Engineering an Fc-Fusion of a Capsule Degrading Enzyme for the Treatment of Anthrax.

Polymers of d-glutamic acid (PDGA) form the capsule of the highly virulent Ames strain of . PDGA is antiphagocytic and weakly immunogenic; it enables the bacteria to evade the innate immune responses. CapD is an enzyme that catalyzes the covalent anchoring of PDGA. CapD is an Ntn-amido hydrolase that utilizes an internal Thr-352 as its nucleophile and general acid and base. An internal cleavage produces a free N-terminal Thr-352 and a short and long polypeptide chain. The chains were circularly permuted (CP) to move Thr-352 to the N-terminus of the polypeptide. We previously showed that a branched PEG-CapD-CP could protect mice (80% survival) against a 5 LD challenge with Ames without the use of antibiotics, monoclonals, or vaccines. In attempts to improve the circulation time of CapD and enhance its avidity to its polymeric substrate, an Fc-domain of a mouse IgG1 was fused to CapD-CP and the linker length and sequence were optimized. The resulting construct, Fc-CapD-CP, then was pegylated with a linear 2 kDa mPEG at S334C to produce mPEG-Fc-CapD-CP. Interestingly, the fusion of the Fc-domain and incorporation of the S334C mutation imparted acid stability, but slightly reduced the (∼ 2-fold lower). , the measured protein concentration in sera was higher for the Fc-fusion constructs compared to the mPEG-Fc-CapD-CP. However, the exposure calculated from measured sera enzymatic activity was higher for the mPEG-CapD-CP. The pegylated Fc-fusion was less active than the PEG-CapD-CP, but detectable in sera at 24 h by immunoblot. Here we describe the engineering of a soluble, active, pegylated Fc-fusion of CapD (mPEG-Fc-CapD-CP) with activity , in serum, and on encapsulated bacteria.