BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

• Keywords
• Cancer cell migration, GAMG glioblastoma, HEK-Blue IL-6, IL-6, IL-6R blockers, Malignant melanoma, Migrastatics, Pancreatic carcinoma, Protein engineering,
• MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Cell Movement * drug effects   MeSH
• Cell Proliferation * drug effects   MeSH
• Receptors, Interleukin-6 * metabolism   MeSH
• Protein Binding drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IL6R protein, human MeSH Browser
• Receptors, Interleukin-6 * MeSH

Extensive experiments performed mostly in a variety of animal models convincingly demonstrated the protective effect of polyclonal or monoclonal antibodies administered by the mucosal route. Because of the independence of the mucosal and systemic compartments of the immune system, antibodies from the circulation are not effectively transported in sufficient quantities into external secretions. Nevertheless, local application of antibodies of the desired specificity to mucosal membranes of the respiratory, gastrointestinal, and female genital tracts protected experimental animals from the subsequent challenge by corresponding viral or bacterial pathogens. Thus, generation of monoclonal antibodies of desired specificity and the selection of delivery systems to extend their otherwise short survival on some mucosal surfaces are essential aims of their usability in humans for the effective prevention of mucosally acquired infectious diseases.

• Keywords
• IgA, Mucosal immunity, monoclonal antibodies, passive protection,
• MeSH
• Gastrointestinal Tract MeSH
• Immune System MeSH
• Antibodies, Monoclonal MeSH
• Mucous Membrane * MeSH
• Immunity, Mucosal * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antibodies, Monoclonal MeSH