In Vivo Comparison of Branched vs Linear Pegylation of a Capsule-Degrading Enzyme for Treatment of Anthrax.

Polymers of d-glutamic acid (PDGA) form capsules of virulent strains of Bacillus anthracis. PDGA is antiphagocytic and weakly immunogenic; it enables the bacteria to evade innate immune responses. Capsule depolymerase (CapD) catalyzes the covalent anchoring of PDGA to the cell wall. There has been interest in developing CapD as a therapeutic to treat antibiotic-resistant anthrax infections. We previously showed that a His-tagged and circularly permuted (CP) CapD modified with a branched 3-prong polyethylene glycol (PEG) molecule termed PEG-CapD(S334C)-CP could protect mice with 80% survival against a 5 LD(50) challenge with virulent B. anthracis. In the current study, we compare the efficacy of tag-free CapD(S334C)-CP modified by either the branched 3-prong or a linear 1-prong PEG. Pharmacokinetic studies in mice showed the branched 3-prong PEG-CapD(S334C)-CP has a slightly higher, but not statistically different, total exposure in animals than the linear 1-prong PEG variant. The T (max) of the 1-prong PEG-CapD(S334C)-CP was 8 times shorter than the 3-prong. Mice infected with spore doses of 10 and 100 LD(50), treated 24 h after infection every 8 h for 7 days, were protected by both the 3-prong (10 LD(50): p = 0.0051; 100 LD(50): p = 0.0463, log-rank analysis) and 1-prong (10 LD(50): p = 0.0009; 100 LD(50): p < 0.0001, log-rank analysis) PEG-CapD(S334C)-CP when compared with control animals. A second iteration similarly showed statistically significant protection by both variants at both challenge doses, and the combined data from both experiments showed no significant difference in efficacy between the 1- and 3-prong enzymes confirming that tag-free PEG-CapD(S334C)-CP is protective against experimental anthrax infection.