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

Open AccessPublished:March 20, 2014DOI:https://doi.org/10.1074/jbc.M114.549832
      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.

      Introduction

      The two most abundant soluble proteins in our body, IgG and albumin, account for more than 80% of the total plasma protein pool and amount to an impressive 12 and 40 mg/ml, respectively, in mouse and man. Although albumin acts as a versatile transporter of an array of small molecules, supports colloidal osmotic pressure, and buffers the pH of the blood, IgG is the major antibody class that protects against invading pathogens. However, IgG and albumin also share two remarkable features. First, they both have prolonged half-lives of 19 days compared with few days or less for other circulating proteins. Second, they are unique among the plasma proteins in that there is a direct relationship between their blood concentrations and their fractional catabolic rates. It has now become apparent that their homeostatic regulation is controlled by a cellular receptor, named the neonatal Fc receptor (FcRn),
      The abbreviations used are:
      FcRn
      neonatal Fc receptor
      hFcRn
      human FcRn
      mFcRn
      mouse FcRn
      DIII
      domain III
      HC
      heavy chain
      HSA
      human serum albumin
      MSA
      mouse serum albumin
      scFv
      single-chain fragment variable
      SPR
      surface plasmon resonance
      Tg
      transgenic
      β2m
      β2-microglobulin
      CSA
      cynomolgus monkey serum albumin.
      which rescues both from intracellular degradation and thus is responsible for their prolonged half-lives (
      • Montoyo H.P.
      • Vaccaro C.
      • Hafner M.
      • Ober R.J.
      • Mueller W.
      • Ward E.S.
      Conditional deletion of the MHC class I-related receptor FcRn reveals the sites of IgG homeostasis in mice.
      ,
      • Chaudhury C.
      • Mehnaz S.
      • Robinson J.M.
      • Hayton W.L.
      • Pearl D.K.
      • Roopenian D.C.
      • Anderson C.L.
      The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan.
      ).
      FcRn is a major histocompatibility class I-related molecule that is built up of a unique transmembrane heavy chain (HC), which noncovalently associates with the common β2-microglobulin (β2m) (
      • Simister N.E.
      • Mostov K.E.
      Cloning and expression of the neonatal rat intestinal Fc receptor, a major histocompatibility complex class I antigen homolog.
      ,
      • Story C.M.
      • Mikulska J.E.
      • Simister N.E.
      A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus.
      ). The extracellular part of the HC consists of three domains (α1, α2, and α3) where the N-terminal α1-α2 domains form eight antiparallel β-pleated strands topped by two long α-helices followed by the membrane proximal α3-domain (
      • Burmeister W.P.
      • Huber A.H.
      • Bjorkman P.J.
      Crystal structure of the complex of rat neonatal Fc receptor with Fc.
      ,
      • West Jr., A.P.
      • Bjorkman P.J.
      Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor.
      ). The β2m subunit is bound to the HC via both the α1-α2 platform and the α3-domain.
      IgG and albumin bind FcRn simultaneously in a noncooperative and strictly pH-dependent manner, with strong binding at pH 6.0 that becomes progressively weaker approaching neutral pH (
      • Chaudhury C.
      • Mehnaz S.
      • Robinson J.M.
      • Hayton W.L.
      • Pearl D.K.
      • Roopenian D.C.
      • Anderson C.L.
      The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan.
      ,
      • Andersen J.T.
      • Dee Qian J.
      • Sandlie I.
      The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH-dependent binding to albumin.
      ,
      • Chaudhury C.
      • Brooks C.L.
      • Carter D.C.
      • Robinson J.M.
      • Anderson C.L.
      Albumin binding to FcRn: distinct from the FcRn-IgG interaction.
      ). These pH-sensitive interactions are regulated by ionic networks at the interfaces and internally in each protein (
      • Burmeister W.P.
      • Huber A.H.
      • Bjorkman P.J.
      Crystal structure of the complex of rat neonatal Fc receptor with Fc.
      ,
      • Kim J.K.
      • Firan M.
      • Radu C.G.
      • Kim C.H.
      • Ghetie V.
      • Ward E.S.
      Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn.
      ,
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ,
      • Schmidt M.M.
      • Townson S.A.
      • Andreucci A.J.
      • King B.M.
      • Schirmer E.B.
      • Murillo A.J.
      • Dombrowski C.
      • Tisdale A.W.
      • Lowden P.A.
      • Masci A.L.
      • Kovalchin J.T.
      • Erbe D.V.
      • Wittrup K.D.
      • Furfine E.S.
      • Barnes T.M.
      Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.
      ). FcRn rescues the proteins from degradation in hematopoietic cells and endothelial cells lining the vascular space by binding IgG and albumin within intracellular endosomal compartments, which then results in transport of the ternary complex to the cell membrane for release of ligands back into the circulation as a consequence of exposure to the neutral pH of the blood (
      • Montoyo H.P.
      • Vaccaro C.
      • Hafner M.
      • Ober R.J.
      • Mueller W.
      • Ward E.S.
      Conditional deletion of the MHC class I-related receptor FcRn reveals the sites of IgG homeostasis in mice.
      ,
      • Kobayashi K.
      • Qiao S.W.
      • Yoshida M.
      • Baker K.
      • Lencer W.I.
      • Blumberg R.S.
      An FcRn-dependent role for anti-flagellin immunoglobulin G in pathogenesis of colitis in mice.
      ,
      • Akilesh S.
      • Christianson G.J.
      • Roopenian D.C.
      • Shaw A.S.
      Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism.
      ).
      Full-length antibodies of the IgG type and IgG Fc fusions are the fastest growing classes of biopharmaceuticals (
