Small protein blockers of human IL-6 receptor alpha inhibit proliferation and migration of cancer cells.

Cell communication and signaling : CCS 2024 May 07

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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. 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. 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. 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. © 2024. The Author(s).

Citation

Yaroslava Groza, Lukáš Lacina, Milan Kuchař, Leona Rašková Kafková, Kateřina Zachová, Olga Janoušková, Radim Osička, Jiří Černý, Hana Petroková, Joanna Maria Mierzwicka, Natalya Panova, Petr Kosztyu, Kristýna Sloupenská, Jan Malý, Jozef Škarda, Milan Raška, Karel Smetana, Petr Malý. Small protein blockers of human IL-6 receptor alpha inhibit proliferation and migration of cancer cells. Cell communication and signaling : CCS. 2024 May 07;22(1):261