Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol

Abstract

Lysostaphin is a 27-kDa endopeptidase that enzymatically disrupts the cell wall of Staphylococcus aureus and is a promising candidate for treating S. aureus blood-borne infections. It would be extremely useful to define conditions that would both increase lysostaphin's in vivo half-life to allow for more effective tissue distribution and reduce its immunogenicity. Conjugation of polyethylene glycol (PEG) to lysostaphin (PEGylation) was investigated as a means to accomplish these goals. Rather than using linear forms of PEG, branched PEGs were chosen as the initial candidates because their large spatial volumes prevent entry of the polymer into the enzyme's active sites, which could potentially reduce enzymatic function. Enzymatic activity for most PEGylated lysostaphins was reduced, but these compounds were still considerably active compared to unconjugated lysostaphin, with conjugates that had lower degrees of PEG modification having greater activity than those with higher degrees. PEGylated lysostaphin injected intravenously had a serum drug half-life of up to 24 h and resulted in much higher plasma drug concentrations than an equal dose of unconjugated lysostaphin, which had a half-life of less than 1 h. Finally, reduced binding affinity was shown for PEGylated lysostaphin in an antilysostaphin capture enzyme-linked immunosorbent assay, with some PEG-lysostaphin conjugates having binding affinities that were reduced more than 10-fold compared to unconjugated lysostaphin. These findings demonstrate that PEGylation of lysostaphin, while diminishing its S. aureus killing activity, results in prolonged serum drug persistence and reduced antibody binding. These features should significantly enhance lysostaphin's therapeutic value as an intravenous "antibiotic" against S. aureus.

FIG. 1.

Conjugation of a 10-kDa mPEG2-NHS ester to lysostaphin. Unconjugated lysostaphin runs as a single band of 27 kDa. All wells were loaded with 300 ng of total protein. Lanes 1 and 4, molecular mass marker; lanes 2 and 3, lysostaphin conjugated at a 10:1 or 2.5:1 molar ratio of PEG in aqueous, borate buffer; lanes 5 to 10, lysostaphin conjugated at a 10:1 molar ratio of PEG in 50% DMSO-50% borate buffer with low (lanes 5 and 6), medium (lanes 7 and 8), and high (lanes 9 and 10) reactant concentrations; lane 11, native lysostaphin.

FIG. 2.

Lysis of HKSA5. Lysostaphin was added at t = 0 h, and bacterial lysis was monitored by measuring the decrease in absorbance with time. High degrees of lysostaphin conjugation with either the 10- or 40-kDa PEG (10K High and 40K High) led to reduced SA5 lysis, whereas enzymatic activity was preserved for low degrees of PEG conjugation (10K Low and 40K Low).

FIG. 3.

Serum pharmacokinetics of PEGylated lysostaphin in mice. PEG-lysostaphin conjugates (200 μl) injected at a dose of 0.8 or 0.2 mg show much higher serum drug concentrations and increased serum drug half-life than a 0.8-mg dose of unconjugated lysostaphin. Each data point is the average lysostaphin concentration from pooled sera of three mice.

FIG. 4.

Lysostaphin capture immunoassay with PEGylated lysostaphin. All of the PEG-lysostaphin samples have reduced antilysostaphin binding activity (from 3 to >10 times less binding). Low degrees of lysostaphin conjugation with both 10- and 40-kDa PEGs did not shield antibody as well as the higher-degree modifications.

FIG. 5.

SDS-PAGE of a 40-kDa PEG conjugate purified into predominantly mono- or di-PEGylated lysostaphin (1-mer and 2-mer). Lane 1, molecular mass marker; lanes 2 and 3, PEG-lysostaphin 1-mer loaded at 1 and 0.3 μg of total protein; lanes 4 and 5, PEG-lysostaphin 2-mer loaded at 1 and 0.3 μg of total protein; lane 6, native lysostaphin (0.3 μg).

FIG. 6.

Enzymatic killing activity in a high inoculum of live SA5 with various concentrations of PEG-lysostaphin (1-mer and 2-mer) or native lysostaphin. A log₁₀ reduction of 4.6 represents complete clearance of bacteria from the culture. Enzyme killing activity is preserved with mono-PEGylated lysostaphin, although it is slightly reduced compared to native lysostaphin. However, lysostaphin activity was virtually eliminated for the 2-mer.