      • Chan A.C.
      • Carter P.J.
      Therapeutic antibodies for autoimmunity and inflammation.
      ). Their remarkably long serum half-life provided by FcRn has surely contributed to their clinical success. Moreover, the central role of FcRn in controlling the pharmacokinetics of IgG has inspired the development of novel Fc-engineered IgG molecules with improved FcRn binding that result in extended half-lives and improved therapeutic efficacy (
      • Ghetie V.
      • Popov S.
      • Borvak J.
      • Radu C.
      • Matesoi D.
      • Medesan C.
      • Ober R.J.
      • Ward E.S.
      Increasing the serum persistence of an IgG fragment by random mutagenesis.
      • Hinton P.R.
      • Johlfs M.G.
      • Xiong J.M.
      • Hanestad K.
      • Ong K.C.
      • Bullock C.
      • Keller S.
      • Tang M.T.
      • Tso J.Y.
      • Vásquez M.
      • Tsurushita N.
      Engineered human IgG antibodies with longer serum half-lives in primates.
      ,
      • Vaccaro C.
      • Zhou J.
      • Ober R.J.
      • Ward E.S.
      Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels.
      ,
      • Zalevsky J.
      • Chamberlain A.K.
      • Horton H.M.
      • Karki S.
      • Leung I.W.
      • Sproule T.J.
      • Lazar G.A.
      • Roopenian D.C.
      • Desjarlais J.R.
      Enhanced antibody half-life improves in vivo activity.
      ). The lesson learned from such studies is that the major engineering challenge is to increase binding affinity at pH 6.0 while at the same time retaining low affinity at nearly neutral pH, so as to allow efficient FcRn-mediated recycling.
      Although the molecular interaction between FcRn and IgG has been studied for decades, the characterization of the albumin-FcRn interaction is just starting (
      • Andersen J.T.
      • Dee Qian J.
      • Sandlie I.
      The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH-dependent binding to albumin.
      ,
      • Chaudhury C.
      • Brooks C.L.
      • Carter D.C.
      • Robinson J.M.
      • Anderson C.L.
      Albumin binding to FcRn: distinct from the FcRn-IgG interaction.
      ,
      • Andersen J.T.
      • Dalhus B.
      • Cameron J.
      • Daba M.B.
      • Plumridge A.
      • Evans L.
      • Brennan S.O.
      • Gunnarsen K.S.
      • Bjørås M.
      • Sleep D.
      • Sandlie I.
      Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor.
      ,
      • Schmidt M.M.
      • Townson S.A.
      • Andreucci A.J.
      • King B.M.
      • Schirmer E.B.
      • Murillo A.J.
      • Dombrowski C.
      • Tisdale A.W.
      • Lowden P.A.
      • Masci A.L.
      • Kovalchin J.T.
      • Erbe D.V.
      • Wittrup K.D.
      • Furfine E.S.
      • Barnes T.M.
      Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.
      ,
      • Andersen J.T.
      • Daba M.B.
      • Sandlie I.
      FcRn binding properties of an abnormal truncated analbuminemic albumin variant.
      ). Still, the knowledge of its prolonged half-life has been utilized for some time to improve the in vivo efficacy of therapeutics. The approaches taken have been to associate or covalently couple a protein of interest by genetic fusion or chemical conjugation to albumin leading to increase in vivo half-life of the fusion compared with the original molecule (
      • Elsadek B.
      • Kratz F.
      Impact of albumin on drug delivery: new applications on the horizon.
      ,
      • Sleep D.
      • Cameron J.
      • Evans L.R.
      Albumin as a versatile platform for drug half-life extension.
      ). The great advantage of extended serum persistence is a more even serum concentration of the drug, lower dosing frequency, and the fact that the doses given may be decreased without compromising pharmacological efficacy. This may well translate into less toxicity and side effects, as well as improved compliance. Using albumin to increase half-life has been shown to be useful for a wide range of different proteins that includes coagulation factors, antibody fragments, interferon 2b, and glucagon-like peptide-1, as reviewed elsewhere (
      • Elsadek B.
      • Kratz F.
      Impact of albumin on drug delivery: new applications on the horizon.
      • Sleep D.
      • Cameron J.
      • Evans L.R.
      Albumin as a versatile platform for drug half-life extension.
      ,
      • Andersen J.T.
      • Cameron J.
      • Plumridge A.
      • Evans L.
      • Sleep D.
      • Sandlie I.
      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.
      ,
      • Santagostino E.
      • Negrier C.
      • Klamroth R.
      • Tiede A.
      • Pabinger-Fasching I.
      • Voigt C.
      • Jacobs I.
      • Morfini M.
      Safety and pharmacokinetics of a novel recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in hemophilia B patients.
      ). Thus, we aimed to develop novel engineered human serum albumin (HSA) variants with improved pH-dependent binding to human FcRn (hFcRn) and enhanced serum half-life that can be used as improved carriers for diagnostics and therapeutics.
      Here, we report on a panel of engineered single HSA variants with substitutions at position 573 within the C-terminal helix of domain III (DIII). All show considerably improved binding to hFcRn. Surprisingly, replacement of Lys-573 with any amino acid resulted in enhanced binding to hFcRn at acidic pH. In particular, K573P, where Lys-573 was replaced by a proline, had more than 12-fold improved affinity toward hFcRn, resulting in extended serum half-life in WT mice, mice transgenic (Tg) for hFcRn and cynomolgus monkeys. Importantly, fusion of an antibody derived single-chain variable fragment (scFv) to the K573P variant did not negatively affect the favorable binding. Thus, the K573P variant may be an ideal carrier for half-life extension of both conjugated and genetically fused diagnostics and therapeutics.

