Development of novel neutralizing single-chain fragment variable antibodies against S100A8

Abstract

Abstract S100A8 plays a critical role in the pathogenesis of several inflammatory diseases and multiple types of cancer. Therefore, targeting the S100A8 function may alleviate the pathogenic process in various diseases. Here, specific single-chain variable fragment (scFv) antibodies targeting recombinant S100A8 (rS100A8) were selected by phage display technique and characterized using cDNA sequencing, immunoassay tests, and molecular docking. The neutralizing activity of scFvs was examined by a cell viability assay in rS100A8-treated macrophages. Furthermore, the modulatory effects of scFvs on the expression of inflammatory markers and apoptosis-related genes in macrophages and human colorectal carcinoma HT-29 cells treated with rS100A8 or dextran sulfate sodium (DSS) were assessed by RT-qPCR and flow cytometry. Based on our results, four scFvs were identified to be capable of detecting rS100A8 in the immunoassay tests. Among the selected scFvs, two clones (SA8-E6 and SA8-E12), alone or in combination, exhibited the highest blocking activity on rS100A8 and potently inhibited S100A8-induced cytotoxicity in macrophages. The use of a SA8-E6 and SA8-E12 (SA8-E6-12) cocktail inhibited the upregulation of TLR4 and RAGE, as well as inflammation and apoptosis-related genes in macrophages and HT-29 cells stimulated with rS100A8. Additionally, SA8-E6-12 exerted a significant inhibitory effect on inflammation and apoptosis induced by the S100A8/A9 complex in DSS-stimulated macrophages. We also demonstrated by molecular docking that the interaction of S100A8 with SA8-E6-12 was mainly restricted to the binding domain involved in the connection of S100A8 to TLR4. Further studies are required to explore the efficacy of these anti-S100A8 scFvs as potential therapeutic agents using in vivo models of S100A8-driven inflammatory diseases.

Keywords

• S100A8
• scFv
• Phage display
• Inflammation
• Apoptosis
• Therapeutics