| DOI | Resolve DOI: https://doi.org/10.1096/fj.201903231R |
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| Author | Search for: Van Faassen, Henk1; Search for: Ryan, Shannon1; Search for: Henry, Kevin A.1; Search for: Raphael, Shalini; Search for: Yang, Qingling1; Search for: Rossotti, Martin A.1; Search for: Brunette, Eric1; Search for: Jiang, Susan1; Search for: Haqqani, Arsalan S.1; Search for: Sulea, Traian1; Search for: Mackenzie, C. Roger1; Search for: Tanha, Jamshid1; Search for: Hussack, Greg1 |
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| Affiliation | - National Research Council of Canada. Human Health Therapeutics
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| Format | Text, Article |
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| Subject | antibody; half-life; nanobody; pH sensitivity; serum albumin; single-domain antibody |
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| Abstract | Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VHHs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VHH (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VHHs maintained in vivo half-life extension capabilities. These VHHs represent a new set of tools for extending protein therapeutic half-life and one (M75) demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life. |
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| Publication date | 2020-04-02 |
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| Publisher | Federation of American Society of Experimental Biology |
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| In | |
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| Language | English |
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| Peer reviewed | Yes |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | 26a0c373-baa2-446e-8926-ec1dba38983c |
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| Record created | 2020-06-19 |
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| Record modified | 2020-06-29 |
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