Figure 6

Tozorakimab potently inhibits the formation of IL-33^ox. (a) The A549 epithelial cell scratch wound healing assay was normalized to the untreated control (data points are a mean of six replicates, n = 4 separate experiments). (b) Surface plasmon resonance binding profiles for tozorakimab Fab fragment binding to chip-immobilized N-terminally tagged wild-type IL-33^red or wild-type IL-33^ox. Both IL-33 constructs were based on the 112–270 residue form. Amine biotinylated ubiquitin served as a protein control. The bar indicates the timing of the 2 min injection of 10 nM monomerized Fab. (c) Impact of recombinant IL-33^ox in the presence of test antibodies on human lung epithelial cell migration. The data shown (mean ± SD of triplicate determinations) are a representative example from two independent experiments. (d) Time course of recombinant IL-33^red oxidation in the presence of antibodies. IL-33^ox levels were detected using IL-33^ox ELISA (mean ± SD of duplicate determinations). (e) Impact of recombinant IL-33^red, oxidized for 24 h in the presence of test antibodies, on human lung epithelial cell migration. The data shown (mean ± SD of triplicate determinations) are a representative example from two independent experiments. (f) A schematic of the proposed mode of action of tozorakimab. IL-33^red, that is rapidly released following epithelial damage, binds tozorakimab with a high affinity and a fast association rate. Tozorakimab directly neutralizes IL-33^red inflammatory activities at the ST2 receptor. The IL-33^red–tozorakimab complex prevents the oxidation of IL-33, IL-33^ox–RAGE/EGFR signalling and epithelial dysfunction. EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; IL-1RAP, interleukin-1 receptor accessory protein; mAb, monoclonal antibody; ox, oxidized; RAGE, receptor for advanced glycation end products; red, reduced; RU, response units; SD, standard deviation.