Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. N-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on O-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of O-glycans. Although O-glycosidase can catalyze the removal of core 1 and core 3 O-linked disaccharides from glycoproteins, analysis of other types of O-glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available O-protease and solid phase platform. This method enables the assignment of O-glycosylated peptides, N-glycan profile, sialyl O-glycopeptides linkage, and mapping of heterogeneous O-glycosylation. For the first time, we can analyze intact O-glycopeptides generated by O-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known O-glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant O-glycosites and setting a basis set of O-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein O-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where O-glycosylation is central.

Antibody-targeted superantigens have a potential to become useful drugs for tumor therapy. However, clinical practice has identified several issues that need to be addressed to optimize such molecules. On the basis of the experience from superantigen products in clinical trials, a novel tumor-targeted superantigen, naptumomab estafenatox (5T4FabV18-SEA/E-120 or ABR-217620) has been designed. Critical properties, such as tumor reactivity, therapeutic window, and seroreactivity were all improved. The engineered 5T4Fab moiety recognizes the 5T4 antigen expressed on a large number of solid tumor forms with an affinity in the order of 1 nM. The fusion protein induces T-cell mediated killing of tumor cells at concentrations around 10 pM. Compared with a construct with a wild-type superantigen, it is more potent in mediating killing of tumor cells but a 10,000-fold less active in mediating killing of MHC class II positive cells. The target epitopes for naturally occurring antibodies toward bacterial superantigens are reduced. Only large excesses of human anti-SEA antibodies neutralize the antitumor effects of the antibody-targeted superantigen. Naptumomab estafenatox induces dramatic reduction of established human tumors in Severe Combined Immunodeficient mice grafted with human lymphocytes. Thus, naptumomab estafenatox is a novel optimized tumor-targeted superantigen currently investigated in clinical trials.

Paquinimod reduces skin fibrosis in an experimental model of SSc, and this effect correlates with local and systemic effects on the immune system.

Objectives To evaluate the changes in disease-related biomarkers and safety of paquinimod, an oral immunomodulatory compound, in patients with systemic sclerosis (SSc). Methods In this open-label, single-arm, multicenter study, SSc patients with a rapidly progressive disease received paquinimod for 8 weeks. Blood and skin biopsies were collected at baseline, during treatment, and at follow-up for the analyses of type I interferon (IFN) activity, chemokine (C-C motif) ligand 2 (CCL2), and the number of myofibroblasts. The safety of paquinimod was evaluated throughout the study. Results Nine SSc patients were enrolled and completed the study treatment with paquinimod at 3 mg/day for 8 weeks. After the treatment, a reduction of type I IFN activity in the plasma from one patient with elevated baseline IFN activity was recorded. A trend towards reduced IFN activity in the skin after treatment was also observed in patients. The serum level of CCL2 was reduced in 7 of 9 patients after paquinimod treatment. There was a median reduction of 10% of the number of myofibroblasts in skin biopsies at week 8 compared to baseline. No change in modified Rodnan skin score and quality of life was detected in the study. Reported adverse events (AEs) were mild to moderate and expected with the most common being arthralgia (n = 3) and headache (n = 3), and C-reactive protein (CRP) increase. Conclusions Analysis of biomarkers before and after treatment suggest reduced type I IFN activity and reduced number of myofibroblasts in lesional skin. Paquinimod was overall well tolerated with mild to moderate and expected AEs. Trial registration ClinicalTrials.gov, NCT01487551. Registered on 7 September 2011

