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 pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)-transformed B cell line 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 which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R) alpha complex with the signaltransducing "beta" chain, gp130, but leave the binding of IL-6 to IL-6R alpha intact. The resulting mutant protein (with substitutions of residues Gln160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL-6R alpha was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL-6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.
de Hon
F D
FD
Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Ehlers
M
M
Rose-John
S
S
Ebeling
S B
SB
Bos
H K
HK
Aarden
L A
LA
Brakenhoff
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002455:Cell Division; D002461:Cell Line, Transformed; D017931:DNA Primers; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D015850:Interleukin-6 / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008113:Liver Neoplasms; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000276:immunology* / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D018123:Receptors, Interleukin / Q000037:antagonists & inhibitors* / Q000502:physiology; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D015850:Interleukin-6; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins