Extending serum half-life of albumin by engineering neonatal Fc receptor (FcRn) binding

Abstract

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.

Keywords: Albumin; Animal Models; Antibody Engineering; Biodegradation; Bioengineering; FC Receptors; Pharmacokinetics; pH Regulation.

FIGURE 1.

Amino acid substitutions in the last C-terminal end of HSA DIII modulate binding to hFcRn. A, an illustration of the crystal structure of full-length HSA with the three domains DI (pink), DII (orange), and DIII (cyan/blue) highlighted. The DIII is split into subdomains DIIIa (cyan) and DIIIb (blue). B, a close-up of DIIIb and the last α-helix in the C-terminal end with the amino acid residues Lys-573, Lys-574, and Gln-580 indicated. The figures were made using the software PyMOL, using coordinates from Protein Data Bank entry 1bm0. C–E, representative SPR sensorgrams showing binding of 1 μm of WT HSA, K574A, and Q580A (C); WT HSA and K573A (D); and WT HSA and HSA K573X (E) to immobilized hFcRn at pH 6.0. Injections were performed at 25 °C, and the flow rate was set to 40 μl/min. The kinetic rate constants were obtained using a simple first order (1:1) bimolecular interaction model (Langmuir) or a steady state affinity model supplied by the BIAevaluation 4.1 software. The kinetic values represent the average of triplicates.

FIGURE 2.

Considerably improved pH-dependent binding to hFcRn of a HSA variant containing K573P. A, a ClustalW amino acid sequence alignment of the last C-terminal α-helix of albumin derived from 20 species. The amino acid residues at positions 573, 574, and 580 are indicated. A lysine or a proline at position 573 is highlighted in blue or red, respectively. B, a representative SPR sensorgrams showing binding of 1 μm of K573P to immobilized hFcRn at pH 6.0 and 7.4. Injections were performed at 25 °C, and the flow rate was 40 μl/min. The kinetic rate constants were obtained using a Langmuir binding model supplied by the BIAevaluation 4.1 software. The kinetic values represent the averages of triplicates.

FIGURE 3.

A panel of single point HSA variants with improved binding to hFcRn. A, WT HSA and HSA variants were produced in S. cerevisiae and subsequently purified on an albumin affinity matrix before analysis by 4–12% (w/v) SDS-PAGE. B–S, representative sensorgrams showing binding of titrated amounts of WT HSA and 573 variants to immobilized hFcRn at pH 5.5. Injections were performed at 25 °C, and the flow rate was 40 μl/min. The kinetic rate constants were obtained using a Langmuir binding model supplied by the BIAevaluation 4.1 software. The kinetic values represent the averages of triplicates.

FIGURE 4.

HSA K573P shows improved binding to mFcRn. A–C, representative sensorgrams showing binding of titrated amounts of WT MSA (A, 10–0.03 μm), WT HSA (B, 100–1.4 μm), and HSA K573P (C, 10–0.03 μm) to immobilized mFcRn at pH 6.0. D–F, representative sensorgrams showing binding of titrated amounts of WT CSA (D, 10–0.03 μm), WT HSA (E, 10–0.03 μm), and HSA K573P (F, 10–0.03 μm) to immobilized cynomolgus monkey FcRn (cmFcRn) at pH 6.0. All injections were performed at 25 °C, and the flow rate was 40 μl/min. The kinetic rate constants were obtained using a Langmuir binding model or a steady state affinity model supplied by the BIAevaluation 4.1 software. The kinetic values represent the averages of triplicates.

FIGURE 5.

HSA K573P shows extended serum half-life in mice and cynomolgus monkeys. A and B, log-linear changes in serum concentrations of WT HSA, K500A, and K573P in WT NMRI mice (A, 18 mice/group, 6 mice/time point) and FcRn^−/− hFcRn Tg32 mice (B, 10 mice/group, 5 mice/time point). HSA variants were administered by intravenous infusions at 10 mg/kg, followed by collecting serum samples from the tail vein before the serum HSA concentrations were determined using an AlphaLISA immunoassay. C, log-linear changes in serum concentrations of WT HSA and K573P in cynomolgus monkeys (2 monkeys/group). HSA variants were administered by intravenous infusions at 1 mg/kg, followed by collecting serum samples before the HSA concentrations were determined using an anti-c-Myc ELISA. The results are means ± standard errors.

FIGURE 6.

Engineering of HSA does not affect binding to IgG. A, ELISA showing binding of hFcRn-GST to titrated amounts of human IgG1 (10–0.07 nm) in the absence or presence of 1000 nm of HSA, MSA, K500A, or K573P. The ELISA was performed at pH 6.0. The numbers given represent the mean of triplicates. B and C, ELISA quantification of the serum concentrations of total mouse IgG WT mice (B) and hFcRn Tg mice (C) before and after injections (0, 24, and 72 h) of WT HSA, K500A, and K573P.

FIGURE 7.

Binding of HSA scFv fusions to hFcRn and a structural overview of the C-terminal end of HSA in complex with hFcRn. A and B, representative sensorgrams showing binding of 1 μm of scFv-WT HSA, scFv-K500A, and scFv-K573P (A) and WT HSA-scFv, K500A-scFv, and K573P-scFv (B) to immobilized hFcRn at pH 6.0. Injections were performed at 25 °C, and the flow rate was 40 μl/min. C, an overview of the crystal structure complex between recombinant hFcRn and a HSA variant with four point substitutions (Protein Data Bank entry 4k71). The three HSA domains, DI, DII, and DIII, are shown in pink, orange, and cyan/blue, respectively. DIII is split into subdomains DIIIa (cyan) and DIIIb (blue). The HC of the hFcRn is shown in green, whereas the β2m subunit is shown in gray. The last α-helix of DIIIb is indicated by an arrow. D, a close-up of the structural area of the complex showing the last α-helix of DIIIb and the interaction of Lys-573 with Glu-69 and Ser-20 of the β2m unit. The neighboring hydrophobic Phe-22 is also indicated. The figures were made using the software PyMOL.