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 (t½ = 22.4 h) compared with its WT counterpart (t½ = 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.
Highlights
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
Tel.: 45-87155831; E-mail: kenh@inano.au.dk. 3 The abbreviations used are: Human serum albumin (HSA), human serum albumin; rHSA, recombinant human albumin; MSA, murine serum albumin; FcRn, neonatal Fc receptor; hFcRn, human FcRn; mouse FcRn (mFcRn), murine FcRn; DAR, drug-to-albumin ratio; by multiple noncovalent binding sites for endogenous molecules such as fatty acids, steroids, and bilirubin [1] and a long circulatory half-life (ϳ19 days) predominately facilitated by engagement with the cellular recycling heterodimeric neonatal Fc receptor (FcRn) [2,3,4,5]. These properties promote albumin as an attractive drug delivery platform (6 –8) that has been utilized for long-acting drugs such as the marketed Levemir and Victoza (Novo Nordisk) that reversibly associate with hydrophobic binding pockets in endogenous HSA by inclusion of fatty acid chains in the drug design
The triple-thiol rHSA 3xAF680 fluorescence–labeled variants (NB, WT, and high FcRn binding (HBII)) were administered by intravenous injection into BALB/cAnNRj mice (n ϭ 7); blood samples were taken at 1 min, 30 min, 4 h, 24 h, 48 h, 72 h, and 96 h; and the level of fluorescence in blood detected by In Vivo Image System (IVIS) (PerkinElmer Life Sciences) bioimaging (Table 7, Fig. 3, and Fig. S4)
Summary
A new class of recombinant human albumin with multiple surface thiols exhibits stable conjugation and enhanced FcRn binding and blood circulation. C. are employees of Albumedix Ltd., who manufacture and supply albumin for pharmaceutical use This present work aims to increase the conjugation capacity by site-selective introduction of additional thiol groups into a AF680, Alexa Fluor 680; NB, null-FcRn binding affinity variant; HB, high FcRn– binding affinity variant; HBI, HB variant I; HBII, HB variant II; DI, DII, and DIII, domains I, II, and III, respectively; GP-HPLC, gel-permeation HPLC; UHPLC, ultrahigh-performance liquid chromatography; DTNB, 5,5-dithiobis-(2-nitrobenzoic acid); TNB, 5-thio-2-nitrobenzoic acid; ROI, region of interest; t1/2, half-life; KD, dissociation constant; BMMS, buffered minimal media sucrose. Difference shows actual measured mass minus theoretical mass. (For the complete sample set, see Table S2)
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