Fusion of Lysostaphin to an Albumin Binding Domain Prolongs Its Half-Life and Bactericidal Activity in the Systemic Circulation

Abstract

Antibacterial lysins are promising proteins that are active against both antibiotic-susceptible and antibiotic-resistant bacterial strains. However, a major limitation of antibacterial lysins is their fast elimination from systemic circulation. PEGylation increases the plasma half-life of lysins but renders them inactive. Here we report the construction of a fusion protein of lysostaphin, a potent anti-staphylococcal lysin, and an albumin-binding domain from streptococcal protein G. The resulting fusion protein was less active than the parent enzyme lysostaphin, but it still retained significant antibacterial activity even when bound to serum albumin. The terminal half-life of the fusion protein in rats was five-fold greater than that of lysostaphin (7.4 vs. 1.5 h), and the area under the curve increased more than 115 times. Most importantly, this increase in systemic circulation time compensated for the decrease in activity. The plasma from rats that received an injection of the fusion protein retained bactericidal activity for up to 7 h, while plasma from rats that received plain lysostaphin lacked any detectable activity after 4 h. To the best of our knowledge, this is the first report of an antibacterial lysin with both improved pharmacokinetic parameters and prolonged bactericidal activity in the systemic circulation.

Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection

“…5B ). Lysostaphin was included as a reference in this experiment, and its alpha-phase half-life was found to be 1.9 h, consistent with previous findings ( 36 , 37 ). The beta-phase half-life values for all tested ABD-fused PGHs were found to be significantly higher than those of the respective parental enzymes, which ranged between 32 and 52 h ( Fig.…”

“…Half-life extension by ABD fusion has been successfully pursued for a number of protein therapeutics ( 49 ). It also constitutes a promising strategy for improvement of pharmacokinetic properties of PGHs, as demonstrated here and in previous proof-of-principle studies ( 37 , 41 ). In our current study, the long-circulating PGH ABD_M23 construct proved superior to its parental M23 enzyme in killing S. aureus in the bloodstream of bacteremic mice, despite its reduced activity in vitro.…”

“…To date, half-life extension of PGHs had been attempted via multiple approaches, including dimerization ( 38 , 45 ), PEGylation ( 36 ), and ABD fusion ( 37 , 41 ). Previous studies were able to show improvements in the PGH pharmacokinetic ( 36 – 38 , 41 , 45 ) or pharmacodynamic ( 37 ) properties but not the therapeutic implications of a prolonged half-life.…”

“…PGHs, due to their proteinaceous nature, generally feature a relatively short serum circulation half-life. For example, the half-life of lysostaphin was previously found to be 1 h in mice ( 36 ) and 1.5 h in rats ( 37 ), that of Cpl-1 was found to be 20.5 min in mice ( 38 ), and that of SAL200 was found to be 0.3 to 9.7 h in monkeys ( 39 ) and 2.4 to 22.8 min in humans ( 40 ). The longest half-life of 23 h was reported for an engineered version of LysK, CSL[Eu], in mice ( 41 ).…”

“…Proteins covalently fused to ABD can form a high-affinity complex with HSA, which is then rescued from renal filtration due to its large hydrodynamic volume and is protected from lysosomal degradation by FcRn recycling ( 49 ). For LysK ( 41 ) and lysostaphin ( 37 ), this resulted in significant extension of their half-lives ( 37 , 41 ) and in other improvements in pharmacodynamics ( 37 ). While these results are encouraging, no studies have shown that half-life extension of PGHs translates into enhanced efficacy in infection models.…”