      DISCUSSION

      In this report we used rational design to engineer a panel of HSA variants with altered hFcRn binding affinities. One candidate, K573P, showed extended serum half-life in normal mice and mice Tg for the human receptor, as well as in cynomolgus monkeys. Importantly, the enhanced affinity of the K573P mutation was retained postfusion of a scFv fragment to either the N- or C-terminal end. This variant is one of a panel of variants where all have increased binding to hFcRn at acidic pH relative to WT HSA. This work thus paves the way for a new generation of HSA fusion therapeutics utilizing the novel engineered HSA scaffolds as drug carriers. Our results indicate that serum half-life can be tailored using the HSA variants developed, which ultimately may lead to superior efficacy of biopharmaceuticals and greater convenience for patients.
      We have previously reported that MSA binds much more strongly to mFcRn than does HSA (
      • Andersen J.T.
      • Cameron J.
      • Plumridge A.
      • Evans L.
      • Sleep D.
      • Sandlie I.
      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.
      ,
      • Andersen J.T.
      • Daba M.B.
      • Berntzen G.
      • Michaelsen T.E.
      • Sandlie I.
      Cross-species binding analyses of mouse and human neonatal Fc receptor show dramatic differences in immunoglobulin G and albumin binding.
      ). This must be considered when mice are utilized as preclinical models to determine the pharmacokinetics of HSA variants and fusions. When small amounts of HSA fusions are injected into mice, they compete for mFcRn binding with large amounts of endogenous MSA (40 mg/ml). Biopharmaceutical fusion to HSA has resulted in improved pharmacokinetics, likely as the result of increased molecular weight above the renal clearance threshold; however, the serum half-lives did not reach that of endogenous albumin, underscoring the importance of efficient FcRn-mediated recycling and the route of clearance of HSA fusions. The nature of the fused protein may also potentially affect receptor binding, which needs to be addressed for each unique fusion. Thus, prior to in vivo evaluations in rodents, the FcRn binding kinetics should be determined.
      Here we demonstrate that single point mutations in HSA have a large effect on the binding kinetics toward FcRn from mouse, cynomolgus monkey, and human. Despite the shortcomings of existing mouse models, we provide evidence that such engineering translates into altered half-life, where K573P and K500A showed increased and decreased half-life, respectively. Notably, whereas WT HSA showed almost a similar half-life as K500A in WT mice, it demonstrated 2-fold longer half-life in the hFcRn Tg mice. This is likely to be due to the fact that this mouse strain heavily overexpresses the receptor, which results in more available FcRn that can engage in binding of injected HSA in the presence of competing MSA. Nevertheless, the K573P variant demonstrated nearly 50% increase in serum persistence, and these encouraging results prompted us to address the half-life of K573P in cynomolgus monkeys. Here it was revealed that the single substitution extended the half-life by 3.4 days (5.4–8.8 days). Thus, it is expected that the effect will be at least as great in humans.
      We have previously shown that swapping of DIII derived from MSA onto DI-DII of HSA gave rise to a hybrid albumin with considerably improved binding toward mFcRn, whereas swapping of DIII of HSA onto DI-DII of MSA reduced binding (
      • Andersen J.T.
      • Cameron J.
      • Plumridge A.
      • Evans L.
      • Sleep D.
      • Sandlie I.
      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.
      ). Further, we swapped the stretch of amino acids corresponding to the C-terminal α-helix of MSA onto HSA (HSA-Cm), which resulted in a 4-fold improved binding compared with WT HSA (
      • Andersen J.T.
      • Cameron J.
      • Plumridge A.
      • Evans L.
      • Sleep D.
      • Sandlie I.
      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.
      ). To our knowledge, this was the first HSA variant reported to have improved pH-dependent binding toward hFcRn. HSA-Cm harbors eight amino acid changes compared with the WT HSA, one of which is K573P. By introducing only K573P into HSA, the binding affinity to mFcRn was improved by more than 20-fold, demonstrating the importance of a proline at this position for optimal binding to mFcRn, and it partly explains why WT HSA binds the mouse receptor poorly.
