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.

Glucagon-like peptide 1 (GLP-1) is a small incretin hormone stimulated by food intake, resulting in an amplification of the insulin response. Though GLP-1 is interesting as a drug candidate for the treatment of type 2 diabetes mellitus, its short plasma half-life of <3 min limits its clinical use. A strategy for extending the half-life of GLP-1 utilizes the long half-life of human serum albumin (HSA) by combining the two via chemical conjugation or genetic fusion. HSA has a plasma half-life of around 21 days because of its interaction with the neonatal Fc receptor (FcRn) expressed in endothelial cells of blood vessels, which rescues circulating HSA from lysosomal degradation. We have conjugated GLP-1 to C34 of native sequence recombinant HSA (rHSA) and two rHSA variants, one with increased and one with decreased binding affinity for human FcRn. We have investigated the impact of conjugation on FcRn binding affinities, GLP-1 potency, and pharmacokinetics, combined with the solution structure of the rHSA variants and GLP-1-albumin conjugates. The solution structures, determined by small-angle X-ray scattering, show the GLP-1 pointing away from the surface of rHSA. Combining the solution structures with the available structural information about the FcRn and GLP-1 receptor obtained from X-ray crystallography, we can explain the observed in vitro and in vivo behavior. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease in the potency of GLP-1 can be explained by a steric hindrance of binding of GLP-1 to its receptor.

Albumin is the most abundant plasma protein involved in the transport of many compounds, such as fatty acids, bilirubin, and heme. The endothelial cellular neonatal Fc receptor (FcRn) has been suggested to play a central role in maintaining high albumin plasma levels through a cellular recycling pathway. However, direct mapping of this process is still lacking. This work presents the use of wild-type and engineered recombinant albumins with either decreased or increased FcRn affinity in combination with a low or high FcRn-expressing endothelium cell line to clearly define the FcRn involvement, intracellular pathway, and kinetics of albumin trafficking by flow cytometry, quantitative confocal microscopy, and an albumin-recycling assay. We found that cellular albumin internalization was proportional to FcRn expression and albumin-binding affinity. Albumin accumulation in early endosomes was independent of FcRn-binding affinity, but differences in FcRn-binding affinities significantly affected the albumin distribution between late endosomes and lysosomes. Unlike albumin with low FcRn-binding affinity, albumin with high FcRn-binding affinity was directed less to the lysosomes, suggestive of FcRn-directed albumin salvage from lysosomal degradation. Furthermore, the amount of recycled albumin in cell culture media corresponded to FcRn-binding affinity, with a ∼3.3-fold increase after 1 h for the high FcRn-binding albumin variant compared with wild-type albumin. Together, these findings uncover an FcRn-dependent endosomal cellular-sorting pathway that has great importance in describing fundamental mechanisms of intracellular albumin recycling and the possibility to tune albumin-based therapeutic effects by FcRn-binding affinity.

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.

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.

The long circulatory half-life of albumin facilitated by the interaction with the cellular recycling neonatal Fc receptor (FcRn) is utilized for drug half-life extension. FcRn engagement effects following covalent attachment of cargo to cysteine 34, however, have not been investigated. Poly(ethylene glycol) polymers were used to study the influence of cargo molecular weight on human FcRn engagement of recombinant wild type (WT) albumin and an albumin variant engineered for increased FcRn binding. Decreased affinity was observed for all conjugates; however, the engineered albumin maintained an affinity above that of unmodified wild type albumin that promotes it as an attractive drug delivery platform.