. The types of low frequency concurrent mutations in candidate oncogenes presented here suggest necessary modifications both to methods for detection of these variants and to general treatment strategies. To date, Sanger sequencing has been effectively used for detection of treatment-relevant somatic mutations. However, in a heterogeneous mixture of cancerous and normal tissue, Sanger sequencing will likely fail to detect low frequency mutations. More sensitive and cost-effective sequencing methods are required to systematically assess the mutation status within cancer pathway genes or at the whole genome level. Furthermore, because patients often develop resistance to targeted therapy over time that is due to the preexistence of low frequency mutations in oncogenes, treatment strategies based on combination therapy might prove to be the most optimal treatment approach for cancer patients. Ultradeep sequencing can characterize intratumor heterogeneity and identify such mutations to ultimately affect treatment decisions. Citation Format: Christopher Morehouse, Liyan Jiang, Jiagi Huang, Wei Zhu, Susana Korolevich, Xiaoxiao Ge, Kim Lehmann, Zheng Lui, Christine Kiefer, Meggan Czapiga, Xinying Su, Philip Brohawn, Yi Gu, Brandon Higgs, Yihong Yao. Low frequency KRAS mutations in colorectal cancer patients and the presence of multiple mutations in oncogenic drivers in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2377. doi:10.1158/1538-7445.AM2014-2377

Background IgG4-related disease (IgG4RD) is a spectrum of systemic indications characterized by fibrosclerosis in various organs, tissue infiltrating IgG4+ plasma cells, and sometimes elevation of IgG4 in the serum. Mikulicz9s disease (MD) is one of multiple IgG4RDs with single organ involvement including lachrymal, parotid, and submandibular glands. The pathogenesis of these diseases has been linked to infection and autoimmunity with a predominance of Th2- and Treg-cell cytokines driving increased levels of eosinophils, IgG4, and IgE, ultimately leading to cell infiltration and organ damage. To date, less is known about the molecular differences between IgG4RD and MD, as well as transcriptome-wide mediators of disease. Objectives We evaluated most altered molecular pathways in the blood of IgG4RD and MD patients. Methods Blood was procured from 13 MD (ages 32-65; 7 Male), 9 IgG4RD (ages 48-80; 9 Male), and 10 healthy control (ages 30-57; 7 Male) Chinese subjects and RNA was sequenced with Illumina HiSeq. Seven MD and 5 IgG4RD patients were treated with ≤20mg prednisone with one other glucocorticoid-sparing agent. In IgG4RD, 342 and 683 genes were over- and under-expressed, respectively and 35 and 33 genes were over- and under-expressed in MD (q 2). Gene signatures were identified from ex vivo stimulation experiments with whole blood. All comparisons were relative to controls unless stated. Results IgG4 was the most over-expressed mRNA in both MD and IgG4RD (fold>32; q IgG4RD =0.001, q MD =0.002), with IgE mRNA among the top 10 most over-expressed (fold>8; q IgG4RD =0.0003, q MD =0.01) and both correlated with each other (r=0.85) and IL-5R mRNA (r IgG4 =0.80; r IgE =0.64), which was suppressed in patients treated with prednisone compared to those not (p IgG4RD =0.008; p MD =0.0001). The same mRNA suppression of IgG4 and IgE was seen in MD patients on prednisone compared to those not (p IgG4 =0.002 and p IgG4RD =0.004; p IgG4RD =0.03; p MD =0.03). Conclusions Our study shows the increase of IgG4 and IgE mRNAs, the activation of Th2, Treg, and other inflammatory cytokine pathways in both diseases compared to controls. A reduced B cell signature in the blood of IgG4RD and MD patients suggests infiltration to disease sites. Prednisone treatment suppresses IgG4 and IgE mRNAs in MD, IL-5R mRNA in both diseases, and activates IL-10 pathways in both diseases compared to treatment naive patients. Further studies need to be conducted to confirm these observations and correlate them with clinical activity. Disclosure of Interest : B. Higgs Shareholder of: Astra Zeneca, Employee of: MedImmune, Y. Liu: None declared, J. Guo: None declared, C. Morehouse Shareholder of: Astra Zeneca, Employee of: MedImmune, Y. Sebastian Shareholder of: Astra Zeneca, Employee of: MedImmune, W. Zhu Shareholder of: Astra Zeneca, Employee of: MedImmune, L. Richman Shareholder of: Astra Zeneca, Employee of: MedImmune, Y. Yao Shareholder of: Astra Zeneca, Employee of: MedImmune, Z. Li: None declared DOI 10.1136/annrheumdis-2014-eular.2255

