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

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.     

Plasma half-life extension of small recombinant antibodies by fusion to immunoglobulin-binding domains

Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBDs) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C terminus of a single-chain Fv and a bispecific single-chain diabody, respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extents for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpG(C3)) followed by domains B and D of SpA, suggesting that SpG(C3) is particularly useful to extend the plasma half-life of small proteins.     

Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.     

The Crystal Structure of the Active Domain of Anopheles Anti-platelet Protein,a Powerful Anti-coagulant,in Complex with an Antibody

Blood clotting is a vitally important process that must be carefully regulated to prevent blood loss on one hand and thrombosis on the other. Severe injury and hemophilia may be treated with pro-coagulants, whereas risk of obstructive clotting or embolism may be reduced with anti-coagulants. Anti-coagulants are an extremely important class of drug, one of the most widely used types of medication, but there remains a pressing need for novel treatments, however, as present drugs such as warfarin have significant drawbacks. Nature provides a number of examples of anti-coagulant proteins produced by blood-sucking animals, which may provide templates for the development of new small molecules with similar physiological effects. We have, therefore, studied an Anopheles anti-platelet protein from a malaria vector mosquito and report its crystal structure in complex with an antibody. Overall the protein is extremely sensitive to proteolysis, but the crystal structure reveals a stable domain built from two helices and a turn, which corresponds to the functional region. The antibody raised against Anopheles anti-platelet protein prevents it from binding collagen. Our work, therefore, opens new avenues to the development of both novel small molecule anti-clotting agents and anti-malarials.     

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.     

Structural Basis for Penetration of the Glycan Shield of Hepatitis C Virus E2 Glycoprotein by a Broadly Neutralizing Human Antibody

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412–423 (E2_412–423) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2_412–423 epitope to 2.0 Å resolution. The conformation of E2_412–423 bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the β-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2_412–423 complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2_412–423 epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.     

Inhibiting alternative pathway complement activation by targeting the factor D exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.     

X-ray Crystal Structures of Monomeric and Dimeric Peptide Inhibitors in Complex with the Human Neonatal Fc Receptor,FcRn

The neonatal Fc receptor, FcRn, is responsible for the long half-life of IgG molecules in vivo and is a potential therapeutic target for the treatment of autoimmune diseases. A family of peptides comprising the consensus motif GHFGGXY, where X is preferably a hydrophobic amino acid, was shown previously to inhibit the human IgG:human FcRn protein-protein interaction (Mezo, A. R., McDonnell, K. A., Tan Hehir, C. A., Low, S. C., Palombella, V. J., Stattel, J. M., Kamphaus, G. D., Fraley, C., Zhang, Y., Dumont, J. A., and Bitonti, A. J. (2008) Proc. Natl. Acad. Sci. U.S.A., 105, 2337–2342). Herein, the x-ray crystal structure of a representative monomeric peptide in complex with human FcRn was solved to 2.6 Å resolution. The structure shows that the peptide binds to human FcRn at the same general binding site as does the Fc domain of IgG. The data correlate well with structure-activity relationship data relating to how the peptide family binds to human FcRn. In addition, the x-ray crystal structure of a representative dimeric peptide in complex with human FcRn shows how the bivalent ligand can bridge two FcRn molecules, which may be relevant to the mechanism by which the dimeric peptides inhibit FcRn and increase IgG catabolism in vivo. Modeling of the peptide:FcRn structure as compared with available structural data on Fc and FcRn suggest that the His-6 and Phe-7 (peptide) partially mimic the interaction of His-310 and Ile-253 (Fc) in binding to FcRn, but using a different backbone topology.     

Structure of the Toxoplasma gondii ROP18 Kinase Domain Reveals a Second Ligand Binding Pocket Required for Acute Virulence

At least a third of the human population is infected with the intracellular parasite Toxoplasma gondii, which contributes significantly to the disease burden in immunocompromised and neutropenic hosts and causes serious congenital complications when vertically transmitted to the fetus. Genetic analyses have identified the Toxoplasma ROP18 Ser/Thr protein kinase as a major factor mediating acute virulence in mice. ROP18 is secreted into the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. However, its precise molecular function and regulation are not fully understood. We have determined the crystal structure of the ROP18 kinase domain, which is inconsistent with a previously proposed autoinhibitory mechanism of regulation. Furthermore, a sucrose molecule bound to our structure identifies an additional ligand-binding pocket outside of the active site cleft. Mutational analysis confirms an important role for this pocket in virulence.     

Recognition of the Thomsen-Friedenreich Pancarcinoma Carbohydrate Antigen by a Lamprey Variable Lymphocyte Receptor

Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs were recently shown to recognize glycans, such as the tumor-associated Thomsen-Friedenreich antigen (TFα; Galβ1–3GalNAcα), with a selectivity rivaling or exceeding that of lectins and antibodies. To understand the basis for TFα recognition by one such VLR (VLRB.aGPA.23), we measured thermodynamic parameters for the binding interaction and determined the structure of the VLRB.aGPA.23-TFα complex to 2.2 Å resolution. In the structure, four tryptophan residues form a tight hydrophobic cage encasing the TFα disaccharide that completely excludes buried water molecules. This cage together with hydrogen bonding of sugar hydroxyls to polar side chains explains the exquisite selectivity of VLRB.aGPA.23. The topology of the glycan-binding site of VLRB.aGPA.23 differs markedly from those of lectins or antibodies, which typically consist of long, convex grooves for accommodating the oligosaccharide. Instead, the TFα disaccharide is sandwiched between a variable loop and the concave surface of the VLR formed by the β-strands of the leucine-rich repeat modules. Longer oligosaccharides are predicted to extend perpendicularly across the β-strands, requiring them to bend to match the concavity of the VLR solenoid.     

Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex_8–16GlcNAc₂ modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex₉GlcNAc₂ at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.     

Dynamics of Inter-heavy Chain Interactions in Human Immunoglobulin G (IgG) Subclasses Studied by Kinetic Fab Arm Exchange

Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange (“Fab arm exchange”). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.     

LUZ-Y,a Novel Platform for the Mammalian Cell Production of Full-length IgG-bispecific Antibodies

The ability of bispecific antibodies to simultaneously bind two unique antigens has great clinical potential. However, most approaches utilized to generate bispecific antibodies yield antibody-like structures that diverge significantly from the structure of archetype human IgG, and those that do approach structural similarity to native antibodies are often challenging to engineer and manufacture. Here, we present a novel platform for the mammalian cell production of bispecific antibodies that differ from their parental mAbs by only a single point mutation per heavy chain. Central to this platform is the addition of a leucine zipper to the C terminus of the C_H3 domain of the antibody that is sufficient to drive the heterodimeric assembly of antibody heavy chains and can be readily removed post-purification. Using this approach, we developed various antibody constructs including one-armed Abs, bispecific antibodies that utilize a common light chain, and bispecific antibodies that pair light chains to their cognate heavy chains via peptide tethers. We have applied this technology to various antibody pairings and will demonstrate the engineering, purification, and biological activity of these antibodies herein.     

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand''s carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.     

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2_hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2_hPg. However, the binding strength of VEK30 (K_D = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (K_D = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH₂ and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2_hPg with nearly the same affinity as PAM (K_D = 1–2 nm). However, addition of two PAM residues (Arg¹²⁶-His¹²⁷) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2_hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg¹¹³, His¹¹⁴, Glu¹¹⁶, Arg¹²⁶, His¹²⁷), mutation of which reduced PAM binding affinity for K2_hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.     

Characterization of the Membrane-targeting C1 Domain in Pasteurella multocida Toxin

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G_q- and G_12/13-dependent pathways through the deamidation of a glutamine residue in the α-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575–1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590–670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT ΔC1(4H)) neither localized to the plasma membrane nor stimulated the G_q/12/13-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT ΔC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.     

The Making of a Sweet Modification: Structure and Function of O-GlcNAc Transferase

O-GlcNAc transferase is an essential mammalian enzyme responsible for transferring a single GlcNAc moiety from UDP-GlcNAc to specific serine/threonine residues of hundreds of nuclear and cytoplasmic proteins. This modification is dynamic and has been implicated in numerous signaling pathways. An unexpected second function for O-GlcNAc transferase as a protease involved in cleaving the epigenetic regulator HCF-1 has also been reported. Recent structural and biochemical studies that provide insight into the mechanism of glycosylation and HCF-1 cleavage will be described, with outstanding questions highlighted.     

Genetic Engineering of the Phosphocarrier Protein NPr of the Escherichia coli Phosphotransferase System Selectively Improves Sugar Uptake Activity

The Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) in prokaryotes mediates the uptake and phosphorylation of its numerous substrates through a phosphoryl transfer chain where a phosphoryl transfer protein, HPr, transfers its phosphoryl group to any of several sugar-specific Enzyme IIA proteins in preparation for sugar transport. A phosphoryl transfer protein of the PTS, NPr, homologous to HPr, functions to regulate nitrogen metabolism and shows virtually no enzymatic cross-reactivity with HPr. Here we describe the genetic engineering of a "chimeric" HPr/NPr protein, termed CPr14 because 14 amino acid residues of the interface were replaced. CPr14 shows decreased activity with most PTS permeases relative to HPr, but increases activity with the broad specificity mannose permease. The results lead to the proposal that HPr is not optimal for most PTS permeases but instead represents a compromise with suboptimal activity for most PTS permeases. The evolutionary implications are discussed.     

Replacing the Factor VIII C1 Domain with a Second C2 Domain Reduces Factor VIII Stability and Affinity for Factor IXa

Factor VIII (FVIII) consists of a heavy chain (A1(a1)A2(a2)B domains) and light chain ((a3)A3C1C2 domains). To gain insights into a role of the FVIII C domains, we eliminated the C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIII_C2C2) as measured by thermal decay at 55 °C of FVIII activity was markedly reduced (∼11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIII_C2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (∼2.8-fold) than that for WT FVIII. Among several anti-FVIII antibodies tested (anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody), only ESH4 inhibited membrane binding of both WT FVIII and FVIII_C2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas the activity of FVIII_C2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (∼10-fold), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIII_C2C2 variant (∼4-fold). Together, the reduced stability plus impaired FIXa interaction of FVIII_C2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.     

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*

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.