Related to Fig. 1. a, Structural alignment of hIL-18 (green)–hIL-18Rα/Rβ (cyan) complex (PDB ID 3WO4) with hIL-18–vIL-18BP (blue) complex (PDB ID 3F62). b, c, Representative surface plasmon resonance (SPR) sensorgrams of mouse WT IL-18 binding to IL-18Rα (b) or IL-18BP (c). IL-18Rα measurements were conducted using a conventional multiple cycle program, whereas IL-18BP measurements were conducted using a single-cycle program. d, Dose–response curves of IL-18BP protein antagonizing IL-18Rα in complex with indicated IL-18 and mutants (E42A, K89A and E42A/K89A). Experiments were performed in duplicate (n = 2). e, Randomized positions of murine IL-18 to create DR_18, with the corresponding degenerate codon and the potential amino acid at each position. f, Summary of the experimental design for directed evolution and yeast selection process to generate DR-18. Yeast libraries were selected for IL-18Rα binding and counter-selected against IL-18BP using magnetic-activated cell sorting (MACS; rounds 1 and 2) and subsequently FACS (rounds 3–5). Blue text (right) indicates positive selection reagent, and red text (left) shows the counter-selection reagent. g, Structural representation of DR-18 mutation positions in IL-18Rα and IL-1BP binding overlap region. Side chains from a minimized set of mutations up to six consensus residues (1N, 50M, 52K, 55E, 56V and 59L) are displayed as stick models. b–d, Data are representative of two independent experiments and are presented as mean ± s.e.m.