Background MEDI-546 is an investigational human IgG1κ monoclonal antibody directed against subunit 1 of the type I interferon receptor (IFNAR1). An open-label single- and multiple-dose phase 1a clinical trial has been conducted for MEDI-546 in adult systemic sclerosis (SSc) subjects (NCT00930683). Objectives To identify serum markers and pathophysiological pathways modulated by MEDI-546 in SSc subjects. Methods Affymetrix whole genome arrays were used to measure transcript expression in whole blood and skin biopsies of SSc subjects, and a 5 gene type I IFN signature was used to determine the pharmacodynamics effects of MEDI-546. Serum levels of 93 proteins, two collagen synthesis markers, along with 6 novel collagen degradation and tissue damage markers were measured in 47 SSc subjects and 30 healthy controls. These serum markers were then used to assess the down-stream biological effects of MEDI-546 by comparing pre- and post-dose samples. Results Whole blood gene expression study demonstrated down-regulation of type I IFN-inducible transcripts across various time points post-administration of MEDI-546. The most extensive down-stream effects after type I IFN neutralization were seen at day 56, and upstream regulator analysis suggested the suppression of T cell receptor, nuclear factor of activated T cells (NFATC2), and CD40L by MEDI-546 administration. The proteomics study identified 8 proteins with decreased serum levels at day 56 among which 6 are type I IFN-inducible and 4 (CXCL10, CD40L, CXCL11, IL2RA) are associated with T cell activation and movement. Reduction of CXCL10 and CD40L levels were significantly correlated with type I IFN gene signature suppression. Furthermore, our results showed that C3M, a novel serum marker of matrix metalloproteinase-degraded type III collagen, is significantly lower in SSc subjects than normal controls, while the classic type III collagen synthesis marker (PIIINP) is significantly higher in SSc subjects. The ratio of PIIINP/C3M showed trends of correlation with SSc disease index MRTSS and HAQDI. MEDI-546 administration significantly decreased serum PIIINP/C3M ratio, suggesting a suppressive effects of MEDI-546 on collagen accumulation of SSc subjects. Skin microarray data also demonstrated the suppression of multiple collagen transcripts, tissue inhibitor of metalloproteinase, and other extracellular matrix-related transcripts by MEDI-546 administration. The down-regulation of PIIINP/C3M ratio was significantly correlated with the suppression of a T cell-associated composite index defined by serum levels of CXCL10, CD40L, CXCL11, and IL2R. Two SSc subjects with the highest decrease of MRTSS score demonstrated high suppression of both T cell-associated protein index and PIIINP/C3M ratio after MEDI-546 administration. Conclusions Our study demonstrated suppressive effects of MEDI-546 on T cell activation and collagen accumulation through which tissue fibrosis may be alleviated. A T cell-associated protein index and a novel type III collagen turnover marker may serve as early response or predictive markers for type I IFN-targeted therapy in SSc subjects in future clinical trials. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2420

Background Leukocyte infiltration plays an important role in the pathogenesis and progression of myositis, and is highly associated with disease severity. Objectives We employed a genomics approach to assess the inflammatory cell infiltration in myositis. Methods Muscle biopsies from 31 myositis patients and 5 normal healthy donors were profiled by microarray in parallel with microRNA (miRNA) expression analyses. Results Several gene signatures, such as the leukocyte index, type 1 interferon (IFN), MHC-1 and immunoglobulin signatures, were developed to characterize myositis patients at the molecular level. The leukocyte index was comprised of genes predominantly associated with the immune function and displayed a strong concordance with the pathological assessment of immune cell infiltration. This leukocyte index was subsequently utilized to evaluate expression changes of transcripts due to leukocyte infiltration in myositis muscle biopsies. The ability to distinguish different sources of altered gene expression in heterogeneous tissues increased our understanding of the complex interactions crucial to the pathogenesis of myositis. Such approach can be applied to various other studies and allowed more accurate interpretation of the underlying biology with gene expression data from heterogenous tissue samples. One application of the leukocyte index