An open-label study to evaluate biomarkers and safety in systemic sclerosis patients treated with paquinimod.

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.

Mapping O-glycosylation Sites Using OpeRATOR and LC-MS.

O-glycosylation is a difficult posttranslational modification to analyze. O-glycans are labile and often cluster making their analysis by LC-MS very challenging. OpeRATOR is an O-glycan specific protease that cleaves the protein backbone N-terminally of glycosylated serine and threonine residues. This enables the generation of glycopeptides of suitable size for mapping O-glycosylation sites in detail by bottom-up LC-MS analysis. In this chapter we demonstrate a simple workflow for in-depth analysis of O-glycosylation sites on heavily glycosylated proteins using OpeRATOR digestion and HILIC-MS/MS analysis.

Antibody Conjugations via Glycosyl Remodeling.

The antibody Fc-glycans are interesting targets for conjugation of cytotoxic compounds due to their localization and their chemical composition. In striving to obtain site-specific conjugates, the antibody Fc-glycans have been explored in numerous ways. Here we present a two-step enzymatic methodology coupled to click-chemistry for conjugation at the core GlcNAc of the Fc-glycan resulting in ADCs that are homogenous with a DAR 2.0, retain antigen binding, and display cytotoxic anti-tumor effects both in vitro and in vivo.

Deciphering Protein O-Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment.

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.

Paquinimod reduces skin fibrosis in tight skin 1 mice, an experimental model of systemic sclerosis.

BACKGROUND: Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune dysfunction. A hallmark of SSc is the excessive accumulation of extracellular matrix in the skin and in internal organs. There is a high and unmet medical need for novel therapies in this disease. The pathogenesis of SSc is complex and still poorly understood, but the innate immune system has emerged as an important factor in the disease. SSc patients show increased numbers of macrophages/monocytes in the blood and in the skin compared to healthy individuals and these cells are important sources of profibrotic cytokines and chemokines. Paquinimod belongs to a class of orally active quinoline-3-carboxamide (quinoline) derivatives with immunomodulatory properties and has shown effects in several models of autoimmune/inflammatory disorders. Paquinimod is currently in clinical development for treatment of SSc. The immunomodulatory effects of paquinimod is by targeting the myeloid cell compartment via the S100A9 protein. OBJECTIVE: In this study we investigate whether targeting of myeloid cells by paquinimod can effect disease development in an experimental model of SSc, the tight skin 1 (Tsk-1) mouse model. METHODS: Seven weeks old female B6.Cg-Fbn1(Tsk)/J (Tsk-1) mice were treated with vehicle or paquinimod at the dose of 5 or 25mg/kg/day in the drinking water for 8 weeks. The effect of paquinimod on the level of skin fibrosis and on different subpopulations within the myeloid cell compartment in skin biopsies were evaluated by using histology, immunohistochemisty, a hydroxyproline assay and real-time PCR. Furthermore, the level of IgG in serum from treated animals was also analysed. The statistical analyses were performed using Mann-Whitney nonparametric two tailed rank test. RESULTS: The results show that treatment with paquinimod reduces skin fibrosis measured as reduction of skin thickness and decreased number of myofibroblasts and total hydroxyproline content. The effect on fibrosis was associated with a polarization of macrophages in the skin from a pro-fibrotic M2 to a M1 phenotype. Paquinimod treatment also resulted in a reduced TGFß-response in the skin and an abrogation of the increased auto-antibody production in this SSc model. CONCLUSIONS: Paquinimod reduces skin fibrosis in an experimental model of SSc, and this effect correlates with local and systemic effects on the immune system.

Naptumomab estafenatox, an engineered antibody-superantigen fusion protein with low toxicity and reduced antigenicity.

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.