Human serum albumin (HSA) is a natural carrier protein possessing multiple ligand binding sites with a plasma half-life ~19days, facilitated by interaction with the human neonatal Fc receptor (FcRn), that promotes it as a highly attractive drug delivery technology. A lack of adequate rodent models, however, is a major challenge in the preclinical development of albumin-linked therapeutics. This work describes the first double transgenic mouse model bearing both human FcRn and HSA genes (hFcRn(+/+), hAlb(+/+)) under the control of an endogenous promoter. Human FcRn was shown by immunohistochemical and qPCR analysis to be ubiquitously expressed in the major organs. Physiological levels of HSA were detected in the blood that exhibited similar FcRn binding kinetics to recombinant or human serum-derived HSA. The circulatory half-life (t1/2) was shown to be dependent on FcRn binding affinity that increased from low affinity (t1/2 29h), to wild type (t1/2 50h), to high affinity (t1/2 80h) variants, that validates the application of the model for optimizing the pharmacokinetics of drug carriers who's circulatory half-life is dependent in some manner upon interaction with endogenous FcRn. This study presents a novel mouse model that better mimics the human physiological conditions and, thus, has potential wide applications in the development of albumin-linked drugs or conventional drugs whose action is influenced by reversible binding to endogenous HSA.

Background: FcRn controls the long serum half-life of albumin. Results: A single amino acid substitution of albumin considerably improved binding to FcRn and extended serum half-life in mice and rhesus monkeys. Conclusion: Serum half-life of albumin may be tailored by engineering the FcRn-albumin interaction. Significance: This study reports on engineered albumin that may be attractive for improving the serum half-life of biopharmaceuticals.A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.

1 Centre for Immune Regulation (CIR) and Department of Biosciences, University of Oslo, N-0316 Oslo, Norway. 2 CIR and Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo, Norway, PO Box 4950, N-0424 Oslo, Norway. 3 Department for Microbiology, Oslo University Hospital Rikshospitalet and University of Oslo, PO Box 4950, Nydalen, N-0424 Oslo, Norway. 4 Department of Medical Biochemistry, Oslo University Hospital Rikshospitalet and University of Oslo, PO Box 4950, Nydalen, N-0424 Oslo, Norway. 5 Novozymes Biopharma UK Ltd., Castle Court, 59 Castle Boulevard, NG7 1FD Nottingham, United Kingdom. 6 Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark.