Darrell Sleep

Albumin as a versatile platform for drug half-life extension.

BACKGROUND Albumin is the most abundant plasma protein, is highly soluble, very stable and has an extraordinarily long circulatory half-life as a direct result of its size and interaction with the FcRn mediated recycling pathway. In contrast, many therapeutic molecules are smaller than the renal filtration threshold and are rapidly lost from the circulation thereby limiting their therapeutic potential. Albumin can be used in a variety of ways to increase the circulatory half-life of such molecules. SCOPE OF REVIEW This article will review the mechanisms which underpin albumins extraordinarily long circulatory half-life and how the understanding of these processes are currently being employed to extend the circulatory half-life of drugs which can be engineered to bind to albumin, or are conjugated to, or genetically fused to, albumin. MAJOR CONCLUSIONS The recent and growing understanding of the pivotal role of FcRn in maintaining the extended circulatory half-life of albumin will necessitate a greater and more thorough investigation of suitable pre-clinical model systems for assessing the pharmacokinetic profiles of drugs associated, conjugated or fused to albumin. GENERAL SIGNIFICANCE Association, conjugation or fusion of therapeutic drugs to albumin is a well-accepted and established half-life extension technology. The manipulation of the albumin-FcRn interaction will facilitate the modulation of the circulatory half-life of albumin-enabled drugs, leading to superior pharmacokinetics tailored to the disease state and increased patient compliance. This article is part of a Special Issue entitled Serum Albumin.

Interdomain zinc site on human albumin

Albumin is the major transport protein in blood for Zn2+, a metal ion required for physiological processes and recruited by various drugs and toxins. However, the Zn2+-binding site(s) on albumin is ill-defined. We have analyzed the 18 x-ray crystal structures of human albumin in the PDB and identified a potential five-coordinate Zn site at the interface of domains I and II consisting of N ligands from His-67 and His-247 and O ligands from Asn-99, Asp-249, and H2O, which are the same amino acid ligands as those in the zinc enzymes calcineurin, endonucleotidase, and purple acid phosphatase. The site is preformed in unliganded apo-albumin and highly conserved in mammalian albumins. We have used 111Cd NMR as a probe for Zn2+ binding to recombinant human albumin. We show that His-67 → Ala (His67Ala) mutation strongly perturbs Cd2+ binding, whereas the mutations Cys34Ala, or His39Leu and Tyr84Phe (residues which may H-bond to Cys-34) have no effect. Weak Cl− binding to the fifth coordination site of Cd2+ was demonstrated. Cd2+ binding was dramatically affected by high fatty acid loading of albumin. Analysis of the x-ray structures suggests that fatty acid binding to site 2 triggers a spring-lock mechanism, which disengages the upper (His-67/Asn-99) and lower (His-247/Asp-249) halves of the metal site. These findings provide a possible mechanism whereby fatty acids (and perhaps other small molecules) could influence the transport and delivery of zinc in blood.

Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor

Albumin is the most abundant protein in blood where it has a pivotal role as a transporter of fatty acids and drugs. Like IgG, albumin has long serum half-life, protected from degradation by pH-dependent recycling mediated by interaction with the neonatal Fc receptor, FcRn. Although the FcRn interaction with IgG is well characterized at the atomic level, its interaction with albumin is not. Here we present structure-based modelling of the FcRn–albumin complex, supported by binding analysis of site-specific mutants, providing mechanistic evidence for the presence of pH-sensitive ionic networks at the interaction interface. These networks involve conserved histidines in both FcRn and albumin domain III. Histidines also contribute to intramolecular interactions that stabilize the otherwise flexible loops at both the interacting surfaces. Molecular details of the FcRn–albumin complex may guide the development of novel albumin variants with altered serum half-life as carriers of drugs.

Structure, Properties, and Engineering of the Major Zinc Binding Site on Human Albumin

Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn2+ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His67 and Asn99 from domain I, His247 and Asp249 from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn2+ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn2+ binding but alter protein stability, whereas mutation of His67 to alanine removes an interdomain H-bond and weakens Zn2+ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures.

Role of Tyr84 in controlling the reactivity of Cys34 of human albumin.

Cys34 in domain I of the three‐domain serum protein albumin is the binding site for a wide variety of biologically and clinically important small molecules, provides antioxidant activity, and constitutes the largest portion of free thiol in blood. Analysis of X‐ray structures of albumin reveals that the loop containing Tyr84 occurs in multiple conformations. In structures where the loop is well defined, there appears to be an H‐bond between the OH of Tyr84 and the sulfur of Cys34. We show that the reaction of 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) with Tyr84Phe mutant albumin is approximately four times faster than with the wild‐type protein between pH 6 and pH 8. In contrast, the His39Leu mutant reacts with DTNB more slowly than the wild‐type protein at pH < 8, but at a similar rate at pH 8. Above pH 8 there is a dramatic increase in reactivity for the Tyr84Phe mutant. We also report 1H NMR studies of disulfide interchange reactions with cysteine. The tethering of the two loops containing Tyr84 and Cys34 not only appears to control the redox potential and accessibility of Cys34, but also triggers the transmission of information about the state of Cys34 throughout domain I, and to the domainI/II interface.

