Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein

IL-18 can he considered a proinflammatory cytokine mediating disease as wel l as an immunostimulatory cytokine that is important for host defense again st infection and cancer. The high-affinity, constitutively expressed, and c irculating IL-18 binding protein (IL-18BP), which competes with cell surfac e receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present stud ies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generate d by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was f ully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared wit h those of the mutants. Mutants E42A and K89A exhibited 2-fold increased ac tivity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4 -fold greater activity. Unexpectedly, IL-18BP failed to neutralize the doub le mutant E42A plus K89A compared with WT IL-18. The K89A mutant was interm ediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 an d K89 in the mature IL-18 peptide is consistent with previous modeling stud ies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.