| DOI | Resolve DOI: https://doi.org/10.1093/jb/mvac088 |
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| Author | Search for: Sheff, Joey1ORCID identifier: https://orcid.org/0000-0002-9269-6863; Search for: Kelly, John1; Search for: Foss, Mary1; Search for: Brunette, Eric1; Search for: Kemmerich, Kristin1; Search for: van Faassen, Henk1; Search for: Raphael, Shalini1; Search for: Hussack, Greg1; Search for: Comamala, Gerard; Search for: Rand, Kasper; Search for: Stanimirovic, Danica B.1 |
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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 | blood–brain barrier; epitope mapping; hydrogen–deuterium exchange mass spectrometry; IGF1R; single-domain antibody |
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| Abstract | Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood–brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen–deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics. |
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| Publication date | 2022-11-08 |
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| Publisher | Oxford Academic |
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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 | 69c3e110-3e98-49f5-96f6-669c4cfca754 |
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| Record created | 2023-09-14 |
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| Record modified | 2023-09-14 |
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