Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid conjugation

Jinhwan Cho,Sung In Lim,Young S Hahn,Inchan Kwon,Byung Seop Yang

doi:10.1038/s41598-017-18029-y

Abstract

Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the conjugation yield likely due to the enhanced solubility of palmitic acid analog.

Highlights

Use of therapeutic proteins to treat debilitating human diseases is exponentially growing and the worldwide market of therapeutic proteins has reached 167 billion dollars in 20101
We investigated whether fatty acid can be conjugated to a permissive site of a therapeutic protein for the enhanced Human serum albumin (HSA) binding capacity without compromising the therapeutic activity
urate oxidase (Uox)-160AzF treated with the DBCO-Rho dye was fluorescent in in-gel analysis

Summary

Introduction

Use of therapeutic proteins to treat debilitating human diseases is exponentially growing and the worldwide market of therapeutic proteins has reached 167 billion dollars in 20101. The conjugation of fatty acid to lysine residues of peptide drugs for diabetes treatment successfully extended the serum half-life in vivo[13,14] This strategy was not always suitable for therapeutic proteins since peptide drugs have few lysine residues but therapeutic proteins usually have many lysine residues. An alternative strategy to extend serum half-life would be beneficial to clinical applications of Uox. For successful site-specific fatty acid conjugation to a therapeutic protein via site-specific incorporation of a non-natural amino acid and click chemistry (Fig. 1), several factors need to be considered. Site-specific fatty acid-conjugation strategy developed here would be applied to the broad class of therapeutic proteins to generate their variants with the HSA binding capacity (Fig. 1)

Methods

Results

Conclusion