Albumin and its application in drug delivery

Introduction: Rapid clearance of drugs from the body results in short therapeutic half-life and is an integral property of many protein and peptide-based drugs. To maintain the desired therapeutic effect patients are required to administer higher doses more frequently, which is inconvenient and risks undesirable side effects. Drug delivery technologies aim to minimise the number of administrations and dose-related toxicity while maximising therapeutic efficacy. Areas covered: This review describes albumin’s inherent biochemical and biophysical properties, which make it an attractive drug delivery platform and the developmental status of drugs that are associated, conjugated or genetically fused with albumin. Albumin interacts with a number of cell surface receptors including gp18, gp30, gp60, FcRn, cubilin and megalin. The importance of albumin’s interaction with the FcRn receptor, the basis for albumin’s long circulatory half-life, is described, as are engineered albumins with improved pharmacokinetics. Albumin naturally accumulates at tumours and sites of inflammation, a characteristic which can be augmented by the addition of targeting ligands. The development of albumin drug conjugates which reply upon this property is described. Expert opinion: Albumin’s inherent biochemical and biophysical properties make it an ideal drug delivery platform. Recent advances in our understanding of albumin physiology and the improvement in albumin-based therapies strongly suggest that albumin-based therapies have a significant advantage over alternative technologies in terms of half-life, stability, versatility, safety and ease of manufacture. Given the importance of the albumin:FcRn interaction, the interpretation of the pharmacokinetic and pharmacodynamic profiles of albumin-based therapeutics with disturbed albumin:FcRn interaction may have to be reassessed. The FcRn receptor has additional functionality, especially in relation to immunology, antigen presentation and delivery of proteins across mucosal membranes, consequently albumin-based fusions and conjugates may have a future role in oral and pulmonary-based vaccines and drug delivery.

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

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.

The production, characterisation and enhanced pharmacokinetics of scFv–albumin fusions expressed in Saccharomyces cerevisiae

An expression system is described for the production of monomeric scFvs and scFv antibody fragments genetically fused to human albumin (at either the N- or C-terminus or both). Based upon strains of Saccharomyces cerevisiae originally developed for the production of a recombinant human albumin (Recombumin) this system has delivered high levels of secreted product into the supernatant of shake flask and high cell density fed-batch fermentations. Specific binding to the corresponding ligand was demonstrated for each of the scFvs and scFv-albumin fusions and pharmacokinetic studies showed that the fusion products had greatly extended circulatory half-lives. The system described provides an attractive alternative to other microbial systems for the manufacture of this type of product.

Single-chain Variable Fragment Albumin Fusions Bind the Neonatal Fc Receptor (FcRn) in a Species-dependent Manner IMPLICATIONS FOR IN VIVO HALF-LIFE EVALUATION OF ALBUMIN FUSION THERAPEUTICS

Background: Albumin is utilized as carrier of biopharmaceuticals. FcRn binding regulates its long half-life. Results: ScFv fusion to HSA only slightly reduces human FcRn binding, whereas HSA and scFv-HSA fusions have very weak binding to rodent FcRn. Conclusion: Rodents have limitations for preclinical evaluation of HSA fusions. Significance: We illuminate design of HSA fusions and highlight cross-species differences to consider prior to preclinical evaluation. Albumin has a serum half-life of 3 weeks in humans. This has been utilized to extend the serum persistence of biopharmaceuticals that are fused to albumin. In light of the fact that the neonatal Fc receptor (FcRn) is a key regulator of albumin homeostasis, it is crucial to address how fusion of therapeutics to albumin impacts binding to FcRn. Here, we report on a detailed molecular investigation on how genetic fusion of a short peptide or an single-chain variable fragment (scFv) fragment to human serum albumin (HSA) influences pH-dependent binding to FcRn from mouse, rat, monkey, and human. We have found that fusion to the N- or C-terminal end of HSA only slightly reduces receptor binding, where the most noticeable effect is seen after fusion to the C-terminal end. Furthermore, in contrast to the observed strong binding to human and monkey FcRn, HSA and all HSA fusions bound very poorly to mouse and rat versions of the receptor. Thus, we demonstrate that conventional rodents are limited as preclinical models for analysis of serum half-life of HSA-based biopharmaceuticals. This finding is explained by cross-species differences mainly found within domain III (DIII) of albumin. Our data demonstrate that although fusion, particularly to the C-terminal end, may slightly reduce the affinity for FcRn, HSA is versatile as a carrier of biopharmaceuticals.

Dissection of the Neonatal Fc Receptor (FcRn)-Albumin Interface Using Mutagenesis and Anti-FcRn Albumin-blocking Antibodies

Background: Albumin has a long serum half-life, which is regulated by FcRn. Results: A cluster of conserved tryptophan residues of FcRn is required for binding to albumin and anti-FcRn albumin blocking antibodies. Conclusion: The FcRn-albumin interaction is pH-dependent but hydrophobic in nature. Significance: This study provides mechanistic insight into how FcRn binds albumin and regulates its long half-life. Albumin is the most abundant protein in blood and plays a pivotal role as a multitransporter of a wide range of molecules such as fatty acids, metabolites, hormones, and toxins. In addition, it binds a variety of drugs. Its role as distributor is supported by its extraordinary serum half-life of 3 weeks. This is related to its size and binding to the cellular receptor FcRn, which rescues albumin from intracellular degradation. Furthermore, the long half-life has fostered a great and increasing interest in utilization of albumin as a carrier of protein therapeutics and chemical drugs. However, to fully understand how FcRn acts as a regulator of albumin homeostasis and to take advantage of the FcRn-albumin interaction in drug design, the interaction interface needs to be dissected. Here, we used a panel of monoclonal antibodies directed towards human FcRn in combination with site-directed mutagenesis and structural modeling to unmask the binding sites for albumin blocking antibodies and albumin on the receptor, which revealed that the interaction is not only strictly pH-dependent, but predominantly hydrophobic in nature. Specifically, we provide mechanistic evidence for a crucial role of a cluster of conserved tryptophan residues that expose a pH-sensitive loop of FcRn, and identify structural differences in proximity to these hot spot residues that explain divergent cross-species binding properties of FcRn. Our findings expand our knowledge of how FcRn is controlling albumin homeostasis at a molecular level, which will guide design and engineering of novel albumin variants with altered transport properties.