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.

Centre of Nanomaterials and Biotechnologies University of J E Purkyně in Ústí nad Labem Pasteurova 3632 15 Ústí nad Labem 400 96 Czech Republic

Department of Clinical and Molecular Pathology Faculty of Medicine and Dentistry Palacky University Olomouc Hněvotínská 3 Olomouc 779 00 Czech Republic

Department of Dermatovenerology 1st Faculty of Medicine Charles University U Nemocnice 2 Prague 2 12000 Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc and University Hospital Olomouc Hněvotínská 3 Olomouc 779 00 Czech Republic

Institute of Anatomy 1st Faculty of Medicine Charles University U Nemocnice 3 Prague 2 12800 Czech Republic

Laboratory of Ligand Engineering Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Research Center Prumyslova 595 Vestec 252 50 Czech Republic

Laboratory of Molecular Biology of Bacterial Pathogens Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 14220 Czech Republic

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Research Center Prumyslova 595 Vestec 252 50 Czech Republic

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