Fusion to a highly stable consensus albumin binding domain allows for tunable pharmacokinetics

• Published in: Protein engineering, design and selection design and selection, 2015-10, Vol.28 (10), p.385-393
• Main Authors: Jacobs, Steven A., Gibbs, Alan C., Conk, Michelle, Yi, Fang, Maguire, Diane, Kane, Colleen, O'Neil, Karyn T.
• Format: Article
• Language: English
• Subjects: Albumins - metabolism; Amino Acid Sequence; Animals; Consensus Sequence; Escherichia coli - genetics; Humans; Mice; Models, Molecular; Molecular Sequence Data; Protein Engineering - methods; Protein Stability; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacokinetics
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzv040

A number of classes of proteins have been engineered for high stability using consensus sequence design methods. Here we describe the engineering of a novel albumin binding domain (ABD) three-helix bundle protein. The resulting engineered ABD molecule, called ABDCon, is expressed at high levels in the soluble fraction of Escherichia coli and is highly stable, with a melting temperature of 81.5°C. ABDCon binds human, monkey and mouse serum albumins with affinity as high as 61 pM. The solution structure of ABDCon is consistent with the three-helix bundle design and epitope mapping studies enabled a precise definition of the albumin binding interface. Fusion of a 10 kDa scaffold protein to ABDCon results in a long terminal half-life of 60 h in mice and 182 h in cynomolgus monkeys. To explore the link between albumin affinity and in vivo exposure, mutations were designed at the albumin binding interface of ABDCon yielding variants that span an 11 000-fold range in affinity. The PK properties of five such variants were determined in mice in order to demonstrate the tunable nature of serum half-life, exposure and clearance with variations in albumin binding affinity.