Visram, Z.; Kestemont, D.; Bauer, B.; Qiao, R.; Durica-Mitic, S.; Majoros-Hashempour, A.; Schmidt, J.; Berdaguer, R.; Krey, K.; Mutti, M.; Zerbs, M.; von Freyberg, M.; Corsini, L.; Badarau, A.

Anti-staphylococcal lytic agents, such as lysostaphin (LSN), a glycyl-glycyl (Gly-Gly) peptidase, have long been considered for the management of chronic and complicated bacterial infections typically resistant to conventional antibiotics, but their use is restricted by poor pharmacokinetic properties. We generated half-life extended lysostaphin constructs by fusing the lysin - either alone or chimerized with an additional enzymatic cysteine, histidine-dependent amidohydrolases/peptidase (CHAP) domain - to the human IgG1 Fc fragment. The Fc-CHAP-LSN constructs retain high potency against Staphylococcus aureus and coagulase negative staphylococcal strains in vitro and are efficacious in S. aureus ex vivo biofilm models and in vivo sepsis models. A detailed investigation of the Fc-CHAP-LSN mode of action revealed that upon binding to the bacterial cell, but not in solution, LSN mediates its own release by cleaving the Gly-Gly CHAP-LSN linker. The bactericidal activity of Fc-CHAP-LSN is driven by the LSN-catalyzed and target cell-dependent release of free LSN. The half-life extended lysin acts as a pro-drug, unveiling a novel mechanism of targeted release and an alternative approach to half-life extension.