comparing miRNA and mRNA expression profiles revealed a complex interaction between miR-146a expression and the regulation of the type 1 IFN pathway in dermatomyositis. Conclusions Collectively, the distinct miRNA and mRNA signatures identified in this study may contribute to the development of new therapeutic targets and provide utility as molecular biomarkers for characterizing inflammatory myopathies. Disclosure of Interest W. Zhu Shareholder of: Astra Zeneca, Employee of: MedImmune, K. Streicher Shareholder of: Astra Zeneca, Employee of: MedImmune, N. Shen Consultant for: MedImmune, B. Higgs Shareholder of: Astra Zeneca, Employee of: MedImmune, C. Morehouse Shareholder of: Astra Zeneca, Employee of: MedImmune, L. Greenlees Shareholder of: Astra Zeneca, Employee of: MedImmune, K. Ranade Shareholder of: Astra Zeneca, Employee of: MedImmune, L. Richman Shareholder of: Astra Zeneca, Employee of: MedImmune, D. Fiorentino: None Declared, B. Jallal Shareholder of: Astra Zeneca, Employee of: MedImmune, S. Greenberg Consultant for: MedImmune, Y. Yao Shareholder of: Astra Zeneca, Employee of: MedImmune

Background Plasma cells (PC), are differentiated B cells that play a major role in humoral immunity as they actively secrete antibodies which identify and neutralize foreign antigens. Normally, plasma cells develop in response to immune challenges such as infection or vaccination. In autoimmune diseases, however, PCs develop which secrete anti-self antibodies that may play a role in disease pathogenesis. Therefore, PCs are potential targets for therapeutic intervention in autoimmune disease. However, consideration must be given to the potential impact on protective immunity of PC targeted therapy. Objectives A recent report has identified long-lived PC in autoimmune patients which secrete Ig specific for self-antigen (1). These autoreactive cells represent a novel therapeutic target. The self-reactive PCs in the study were identified by commonly known cellular markers including the surface expression of CD19. CD19 is a molecule that is present on B cells from the earliest stages of development through the memory and plasma cell stage. Therefore, our goal was to characterize in detail the expression of CD19 on plasmablasts and plasma cells from human blood and tissues, and relate the phenotype of PCs from different tissues to production of antigen-specific immunoglobulin. Methods PCs were isolated from peripheral blood, tonsil, spleen, and bone marrow (BM) from human donors and characterized for surface antigen expression by flow cytometry. Samples of sorted cells were further characterized by mRNA expression profiling and by ELISPOT to determine total as well as antigen-specific production of immunoglobulin. Results Here we show that CD19 is expressed on normal PC found in human blood, tonsil, spleen, and bone marrow. However, we have identified a population of CD19- PC which exists in the spleen and bone marrow as a sub-population of PC in these tissues. We show that the bone marrow CD19- PC and CD19+ PC have nearly identical morphology, immunophenotype, mRNA expression, and function. However the CD19- PC fraction has a significantly increased frequency of cels which secrete antibodies specific for tetanus/diptheria toxin and influenza vaccine antigens when compared to CD19+ PC in human BM. Conclusions The results demonstrate that most PC from human blood and tissue are CD19+, although cell surface levels of CD19 can vary. Importantly, there exists a distinct CD19- population of PC in spleen and BM. These cells appear to be of a long-lived memory phenotype and secrete antibodies to vaccine antigens. The results have significant implications for the targeting of PC in autoimmune disease, especially with depleting antibodies against CD19, such as MEDI-551. References Mahevas M. et al (2013) B cell depletion in immune thrombocytopenia reveals splenic long-lived plasma cells. J Clin Invest 123(1):432-42. Disclosure of Interest None Declared

