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- W2225003672 abstract "<h3>Background</h3> Ras proteins are present in rheumatoid arthritis (RA) synovial tissues, and are preferentially expressed in the intimal lining layer. Activation of Ras effecter pathways in synovial tissues, including MAP kinases, PI3K, and nuclear factor-κB, is enhanced in RA. We developed affibody molecules with the ability to selectively inhibit the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage. <h3>Objectives</h3> The present study investigated the effects of affibody molecules on synovial cell functions using human synovial fibroblast cell line. <h3>Methods</h3> Affibodys (Mw 6 kDa) were derived from one of the domains of the cell wall protein Protein A of <i>Staphylococcus aureus</i>. Using phage display library, three affibody clones which can binds to H-Ras [His6-Z<sub>(Ras521)2</sub>, His6-Z<sub>(Ras122)2</sub>, His6-Z<sub>Raf322</sub>] were screened from combinatorial library. To evaluate the specificity and affinity of the selected affibody molecules towards their respective targets, we performed real-time biospecific interaction experiments. To examine the effects of affibodies on synovial cell functions, we used a human synovial fibroblast cell line (MH7A). The proliferation was examined by a modified MTT assay. IL-6 and PGE2 productions by MH7A cells were determined by ELISA. <h3>Results</h3> Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1. They were shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner by real-time biospecific interaction experiments. Transformation of plasmids encoding His6-Z<sub>(Ras521)2</sub>, into MH7A cells induced significant amounts of His6-Z<sub>(Ras521)2</sub> proteins, as shown by Western blot analysis, and significantly inhibited the proloferation and suppressed the productions of IL-6 and PGE2 stimulated by TNF-alpha. <h3>Conclusions</h3> We have successfully selected affibody molecules specifically binding to H-Ras with high affinity. We also demonstrated that signaling through the H-Ras–Raf pathway by MH7A cells can be blocked, thereby inhibiting the productions of IL-6 and PGE2 stimulated by TNF-alpha. Molecular targeting of H-Ras-Raf-1 signaling by affibody molecules can be useful as a regulator of synovial cell functions in patients with RA. <h3>Disclosure of Interest</h3> None declared <h3>DOI</h3> 10.1136/annrheumdis-2014-eular.2553" @default.
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- W2225003672 date "2014-06-01" @default.
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- W2225003672 title "AB0134 Affibody Molecules Inhibiting the Interaction between RAS and RAF Suppress the Proliferation and the Production of Inflammatory Mediators by Synovial Cells" @default.
- W2225003672 doi "https://doi.org/10.1136/annrheumdis-2014-eular.2553" @default.
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