Burtea Carmen, Laurent Sophie, Sanli Tuba, Fanfone Deborah, Devalckeneer Aude, Sauvage Sébastien, Beckers Marie-Claire, Rorive Sandrine, Salmon Isabelle, Vander Elst Luce, Lauwerys Bernard R, Muller Robert N
Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons, Avenue Maistriau 19, Mendeleev Building, Mons, B-7000, Belgium.
Center for Microscopy and Molecular Imaging, 8, rue Adrienne Bolland, Gosselies, 6041, Belgium.
Arthritis Res Ther. 2016 Oct 12;18(1):230. doi: 10.1186/s13075-016-1133-8.
Interleukin-7 receptor alpha (IL-7Rα) represents a biomarker with potential applications in rheumatoid arthritis (RA) diagnosis and therapy. We have therefore searched by phage display potential IL-7Rα specific peptides with the primary goal being to develop in vivo molecular imaging tools.
IL-7Rα-targeted peptides were searched within a disulfide-constrained combinatorial phage displayed library of random linear heptapeptides. The apparent dissociation constant (K) and half maximal inhibition constant (IC) were estimated for phage clones and synthesized peptides by ELISA. We used 5-Aza-2'-deoxycytidine (ADC)-stimulated Jurkat cells and human synovial tissue from patients with RA for in vitro characterization of peptides. For molecular imaging studies performed by magnetic resonance imaging (MRI), experimental arthritis was induced in DBA/1 male mice by immunization with an emulsion of complete Freund's adjuvant and type II collagen from chicken sternal cartilage.
After several steps of phage display and peptide screening, two IL-7Rα-specific heptapeptides (P258 and P725) were selected from the initial library, based on their affinity for the target (extracellular domain of IL-7Rα, which contains a fibronectin type III repeat-like sequence). P258 (a linear peptide obtained by removing the Cys-constraint) had the lowest affinity for fibronectin itself and was therefore proposed for molecular imaging. After grafting to ultra-small superparamagnetic particles of iron oxide (USPIO), P258 produced a strong negative contrast on MRI in mice with collagen-induced arthritis (CIA), even at 2 hours post injection. The co-localization of USPIO-P258 with IL-7Rα-expressing cells in the synovial tissue from CIA mice and its ability to discriminate the level of IL-7R expression and the disease severity confirmed its efficacy as an in vivo IL-7Rα imaging agent. Interestingly, the cyclic peptide (P725), which was less adequate for molecular imaging because of higher affinity for fibronectin, had a strong ability to compete with IL-7 for the IL-7Rα binding sites, making it a potential candidate for blocking applications. Accordingly, P725 prevented the signal transducer and activator of transcription 5 (STAT5) activation induced by IL-7 in ADC-stimulated Jurkat cells.
The two peptides identified in this work demonstrate that IL-7Rα targeting in RA presents potential applications for in vivo molecular imaging and putative blocking purposes.
白细胞介素-7受体α（IL-7Rα）是一种在类风湿关节炎（RA）诊断和治疗中具有潜在应用价值的生物标志物。因此，我们通过噬菌体展示技术寻找潜在的IL-7Rα特异性肽，主要目标是开发体内分子成像工具。
在一个由随机线性七肽组成的二硫键约束组合噬菌体展示文库中搜索靶向IL-7Rα的肽。通过酶联免疫吸附测定（ELISA）估算噬菌体克隆和合成肽的表观解离常数（K）和半数最大抑制浓度（IC）。我们使用5-氮杂-2'-脱氧胞苷（ADC）刺激的Jurkat细胞和RA患者的人滑膜组织对肽进行体外特性鉴定。为了通过磁共振成像（MRI）进行分子成像研究，用完全弗氏佐剂和来自鸡胸软骨的II型胶原乳剂免疫DBA/1雄性小鼠，诱导实验性关节炎。
经过噬菌体展示和肽筛选的几个步骤，基于对靶标（IL-7Rα的细胞外结构域，其包含纤连蛋白III型重复样序列）的亲和力，从初始文库中选择了两种IL-7Rα特异性七肽（P258和P725）。P258（一种通过去除半胱氨酸约束获得的线性肽）对纤连蛋白本身的亲和力最低，因此被提议用于分子成像。嫁接到超小超顺磁性氧化铁颗粒（USPIO）上后，P258在胶原诱导的关节炎（CIA）小鼠的MRI上产生了强烈的负性对比，即使在注射后2小时也是如此。CIA小鼠滑膜组织中USPIO-P258与表达IL-7Rα的细胞的共定位及其区分IL-7R表达水平和疾病严重程度的能力证实了其作为体内IL-7Rα成像剂的有效性。有趣的是，由于对纤连蛋白的亲和力较高而不太适合分子成像应用的环肽（P725）具有很强的与IL-7竞争IL-7Rα结合位点的能力，使其成为阻断应用的潜在候选物。因此，P725可阻止IL-7在ADC刺激的Jurkat细胞中诱导的信号转导和转录激活因子5（STAT5）激活。
在本研究中鉴定的这两种肽表明，在RA中靶向IL-7Rα在体内分子成像和假定的阻断目的方面具有潜在应用价值。
