A new class of recombinant human albumin with multiple surface thiols exhibits stable conjugation and enhanced FcRn binding and blood circulation

Abstract

Human serum albumin is an endogenous ligand transport protein whose long circulatory half-life is facilitated by engagement with the human cellular recycling neonatal Fc receptor (hFcRn). The single free thiol located at Cys-34 in domain I of albumin has been exploited for monoconjugation of drugs. In this work, we increased the drug-to-albumin ratio potential by engineering recombinant human albumin (rHSA) variants with varying hFcRn affinity to contain three free, conjugation-competent cysteines. Structural analysis was used to identify positions for cysteine introduction to maximize rHSA stability and formation of the conjugated product without affecting hFcRn binding. The thiol rHSA variants exhibited up to 95% monomeric stability over 24 months and retained hFcRn engagement compared with a WT unconjugated control demonstrated by Biolayer Interferometry. The additional cysteines were further introduced into a panel of rHSA variants engineered with different affinities for hFcRn. After conjugation with three Alexa Fluor 680 (AF680) fluorophores, hFcRn binding was similar to that of the original triple-thiol nonconjugated rHSA variants (0.88 and 0.25 μm for WT albumin with or without 3xAF680 respectively, and 0.04 and 0.02 μm for a high hFcRn-binding variant with or without 3xAF680, respectively). We also observed a 1.3-fold increase in the blood circulatory half-life of a high hFcRn-binding triple-thiol variant conjugated with AF680 (t_½ = 22.4 h) compared with its WT counterpart (t_½ = 17.3 h) in mice. Potential high drug-to-albumin ratios combined with high hFcRn engagement are attractive features of this new class of albumins that offer a paradigm shift for albumin-based drug delivery.

Keywords: Fc receptor; albumin; cysteine-mediated cross-linking; drug delivery; multidrug transporter; multifunctional protein; neonatal Fc receptor; pharmacokinetics; protein engineering; site-directed mutagenesis.

Figure 1.

Rotated views of the crystal structure of the engineered triple-thiol human serum albumin in complex with the FcRn receptor. The β-2-microglobulin/FcRn complex is shown in gold, and human serum albumin is shown in red. The location of the naturally occurring cysteine (Cys-34) and the two positions where cysteines with reactive thiol groups have been inserted (K93C and E294C) are shown in dark blue and teal, respectively. Interface residues within a distance of 5 Å from albumin are marked in green on the surface representation of the FcRn receptor. Modified from Protein Data Bank entry 4N0F.

Figure 2.

Representative fitted binding affinity curves (black) from three replicates for triple-thiol rHSA variants binding to hFcRn from Biolayer Interferometry measurements. The two-fold dilution series from 3 to 0.1875 μm albumin is shown. Raw data curves are depicted in gray. The vertical dashed line indicates transition between association phase and dissociation phase. Top (left to right), standard WT HSA control (single-thiol), triple-thiol WT rHSA, and triple-thiol WT rHSA 3xAF680. Middle (left to right), triple-thiol NB rHSA and triple-thiol NB rHSA 3xAF680. Bottom (left to right), triple-thiol HBII rHSA and triple-thiol HBII rHSA 3xAF680.

Figure 3.

Plasma levels of fluorescently labeled 3xAF680-triple-thiol rHSA variants at different time points (elimination phase: 24–96 h). Fluorescence was measured using the IVIS scanner and normalized to fluorescence level at 1 min (not depicted). Graphs show mean fluorescence intensity (●, NB; ■, WT; ▴, HBII, n = 7) with fitted exponential curves. Error bars, S.D. (For 1 min to 96 h, see Fig. S4.)