A Novel Albumin Conjugation Platform via Covalent Protein Ligation for Long-Acting Therapeutic Proteins

Abstract

Therapeutic proteins, pivotal for treating diverse human diseases, often face challenges such as rapid serum clearance due to renal filtration and enzymatic degradation. To address this issue and enable prolonged therapeutic efficacy, techniques to extend the serum half-life of therapeutic proteins are crucial. Human serum albumin (HSA) has been used as a half-life extender due to its long intrinsic half-life attributed to Fc receptor (FcRn)-mediated recycling. In this study, we employed the SpyTag/SpyCatcher (ST/SC) system, known for forming irreversible isopeptide bonds, to conjugate HSA and therapeutic proteins. The AlbuCatcher platform, a conjugate of HSA and SC, was proposed aiming to provide an easy method to conjugate an ST-fused therapeutic protein to HSA. To minimize activity loss, site-specific HSA conjugation to SC was achieved using the inverse electron-demand Diels-Alder (IEDDA) reaction. Using urate oxidase (Uox) as a model protein with a short half-life, ST was fused to generate Uox-ST. Then, HSA-conjugated Uox (Uox-HSA) was successfully prepared via the Uox-ST/AlbuCatcher reaction, demonstrating a substantial reaction yield (>95%). The in vitro enzyme activity of Uox-ST and Uox-HSA conjugate was comparable to that of unmodified Uox. Moreover, pharmacokinetics studies in mice revealed the serum half-life of the Uox-HSA conjugate to be ~18 h, which was 9.8 h longer than that of the native Uox. This extended half-life clearly shows the effectiveness of the AlbuCatcher platform in enhancing therapeutic protein pharmacokinetics.