      Furthermore, we showed that a lysine at position 573, as found in WT HSA, is the residue that gives the lowest affinity toward hFcRn, because replacement of this charged residue with any other amino acid improves binding at pH 6.0. Some variants also showed low, but detectable binding at pH 7.4. As of now, one cannot easily predict how this will affect the half-life of variants with very strong binding at acidic pH. Moreover, the fact that all species have a proline at position 573, except human and orangutan, is an interesting observation. A proline at this position in the context of the endogenous HSA would result in extended half-life beyond 19–20 days. This would not necessarily have been favorable, because albumin serves as a carrier, not only for nutrients, but also toxins and waste products such as heme and bilirubin (
      • Zunszain P.A.
      • Ghuman J.
      • Komatsu T.
      • Tsuchida E.
      • Curry S.
      Crystal structural analysis of human serum albumin complexed with hemin and fatty acid.
      ,
      • Zunszain P.A.
      • Ghuman J.
      • McDonagh A.F.
      • Curry S.
      Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IXα.
      ); however, in a therapeutic context, extended half-life is desirable.
      Interestingly, K573E, which showed 2.4-fold improved binding to hFcRn, also exists as a very rare mutation in humans, first identified in a homozygote male (
      • Vanzetti G.
      • Porta F.
      • Prencipe L.
      • Scherini A.
      • Fraccaro M.
      A homozygote for a serum albumin variant of the fast type.
      ). It is expected that this variant has a longer serum half-life in such individuals. However, no reports exist of pathogenic manifestations.
      HSA is increasingly utilized as a versatile carrier for therapeutic and diagnostic agents (
      • Elsadek B.
      • Kratz F.
      Impact of albumin on drug delivery: new applications on the horizon.
      ,
      • Sleep D.
      • Cameron J.
      • Evans L.R.
      Albumin as a versatile platform for drug half-life extension.
      ,
      • Andersen J.T.
      • Sandlie I.
      The versatile MHC class I-related FcRn protects IgG and albumin from degradation: implications for development of new diagnostics and therapeutics.
      ). This technology allows for genetic fusion to HSA by fusion to the N- or C-terminal end of a protein of interest (Albufuse® technology), or alternatively, by chemical conjugation to a free cysteine residue within DI (Cys-34). These strategies have been broadly applied for half-life extension of small protein and chemical drugs (reviewed in Refs.
      • Elsadek B.
      • Kratz F.
      Impact of albumin on drug delivery: new applications on the horizon.
      ,
      • Sleep D.
      • Cameron J.
      • Evans L.R.
      Albumin as a versatile platform for drug half-life extension.
      , and
      • Andersen J.T.
      • Sandlie I.
      The versatile MHC class I-related FcRn protects IgG and albumin from degradation: implications for development of new diagnostics and therapeutics.
      ). Moreover, great benefits may be achieved by this technology for small proteins such as drugs for diabetes control, coagulation factors, hormones, or cytokines.
      Importantly, we recently showed that fusion of a peptide or an scFv fragment to either ends of HSA has only a minor negative impact on binding to the receptor, where the most pronounced effect was detected for C-terminal fusions, although still minor (
      • Andersen J.T.
      • Cameron J.
      • Plumridge A.
      • Evans L.
      • Sleep D.
      • Sandlie I.
      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.
      ). This may easily be compensated by introducing mutations, such as K573P, which will result in more than 12-fold better hFcRn binding at acidic pH than the WT counterpart that consequently will result in extended half-life. This may lead to superior pharmacokinetics tailored to disease state and improved patient compliance.

      Acknowledgments

      We are grateful to Sathiaruby Sivaganesh for excellent technical assistance and all our colleagues at the Novozymes Biopharma sites in UK and Denmark.

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