Neutralizing monoclonal antibodies specific for human interleukin-6 (I
L-6) bind two distinct sites on the IL-6 protein (sites I and II). The
ir 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 is
olation 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 I
I-specific monoclonal antibody. One such site II mutant protein (with
double substitution of Gln-160 with Glu and Thr-163 with Pro) was foun
d to be an antagonist of human IL-6. It was inactive on human CESS cel
ls, weakly active on human HepG2 cells, but active on mouse B9 cells.
It could specifically antagonize the activity of wild-type IL-6 on CES
S 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 bi
nding to CESS cells, however, was abolished, suggesting that the mutan
t protein is inactive because the complex of the 80-kDa IL-6 receptor
and the mutant protein cannot associate with the signal transducer gp1
30. The human IL-6 antagonist protein may be potentially useful as a t
herapeutic agent.