Extending Serum Half-life in Vivo of Therapeutic Protein using Albumin-Binding Ligands

Abstract

Therapeutic proteins are indispensable for the treatment of various human diseases. However, intrinsic short serum half-lives of proteins remain significant hurdles for developing new therapeutic proteins or expanding applications of existing ones. In the case of urate oxidase (Uox), a therapeutic protein used for the treatment of hyperuricemia, due to its short half-life, its application for gout treatment requires prolongation of its half-life in vivo. First, conjugation of a fatty acid (FA), a human serum albumin (HSA) ligand, to therapeutic proteins/peptides is an emerging strategy to prolong serum protein half-life via neonatal Fc receptor (FcRn)–mediated recycling. Fatty acid conjugation is effective for peptides and small proteins (less than 28 kDa), but not for Uox (140 kDa). I hypothesized that 1) the intramolecular distance in the conjugate of FA and Uox is a critical factor for effective FcRn-mediated recycling, and 2) small proteins do not cause substantial competition for FcRn binding to albumin, resulting in an extended serum half-life. To control the intramolecular distance in the conjugate, I varied linker lengths between Uox and palmitic acid (PA). These results demonstrate that the intramolecular distance in the conjugate of Uox and FA governs the stable formation of FcRn/HSA/FA-conjugated protein and serum half-life extension in vivo. Unlike the FA-conjugated large protein, the small proteins conjugated with the same set of linkers exhibited comparable FcRn binding to albumin and the extended serum half-lives. Second, injectable hydrogels were used as carriers for the controlled release of therapeutic proteins as well as for convenient spatial release control. However, native interactions between therapeutic proteins and hydrogels are often not strong enough for extended-release of therapeutic proteins. Therefore, implementing selective and strong interactions with hydrogels and therapeutic proteins is required. Herein, I investigated whether strong and selective interactions between HSA and albumin binding peptide (ABP) could be used to achieve a substantially extended release of therapeutic protein from injectable gels. The serum activity half-life of Uox-HSA conjugate injected with the ABP-outfitted PEG-PAEU hydrogels was 96 h, which was 88 times longer than that of the native Uox without any type of hydrogel carrier. Third, in order to improve conjugation yield and biocompatibility of the injectable gel delivery system, I chose the elastin-like polypeptide (ELP) as a based material for delivery vehicle preparation. ELP is a biopolymer consisting of a repeated [V‐P‐G‐X‐G, where X can be any amino acid except proline] pentapeptide sequences inspired by native human elastin. ELP has been investigated as in situ‐forming and injectable depots for drug delivery. Herein, the objective of this study was to develop a biocompatible, injectable, and genetically encodable ELP coacervate that could provide for sustained release of a therapeutic protein. As a result, HSA-conjugated therapeutic protein carried by the albumin ligand-conjugated ELP coacervate exhibits improved sustained-release and extended half-life. The significance of this dissertation research lies in successful prolongation of the serum half-life of Uox using an appropriate linker between FA and Uox. In addition, our results supported that the protein size in the FA-protein conjugate is a crucial factor for effective binding of FcRn with serum albumin, leading to protracted serum half-life in vivo. I believe my findings can contribute to the successful design of other FA conjugated therapeutic proteins for serum half-life extension. Based on ABP-conjugated injectable hydrogels with strong HSA affinity, this approach may also be applied to other albumin-based therapeutic models for clinical applications. Finally, it was demonstrated that albumin binding ligand-conjugated ELP coacervate can served biocompatible, injectable, and genetically encodable polypeptide based carrier for drug delivery.