Abstract
Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.
Highlights
Service, Ams~erdam1066 CX, The Nether~unds,the ~ ~ n s ~Pai s~teurtdu ~ ~ ~ ~ n t118,0,B e ~ ~~u am~n,d ethe~ l e s Nnstitut f i r Biochemie der ~heinisch Wes~ful~sc~hecnh n ~ c h e n ~ A~ahehsenc, hAaueh~en~ - 5Ger~man~y, Neutralizing monoclonal antibodies specific for hu- recent review, see Ref. 14;Refs. 12 and 13). man interleukin-6 fIL-6) bind two distinct sites on the In a variety of human inflammatory, autoimmune, and neoIL6 protein
We show forthe firsttime that receptor binding and signal transduction of human IL-6 (hIL-6) can be uncoupled
This is evidenced by the following observations. 1)The Q160E,T163P
Summary
Antibodies and Cytokines erlands Foundation for FundamentRalesearch.The costs of publication of this article were defrayed in part by the payment of page charges. CESS Assay-B cell stimulatory fador-2 activity of rhIL-6 variants was measured as described [32]. Described in thetext and under “Materials and Methods.“ The specific activity in mouse B9 and CESS assays was measured in SDS extracts of E. coli carrying plasmids encodingthe rhIL-6 site I1mutant proteins as described under “Materialsand Methods.”. At by gel filtration c ~ omato ~ a p ohny a PD-10 column(Phannacia), sterile-filtered, and stored a t 4 “C.The specific radioactivity of the preparations was determined by self-displacement analysis and was corrected for maximal binding capacity as described by Calvo et al [39] This concentration, the control mAb, mAb14(111), inhibited receptor binding, So, whereas mAb16(1I) inhibited biological activity of IL-6moreefficiently than mAbBl(I), when. We had observed that binding of mAblG(I1)to rhIL-6 was abolished by substitution of Tq-158(27).,this substitution protein
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