Interleukin (IL)-33 is a broad-acting alarmin cytokine that can drive inflammatory responses following tissue damage or infection and is a promising target for treatment of inflammatory disease. Here, we describe the identification of tozorakimab (MEDI3506), a potent, human anti-IL-33 monoclonal antibody, which can inhibit reduced IL-33 (IL-33 red ) and oxidized IL-33 (IL-33 ox ) activities through distinct serum-stimulated 2 (ST2) and receptor for advanced glycation end products - epidermal growth factor receptor (RAGE-EGFR complex) signalling pathways. We hypothesized that a therapeutic antibody would require an affinity higher than that of ST2 for IL-33, with an association rate greater than 10 7 M −1 s −1 , to effectively neutralize IL-33 following rapid release from damaged tissue. An innovative antibody generation campaign identified tozorakimab, an antibody with a femtomolar affinity for IL-33 red and a fast association rate (8.5 × 10 7 M −1 s −1 ), which was comparable to soluble ST2. Tozorakimab potently inhibited ST2-dependent inflammatory responses driven by IL-33 in primary human cells and in a murine model of lung epithelial injury. Additionally, tozorakimab prevented the oxidation of IL-33 and its activity via the RAGE/EGFR signalling pathway, thus increasing in vitro epithelial cell migration and repair. Tozorakimab is a novel therapeutic agent with a dual mechanism of action that blocks IL-33 red and IL-33 ox signalling, offering potential to reduce inflammation and epithelial dysfunction in human disease.

Broncho-alveolar lavage (BAL) is routinely used for collection of cells from the alveolar space and for sampling of epithelial lining fluid (ELF) in lungs in preclinical studies. Urea is present in all tissue at the same concentration and therefore is commonly used as a dilution marker of the BAL fluid to calculate back to compound concentration in ELF. The aim of the present study was to investigate the accuracy of ELF concentration determination and the possible extraction from surrounding tissue of compounds with diverse physicochemical properties, using different BAL volumes and repeated BAL procedure. Propranolol, diazepam, indomethacin, AZD4721, and anakinra were delivered as an intravenous (IV) bolus dose followed by an IV infusion for 4 h to reach steady state. BAL was performed with 1 mL, 4 mL, or repeatedly with 4 mL and samples from BAL, plasma and lung tissue were taken for bioanalysis. Repeated BAL procedure revealed significant extraction from tissue as the concentration in sequential BAL fluid samples decreased slowly: >60% of the concentration of propranolol in the first BAL fluid sample was still present in the fifth sample of BAL fluid. BAL procedure with 4 mL resulted in more extraction of urea as well as compounds from tissue and/or blood compared to BAL procedure with 1 mL. Small molecules (propranolol, diazepam, indomethacin, AZD4721) were extracted from tissue to a higher degree than the large protein drug anakinra. The present study clearly demonstrates that BAL procedure results in extraction of urea and compounds from surrounding tissue and the procedure is therefore not fit for the purpose of determination of compound concentrations in ELF.