RPtag as an Orally Bioavailable, Hyperstable Epitope Tag and Generalizable Protein Binding Scaffold.

Antibodies are the most prolific biologics in research and clinical environments because of their ability to bind targets with high affinity and specificity. However, antibodies also carry liabilities. A significant portion of the life-science reproducibility crisis is driven by inconsistent performance of research-grade antibodies, and clinical antibodies are often unstable and require costly cold-chain management to reach their destinations in active form. In biotechnology, antibodies are also limited by difficulty integrating them in many recombinant systems due to their size and structural complexity. A switch to small, stable, sequence-verified binding scaffolds may overcome these barriers. Here we present such a scaffold, RPtag, based on a ribose-binding protein (RBP) from extremophile Caldanaerobacter subterraneus. RPtag binds an optimized peptide with pM affinity, is stable to extreme temperature, pH, and protease treatment, readily refolds after denaturation, is effective in common laboratory applications, was rationally engineered to bind bioactive PDGF-ß, and was formulated as a gut-stable orally bioavailable preparation.

Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt's Macular Degeneration Models.

Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.