Cellular recycling-driven in vivo half-life extension using recombinant albumin fusions tuned for neonatal Fc receptor (FcRn) engagement

Larsen, Mette Bach; Rawsthorne, Helen; Schelde, Karen Kræmmer; Dagnæs‐Hansen, Frederik; Cameron, Jason; Howard, Kenneth A.

doi:10.1016/j.jconrel.2018.07.023

Cited by 33 publications

(32 citation statements)

References 37 publications

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“…The circulatory half-life of the 3xAF680-conjugated triplethiol HBII rHSA variant was increased to 22.4 h in this present work, which is a 1.3-fold change compared with 3xAF680-conjugated triple-thiol WT rHSA. This is comparable with the 1.65-fold change observed between WT and HBII rHSA fusions in the work of Larsen et al (16).…”

Section: Tablesupporting

confidence: 91%

“…The K93C and E294C mutations were combined with null FcRn-binding (NB) (3, 9) and a high FcRn-binding hFcRn HBII variant described previously (16). The thiol variants were conjugated with Alexa Fluor 680 (AF680) dye, and the labeling efficiency was determined by the ratio of absorbance of the fluorophore at 680 nm and albumin at 280 nm.…”

Section: Conjugation With Alexa Fluor 680mentioning

confidence: 99%

“…The K93C and E294C mutations were further introduced into rHSA variants either engineered for null-FcRn binding (NB; H464Q/ K500A) or for high FcRn binding (HBII; E492G/K573P/K574H/ Q580K). The NB rHSA variant contains the K500A mutation combined with a key histidine residue known to reduce FcRn binding (9), where the HBII rHSA variant is a combination variant comprising K573P and additional mutated positions from domain III in regions that have previously been shown to impact FcRn binding (3,9) and exhibit a long circulatory halflife (16). Neither of the triple-thiol NB rHSA variants (with or without 3xAF680) exhibited hFcRn binding, whereas the triplethiol HBII rHSA variant (with or without 3xAF680) bound with Ͼ10-fold higher affinity to hFcRn compared with triple-thiol WT rHSA variants (with or without 3xAF680).…”

Section: Tablementioning

confidence: 99%

“…S. cerevisiae transformations and albumin purifications were performed as described above. The triple-thiol rHSA variant was further modified to create expression cassettes for proteins that also exhibit modified hFcRn-binding affinity: NB, HBI, and HBII, described previously (16). S. cerevisiae transformations and albumin purification using AlbuPure matrix and diethylaminoethyl-Sepharose Fast Flow matrix (GE Healthcare) were performed as described previously (9,25).…”

Section: Combining Thiol Variants With Rhsa Variants With Different Hmentioning

confidence: 99%

“…This present work aims to increase the conjugation capacity by site-selective introduction of additional thiol groups into a range of rHSA variants with different FcRn-binding affinities described previously (3,15,16).…”

mentioning

confidence: 99%

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

Schelde

Nicholls

Dagnæs‐Hansen

et al. 2019

Journal of Biological Chemistry

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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 M for WT albumin with or without 3xAF680 respectively, and 0.04 and 0.02 M 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 (t1 ⁄ 2 ‫؍‬ 22.4 h) compared with its WT counterpart (t1 ⁄ 2 ‫؍‬ 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.

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Section: Tablesupporting

confidence: 91%

Section: Conjugation With Alexa Fluor 680mentioning

confidence: 99%

Section: Tablementioning

confidence: 99%

Section: Combining Thiol Variants With Rhsa Variants With Different Hmentioning

confidence: 99%

mentioning

confidence: 99%

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

Schelde

Nicholls

Dagnæs‐Hansen

et al. 2019

Journal of Biological Chemistry

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Albumin-Binding Fatty Acid–Modified Gapmer Antisense Oligonucleotides for Modulation of Pharmacokinetics

Cai

Lou

Wengel

et al. 2020

Methods in Molecular Biology

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Prolonged circulation and modulation of the pharmacokinetic profile are important to improve the clinical potential of antisense oligonucleotides (ASOs). Gapmer ASOs demonstrate excellent nuclease stability and robust gene silencing activity without the requirement of transfection agents. A major challenge for in vivo applications, however, is the short blood circulatory half-life. This work describes utilisationutilization of the long circulation of serum albumin to increase the blood residence time of gapmer ASO. The method introduces fatty acid modifications into the gapmer ASOs design to exploit the binding and transport property of serum albumin for endogenous ligands. The level of albumin-/gapmer ASOs interaction, blood circulatory half-life and biodistribution was dependent on number, position, and fatty acid type (palmitic or myristic acid) within the gapmer ASO sequence and either phosphorothioate or phosphodiester backbone modifications. This work offers a strategy to optimiseoptimize gapmer ASO pharmacokinetics by a proposed endogenous assembly process with serum albumin that can be tuned by gapmer ASO design modifications.

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Albumin: An Emerging Opportunity in Drug Delivery

Rahimizadeh

Yang

Lim

2020

Biotechnol Bioproc E

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Cellular recycling-driven in vivo half-life extension using recombinant albumin fusions tuned for neonatal Fc receptor (FcRn) engagement

Cited by 33 publications

References 37 publications

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

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

Albumin-Binding Fatty Acid–Modified Gapmer Antisense Oligonucleotides for Modulation of Pharmacokinetics

Albumin: An Emerging Opportunity in Drug Delivery

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