Background Production of pathogenic autoantibodies by self-reactive plasma cells (PC) is a hallmark of autoimmune diseases; thus, PC levels could be associated with efficacy of B-cell depleting therapies. Additionally, investigating the prevalence of PC in autoimmune disease and their relationship with known pathogenic pathways may increase our understanding of the role of PC in disease progression and treatment response. Objectives Flow cytometry methods that are commonly used to enumerate and describe PC are difficult to implement routinely in the clinic and almost impossible in large clinical trials because of the fragility of PC. This difficulty has hampered thorough assessment of the effect of therapeutics on PC. For this reason, we first developed a highly sensitive and specific gene expression signature that can be easily implemented in the clinic, and then applied this signature to assess the impact of a novel therapeutic on PC and the prevalence of PC in different autoimmune diseases. Methods Whole genome microarray analysis of sorted cellular fractions identified a panel of genes, IGHA, IGJ, IGKC, IGKV, and TNFRSF17, whose expression is highly significantly enriched in PC. We combined expression levels of these genes to create a signature score that estimates PC counts in blood and tissue. The sensitivity of this PC signature score was assessed through ex vivo experiments with sorted cells. The PC signature was used to monitor changes in PC levels following anti-CD19 therapy; evaluate the relationship between PC and other autoimmune disease-related genes; and estimate PC levels in affected blood and tissue from multiple autoimmune diseases. Results Results indicated that the PC signature was highly sensitive and capable of detecting as few as 300 PCs. In scleroderma patients enrolled in a Phase I trial with MEDI-551, an anti-CD19 antibody with enhanced effector function, the PC signature was reduced over 90% following MEDI-551 treatment and this reduction was highly correlated (r=0.72, p=0.002) between blood and skin. Comparing the distribution of the PC signature in tissue and whole blood of patients with various autoimmune diseases revealed 30-35% of systemic lupus erythematosus, rheumatoid arthritis, and scleroderma patients with increased PC levels. Conclusions Our results demonstrate the utility of this newly developed gene expression-based PC signature. In addition to providing a robust and straightforward way to accurately measure PC levels in the clinic, the signature may be especially useful to tailor the treatment of autoimmune diseases by identifying those patients who may most benefit from PC-targeted therapies. Disclosure of Interest K. Streicher Shareholder of: MedImmune; AstraZeneca, C. Morehouse Shareholder of: MedImmune; AstraZeneca, C. Groves Shareholder of: MedImmune; AstraZeneca, B. Rajan Shareholder of: MedImmune; AstraZeneca, F. Pilataxi Shareholder of: MedImmune; AstraZeneca, K. Lehmann Shareholder of: MedImmune; AstraZeneca, P. Brohawn Shareholder of: MedImmune; AstraZeneca, B. Higgs Shareholder of: MedImmune; AstraZeneca, K. McKeever Shareholder of: MedImmune; AstraZeneca, S. Greenberg: None Declared, D. Fiorentino: None Declared, L. Richman: None Declared, B. Jallal Shareholder of: MedImmune; AstraZeneca, R. Herbst Shareholder of: MedImmune; AstraZeneca, Y. Yao Shareholder of: MedImmune; AstraZeneca, K. Ranade Shareholder of: MedImmune; AstraZeneca

Background Systemic sclerosis (SSc) is characterized by excessive extracellular matrix (ECM) deposition in the skin, as well as autoantibody production, release of various cytokines, and T-cell activation. Several lines of evidence indicate a potential role for B cells in the pathogenesis of SSc through effects on autoantibody production, T-cell activation, and fibrosis, among others. Accordingly, targeting B cells could effectively reduce ECM deposition and the inflammatory background of this disease. Objectives In animal models of SSc, decreased B-cell function following CD19 inhibition led to reduced skin thickness and a reduction in autoantibody production, which is consistent with the expression of CD19 on plasma cells (PC), the major source of antibody production. Therefore, our goal was to investigate the effect of CD19 inhibition on PC levels and collagen expression in the skin of SSc patients. Methods We evaluated skin biopsies obtained from a Phase I clinical trial of MEDI-551, an anti-CD19 antibody expected to deplete PC and B cells. Skin biopsies were collected pre-treatment (baseline) and at 29 days post-treatment with either MEDI-551 or placebo. To evaluate PC, we used whole genome microarray analysis of sorted cellular fractions to identify a panel of genes expressed predominantly in PC. To account for the multiple collagen types involved in skin fibrosis, a collagen score was calculated as the median expression of three collagen genes, COL1A1, COL3A1, and COL5A1. Results In patients with SSc, the collagen score was positively correlated with the modified Rodnan skin score (mRSS) disease activity measure at baseline (r=0.67, p=0.004), highlighting the role of collagen expression in SSc disease severity. Additionally, we identified a positive correlation between the PC signature and the collagen score at baseline (r=0.60, p=0.001). Following anti-CD19 treatment, the collagen score and the PC signature were both inhibited in skin as much as 90%, with a median inhibition of 35% and 50%, respectively. No change in the PC signature or collagen score was observed following placebo treatment. Interestingly, the inhibition of the PC signature was positively correlated with inhibition of the collagen score in all patients (r=0.80, p=0.001). Conclusions The positive correlation observed between inhibition of the PC signature and inhibition of this collagen score in scleroderma skin supports a role for PC in the pathogenic mechanism of this disease. These preliminary results need to be validated in a larger cohort of patients to determine if changes in PC signature and the collagen score could predict those who respond to treatment with PC-depleting therapeutics. Disclosure of Interest K. Streicher Shareholder of: MedImmune; AstraZeneca, C. Morehouse Shareholder of: MedImmune; AstraZeneca, C. Groves Shareholder of: MedImmune; AstraZeneca, B. Rajan Shareholder of: MedImmune; AstraZeneca, F. Pilataxi Shareholder of: MedImmune; AstraZeneca, K. Lehmann Shareholder of: MedImmune; AstraZeneca, P. Brohawn Shareholder of: MedImmune; AstraZeneca, B. Higgs Shareholder of: MedImmune; AstraZeneca, K. McKeever Shareholder of: MedImmune; AstraZeneca, L. Richman: None Declared, B. Jallal Shareholder of: MedImmune; AstraZeneca, R. Herbst Shareholder of: MedImmune; AstraZeneca, Y. Yao Shareholder of: MedImmune; AstraZeneca, K. Ranade Shareholder of: MedImmune; AstraZeneca

Background Injury of muscle of inflammatory myopathies often results in collagen build-up and a variable degree of fibrosis, which may affect muscle functions. Accompanied with autoimmune responses, elevated type I interferons may be a key components causing muscle damage. Given this persistent inflammatory response in diseased sites of muscle, TGF-β has been identified as the major driver for promoting muscle fibrosis as response for repairing the affected tissues. In addition to promoting collagen synthesis in vitro, it also inhibits collagen degradation. Few efforts have been made to investigate the molecular characteristics of collagens in inflammatory myopathies to date. Objectives We constructed a molecular signature of collagens in dermatomyositis (DM) and polymyositis (PM), and tried to determine the correlations between collagen expression and disease activity, TGF-β expression-a key regulator of collagen metabolism, and type I interferon activation. Methods Gene expression profiling was used to identify differentially expressed collagen genes and to develop a collagen gene signature to reflect status of collagen deposition. Relationships between the collagen gene signature and disease activity measured by MMT8 or CLIHAQDI, TGF-β expression, and type I interferon gene signature were examined. Masson’s Trichrome staining for examining collagen deposition in muscles was also conducted to further confirm the association of collagen gene signature and collagen deposition in DM and PM. Results Elevated transcript expression of collagen subtypes were identified and a collagen gene signature score in muscle was significantly correlated with: 1) disease activity measurements of subjects with PM (MMT8: r = -0.8, p Conclusions A molecular signature for collagens was characterized in DM and PM. Correlation between this signature and disease activity suggests potential utility as a prognostic biomarker. In addition, a significant correlation between this signature and TGF-β expression suggests that inhibiting TGF-β signaling may reduce collagen deposition and fibrosis, providing a potential intervention point for inflammatory myopathies. Disclosure of Interest Z. Liu Shareholder of: Astra Zeneca, Employee of: MedImmune, C. Morehouse Shareholder of: Astra Zeneca, Employee of: MedImmune, B. Higgs Shareholder of: Astra Zeneca, Employee of: MedImmune, M. Czapiga Shareholder of: Astra Zeneca, Employee of: MedImmune, M. Rebelatto Shareholder of: Astra Zeneca, Employee of: MedImmune, P. Brohawn Shareholder of: Astra Zeneca, Employee of: MedImmune, L. Richman Shareholder of: Astra Zeneca, Employee of: MedImmune, D. Fiorentino: None Declared, B. Jallal Shareholder of: Astra Zeneca, Employee of: MedImmune, S. Greenberg Consultant for: MedImmune, Y. Yao Shareholder of: Astra Zeneca, Employee of: MedImmune