Background Paquinimod (ABR-215757) is an oral small molecule compound that belongs to the quinoline-3-carboxamide derivatives, a class of structurally related compounds with immunomodulatory properties. Paquinimod has shown beneficial effects in several autoimmune/inflammatory disease models, including experimental models of systemic sclerosis (SSc). Paquinimod binds the S100A9 protein and disrupts its binding to the pro-inflammatory receptors RAGE and TLR4 (1). Mechanistic studies have shown that paquinimod interferes with accumulation of myeloid cells during inflammation (2, 3). Previous clinical experience consists of two phase I dose escalation safety studies and one exploratory study in SLE patients (4). Objectives The aims of the present study in SSc patients were to evaluate safety and changes in disease related biomarkers. Methods Nine patients with a disease duration of 2.0±2.0 (mean ± SD) years and diffuse SSc were treated with paquinimod at 3.0 mg/day for eight weeks in an international, open label, multi-centre study (ClinicalTrials.gov Identifier: NCT01487551). The primary endpoint was changes in disease related biomarkers. Skin biopsies were assessed histologically for α-SMA positive myofibroblast counts. Expression of a panel of extracellular matrix genes and pro-fibrotic genes were analysed by real-time PCR. Secondary end-points included adverse events (AE), extent of skin fibrosis (mRSS) and Quality of Life (QoL) assessments. Results Paquinimod was well tolerated and all patients completed the eight week treatment period. Except for one SAE (peripheral ischemia, considered severe and unrelated to study medication) only mild or moderate AEs, most commonly arthralgia (n=3) and headache (n=3), were reported. Baseline mRSS was 28±10 (mean ± SD) points and as expected, no change in mRSS and QoL measures were observed in this short-term clinical trial. A small but significant reduction of the number of myofibroblasts in the skin was observed after paquinimod treatment compared to baseline (8%, p=0.023). Down-regulation of a number of pro-fibrotic genes such as CCR2, PAI-1, TIMP3, CAV1, CTGF and FN1 was evident after eight weeks of treatment. While no consistent effects on extracellular matrix genes were observed, the expression of several type I IFN-regulated genes also declined in the majority of the patients. The effects on biomarkers are in line with preclinical data in Tsk-1 mice and support that paquinimod could be effective by targeting the innate immune system in SSc. Conclusions Paquinimod was well tolerated and effects on biomarkers relevant for SSc were observed during treatment. References Björk et al., PLoS Biol. 2009;7(4):e97. Deronic et al., International Immunopharmacology 2014; 18:290-297. Helmersson et al., Am J Pathol. 2013;182(5):1671-80. Bengtsson et al., Arthritis Rheum 2012; 64:1579-88. Disclosure of Interest R. Hesselstrand Consultant for: Active Biotech., J. Distler: None declared, G. Riemekasten: None declared, M. Törngren Shareholder of: Active Biotech.,...

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Background Paquinimod (ABR-215757), a new oral small molecular drug, belongs to the quinoline-3-carboxamide derivatives, a class of structurally related compounds (1). It targets the S100A9 protein and disrupts its binding to the pro-inflammatory receptors; Receptor for Advanced Glycation Endproducts (RAGE) and Toll-like Receptor 4, (TLR4) (2). Both RAGE and TLR4 are involved in the pathogenesis of many autoimmune and inflammatory diseases and paquinimod has shown convincing beneficial effects in several autoimmune/inflammatory disease models. Preclinical studies have shown that paquinimod affects the infiltration of myeloid cells into sites of inflammation (3). Paquinimod is in development for treatment of Systemic Sclerosis (SSc). SSc is a rare chronic autoimmune connective tissue disease characterized by excessive extracellular matrix deposition in the skin and visceral organs. Effects on several biomarkers relevant for SSc have been observed in an exploratory clinical trial of paquinimod (ClinicalTrials.gov Identifier: NCT01487551) in patients with SSc. Objectives The purpose of this study was to evaluate the effects of paquinimod on skin fibrosis in the tight skin 1 (Tsk-1) mouse model, a common model for SSc. Methods Tsk-1 mice were treated for 8 weeks, starting at the age of 7 weeks, with paquinimod ad lib. at 5 and 25 mg/kg/day or with vehicle only. The hypodermal thickness was analyzed in skin sections stained with haematoxylin and eosin. The collagen content in skin was also determined by hydroxyproline assay. Furthermore, the number of α-smooth muscle actin (αSMA) positive myofibroblasts, in the skin sections were determined by immunohistochemical staining. Total IgG levels in serum were measured by ELISA and gene expression in skin biopsies was analyzed by real-time PCR. Results Treatment of Tsk-1 mice with paquinimod resulted in a significant (p<0.05) reduction of 24±6% (mean ± SEM) of the hypodermal thickness, a 22±6% (mean ± SEM) reduction of the hydroxyproline content, a 19±4% (mean ± SEM) decrease in the accumulation of myofibroblasts, and a 59±6% (mean ± SEM) reduction in total levels of serum IgG compared to vehicle treated mice. Paquinimod treated mice also had decreased gene expression of profibrotic biomarkers, such as Col1a2, Ccr2, Ctgf, Il-13 and Fn1 in the skin compared to vehicle treated mice. Conclusions Our results show that paquinimod reduces development of skin fibrosis in the Tsk-1 model measured as hypodermal thickness, hydroxyproline content and number of myofibroblasts in the skin. Paquinimod treatment also resulted in a reduction in the expression of profibrotic genes in the skin. References Jönsson et al. J Med Chem. 2004; 47:2075-88. Björk et al. PLoS Biol. 2009 Apr 28; 7(4):e97. Deronic et al. Int Immunopharmacol. 2014; 18:290-297. Disclosure of Interest M. Stenström Shareholder of: Active Biotech., Employee of: Active Biotech., H. Carlsson Nyhlén Shareholder of: Active Biotech., Employee of: Active Biotech., M. Nilsson Shareholder of: Active Biotech., Em...

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