DEVELOPMENT OF AN INTERLEUKIN (IL)-6 RECEPTOR ANTAGONIST THAT INHIBITS IL-6-DEPENDENT GROWTH OF HUMAN MYELOMA CELLS

The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the path ogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitor s of IL-6 may have clinical applications. We previously succeeded in d eveloping receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)-transformed B cell lin e CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in whic h two different mutations are combined that individually disrupt the a ssociation of the IL-6/IL-6 receptor (R)alpha complex with the signalt ransducing ''beta'' chain, gp130, but leave the binding of IL-6 to IL- 6R alpha intact. The resulting mutant protein (with substitutions of r esidues Gln160 to Glu, Thr163 to Pro, and replacement of human residue s Lys42-Ala57 with the corresponding residues of mouse IL-6) was inact ive on XG-1 cells and weakly antagonized wild-type IL-6 activity on th ese cells. By introducing two additional substitutions (Phel71Leu, Ser 177Arg), the affinity of the mutant protein for IL-6R alpha was increa sed fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized th e activity of IL-6, but not that of leukemia inhibitory factor, oncost atin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonst rating its specificity for IL-6. These data demonstrate the feasibilit y of developing specific IL-6R antagonists. The availability of such a ntagonists may offer an approach to specifically inhibit IL-6 activity in vivo.