Human serum albumin is an endogenous ligand transport protein whose long circulatory half-life is facilitated by engagement with the human cellular recycling neonatal Fc receptor (hFcRn). The single free thiol located at Cys-34 in domain I of albumin has been exploited for monoconjugation of drugs. In this work, we increased the drug-to-albumin ratio potential by engineering recombinant human albumin (rHSA) variants with varying hFcRn affinity to contain three free, conjugation-competent cysteines. Structural analysis was used to identify positions for cysteine introduction to maximize rHSA stability and formation of the conjugated product without affecting hFcRn binding. The thiol rHSA variants exhibited up to 95% monomeric stability over 24 months and retained hFcRn engagement compared with a WT unconjugated control demonstrated by Biolayer Interferometry. The additional cysteines were further introduced into a panel of rHSA variants engineered with different affinities for hFcRn. After conjugation with three Alexa Fluor 680 (AF680) fluorophores, hFcRn binding was similar to that of the original triple-thiol nonconjugated rHSA variants (0.88 and 0.25 m for WT albumin with or without 3xAF680 respectively, and 0.04 and 0.02 m for a high hFcRn-binding variant with or without 3xAF680, respectively). We also observed a 1.3-fold increase in the blood circulatory half-life of a high hFcRn-binding triple-thiol variant conjugated with AF680 (t(1/2) = 22.4 h) compared with its WT counterpart (t(1/2) = 17.3 h) in mice. Potential high drug-to-albumin ratios combined with high hFcRn engagement are attractive features of this new class of albumins that offer a paradigm shift for albumin-based drug delivery.

Baker’s yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins. For the manufacture of heterologous proteins with activities deleterious to the host it can be desirable to minimise production during the growth phase and induce production late in the exponential phase. Protein expression by regulated promoter systems offers the possibility of improving productivity in this way by separating the recombinant protein production phase from the yeast growth phase. Commonly used inducible promoters do not always offer convenient solutions for industrial scale biopharmaceutical production with engineered yeast systems.

The long blood circulation time of albumin has been clinically utilized as a half-life extension technology for improved drug performance. The availability of one free thiol for site-selective chemical conjugation offers an alternative approach to current genetic fusion and association-based products. This special report highlights important factors for successful conjugation that allows the reader to design and evaluate next-generation albumin conjugates. Albumin type, available conjugation chemistries, linker length, animal models and influence of conjugation on albumin pharmacokinetics and drug activity are discussed.

Background: FcRn regulates the long serum half-life of albumin. Results: The C-terminal DIII of HSA is the principal domain for FcRn binding, whereas two loops in DI at the N terminus modulate the interaction. Conclusion: DI of albumin contributes to optimal FcRn binding. Significance: We highlight the importance of DI for pH-dependent binding to FcRn.Albumin is an abundant blood protein that acts as a transporter of a plethora of small molecules like fatty acids, hormones, toxins, and drugs. In addition, it has an unusual long serum half-life in humans of nearly 3 weeks, which is attributed to its interaction with the neonatal Fc receptor (FcRn). FcRn protects albumin from intracellular degradation via a pH-dependent cellular recycling mechanism. To understand how FcRn impacts the role of albumin as a distributor, it is of importance to unravel the structural mechanism that determines pH-dependent binding. Here, we show that although the C-terminal domain III (DIII) of human serum albumin (HSA) contains the principal binding site, the N-terminal domain I (DI) is important for optimal FcRn binding. Specifically, structural inspection of human FcRn (hFcRn) in complex with HSA revealed that two exposed loops of DI were in proximity with the receptor. To investigate to what extent these contacts affected hFcRn binding, we targeted selected amino acid residues of the loops by mutagenesis. Screening by in vitro interaction assays revealed that several of the engineered HSA variants showed decreased binding to hFcRn, which was also the case for two missense variants with mutations within these loops. In addition, four of the variants showed improved binding. Our findings demonstrate that both DI and DIII are required for optimal binding to FcRn, which has implications for our understanding of the FcRn-albumin relationship and how albumin acts as a distributor. Such knowledge may inspire development of novel HSA-based diagnostics and therapeutics.

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.