Intramolecular distance in the conjugate of urate oxidase and fatty acid governs FcRn binding and serum half-life in vivo.

Therapeutic proteins are indispensable for treatment of various human diseases. However, intrinsic short serum half-lives of proteins are still big hurdles for developing new therapeutic proteins or expanding applications of existing ones. Urate oxidase (Uox) is a therapeutic protein clinically used for treatment of hyperuricemia. Due to its short half-life, its application for gout treatment requires prolonging the half-life in vivo. Conjugation of a fatty acid (FA), a serum albumin (SA) ligand, to therapeutic proteins/peptides is an emerging strategy to prolong serum half-life presumably via neonatal Fc receptor (FcRn)-mediated recycling. FA conjugation was proven effective for peptides and small proteins (less than 28 kDa), but not for Uox (140 kDa). We hypothesized that the intramolecular distance in the conjugate of FA and Uox is a critical factor for effective FcRn-mediated recycling. In order to control the intramolecular distance in the conjugate, we varied linker lengths between Uox and palmitic acid (PA). There was a linear correlation between the linker length and serum half-life of PA-conjugated Uox (Uox-PA) conjugates. The longer linker led to about 7-fold greater extension of serum half-life of Uox in mice than the unmodified Uox. The trend in serum half-life extension matched well with that in the tertiary structure formation of FcRn/SA/Uox-PA in vitro. These results demonstrate that the intramolecular distance in the conjugate of Uox and FA governs the stable formation of FcRn/SA/FA-conjugated protein and serum half-life extension in vivo. These findings would also contribute to development of effective FAconjugated therapeutic proteins.