Abstract
BackgroundInterleukin-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.MethodsIL-7Rα-targeted peptides were searched within a disulfide-constrained combinatorial phage displayed library of random linear heptapeptides. The apparent dissociation constant (Kd) and half maximal inhibition constant (IC50) 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.ResultsAfter 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1133-8) contains supplementary material, which is available to authorized users.
Highlights
Interleukin-7 receptor alpha (IL-7Rα) represents a biomarker with potential applications in rheumatoid arthritis (RA) diagnosis and therapy
Identification and characterization of the candidate phage clones A disulfide constrained heptapeptide phage display library was screened against the extracellular domain of interleukin-7 receptor alpha (IL-7Rα) (E21–D239), which contains an FN type III repeat-like sequence (A131–I231)
The results show that the second and third rounds of panning present an optimal binding to IL-7Rα, whereas the fourth round had lost its affinity
Summary
Interleukin-7 receptor alpha (IL-7Rα) represents a biomarker with potential applications in rheumatoid arthritis (RA) diagnosis and therapy. Interleukin-7 (IL-7) is an anti-apoptotic cytokine, essential for T cell proliferation, development and homeostasis. It is involved in B cell development. IL-7Rα is composed of a 195 amino acid intracellular domain, a 25 amino acid transmembrane domain and an extracellular region comprising 219 amino acids. A Trp-Ser-X-Trp-Ser (WSXWS) motif is located close to the transmembrane domain of the extracellular region of IL-7Rα, which contains a fibronectin (FN) type III-like domain. IL-7 binding to its receptor triggers several signaling cascades, i.e., Jak/STAT, PI3K, Ras and mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK), being essential for lymphocyte survival, homeostasis and differentiation [2,3,4]
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