Biophysical Methods Applied in Early Discovery of a Biotherapeutic: Case Study of an Egfr‐Igf1R Bispecific Adnectin

Doyle, Michael L.; Bryson, James W.; Lafont, Virginie; Lin, Zheng; Morin, Paul E.; Schneeweis, Lumelle A.; Yamniuk, Aaron P.; Yanchunas, Joseph

doi:10.1002/9781118354698.ch1

Cited by 1 publication

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“…The free enzyme CapD S334C -CP is highly soluble even in dilute buffers such as 0.5× PBS pH 7. 54 For enzymes that undergo endosomal trafficking, the low pH analysis may be useful.…”

Section: ■ Discussionmentioning

confidence: 99%

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

Matharoo

Chua

Park³

et al. 2022

ACS Infect. Dis.

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Polymers of d-glutamic acid (PDGA) form the capsule of the highly virulent Ames strain of B. anthracis. 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-CapDS334C-CP could protect mice (80% survival) against a 5 LD50 challenge with B. anthracis Ames without the use of antibiotics, monoclonals, or vaccines. In attempts to improve the in vivo circulation time of CapD and enhance its avidity to its polymeric substrate, an Fc-domain of a mouse IgG1 was fused to CapDS334C-CP and the linker length and sequence were optimized. The resulting construct, Fc-CapDS334C-CP, then was pegylated with a linear 2 kDa mPEG at S334C to produce mPEG-Fc-CapDS334C-CP. Interestingly, the fusion of the Fc-domain and incorporation of the S334C mutation imparted acid stability, but slightly reduced the k cat (∼ 2-fold lower). In vivo, the measured protein concentration in sera was higher for the Fc-fusion constructs compared to the mPEG-Fc-CapDS334C-CP. However, the exposure calculated from measured sera enzymatic activity was higher for the mPEG-CapDS334C-CP. The pegylated Fc-fusion was less active than the PEG-CapDS334C-CP, but detectable in sera at 24 h by immunoblot. Here we describe the engineering of a soluble, active, pegylated Fc-fusion of B. anthracis CapD (mPEG-Fc-CapD-CP) with activity in vitro, in serum, and on encapsulated bacteria.

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“…The free enzyme CapD S334C -CP is highly soluble even in dilute buffers such as 0.5× PBS pH 7. 54 For enzymes that undergo endosomal trafficking, the low pH analysis may be useful.…”

Section: ■ Discussionmentioning

confidence: 99%