Leslie Evans

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.

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.

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.

Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter

The methylotrophic yeast H. polymorpha is a popular system for the expression of recombinant proteins using the strong and regulatable methanol oxidase (MOX) promoter. Here we show that the constitutive PMA1 promoter can programme the expression of two heterologous proteins, glucose oxidase and human serum albumin. A constitutive promoter provides a useful additional facility to the H. polymorpha expression system because it allows a simplified fermentation regime, avoids the use of methanol, which is both toxic and an explosive hazard, and allows more flexibility for ectopic gene expression during the course of academic studies. A fragment previously isolated in a promoter screen, using glucose oxidase (GOD) as a reporter gene, was shown to consist of the promoter region and the first 659 bp of the H. polymorpha PMA1 gene, encoding the plasma membrane H+‐ATPase. When the PMA1 promoter was optimally aligned with the GOD coding region, it produced 185 mg/l glucose oxidase in high cell density fed batch fermentations, whereas in previous experiments using the MOX promoter, a yield of 500 mg/l was recovered. The PMA1 promoter was also used to express recombinant human serum albumin (rHA) in H. polymorpha. In high cell density fermentations the PMA1 promoter produced 460 mg/l rHA, whereas 280 mg/l rHA was obtained using the MOX promoter. Taken together, these experiments show that the HpPMA1 programmes the constitutive expression of recombinant proteins and provides a yield comparable to that from the MOX promoter. Copyright

Disruption of the Saccharomyces cerevisiae YAP3 Gene Reduces the Proteolytic Degradation of Secreted Recombinant Human Albumin

Expression of recombinant human albumin (rHA) in Saccharomyces cerevisiae resulted in secretion of both mature albumin and a 45 kDa degradation product, comprising an N‐terminal fragment of rHA with heterogeneous C‐termini between residues 403 and 409 (Geisow et al., 1991). Site‐directed mutagenesis of the human albumin gene (HA) to change Arg410 to Ala (R410A) caused a significant reduction in the amount of fragment produced. Mutation of the adjacent dibasic site Lys413 Lys414 had little effect in isolation, but in combination with the R410A mutation resulted in a further reduction in the amount of rHA fragment produced. This reduction could be duplicated with nature‐identical rHA by disruption of the gene for an aspartyl protease (YAP3), alone or in conjunction with disruption of the KEX2 gene. Disruption of KEX2 alone did not result in any improvement in the degree of degradation of the rHA. Reduced degradation was also observed when an rHA‐human growth hormone fusion protein was secreted from a yap3 strain, suggesting that such strains may have a general utility for heterologous protein secretion.

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.

Yeast 2 µm plasmid copy number is elevated by a mutation in the nuclear gene UBC4

The copy number of the Saccharomyces cerevisiae endogenous 2 µm plasmid is under strict control to ensure efficient propagation to the daughter cell without significantly reducing the growth rate of the mother or the daughter cell. A recessive mutation has been identified that resulted in an elevated but stable 2 µm plasmid copy number, which could be complemented by a genomic DNA clone containing the UBC4 gene, encoding an E2 ubiquitin‐conjugating enzyme. A ubc4::URA3 deletion resulted in the same elevated 2 µm plasmid copy number. An analysis of the endogenous 2 µm transcripts revealed that the steady‐state abundance of REP1, REP2, FLP and RAF were all increased 4–5‐fold in the mutant. Analysis of the mutant ubc4 allele identified a single base pair mutation within the UBC4 coding region, which would generate a glutamic acid to lysine amino acid substitution within a region of conserved tertiary structure located within the first α‐helix of Ubc4p. These investigations represent the first molecular characterization of a mutation within a Saccharomyces cerevisiae nuclear gene shown to affect 2 µm steady‐state plasmid copy number and implicate the ubiquitin‐dependent proteolytic pathway in host control of 2 µm plasmid copy number. Copyright

Enhanced protein expression through strain selection, gene disruption, improved vector design and co-expression of endogenous chaperones

Results An analysis of a series of haploid laboratory yeast strains revealed significant intra-strain variability and unstable plasmid segregation. By combining classic chemical mutagenesis and selection a family of highly efficient Saccharomyces cerevisiae strains has been developed for the commercial production of biopharmaceutical products. When combined with a stable [1], high copy number [2], episomal expression vector system and a strong constitutive promoter, secreted recombinant protein expression titres in excess of 4 g/L were achieved (see Figure 1). Specific genetic modifications to the host were also introduced to increase product yield and control posttranslational modifications, such as proteolysis and glycosylation.

Generation of a double transgenic humanized neonatal Fc receptor (FcRn)/albumin mouse to study the pharmacokinetics of albumin-linked drugs.

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 whos 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.