Epitope mapping of a blood-brain barrier crossing antibody targeting the cysteine-rich region of IGF1R using hydrogen-exchange mass spectrometry enabled by electrochemical reduction

Joey Sheff*, John Kelly, Mary Foss, Eric Brunette, Kristin Kemmerich, Henk van Faassen, Shalini Raphael, Greg Hussack, Gerard Comamala, Kasper Rand, Danica B. Stanimirovic

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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.

Original languageEnglish
JournalJournal of Biochemistry
Volume173
Issue number2
Pages (from-to)95-105
ISSN0021-924X
DOIs
Publication statusPublished - 2023

Keywords

  • blood–brain barrier
  • epitope mapping
  • hydrogen–deuterium exchange mass spectrometry
  • IGF1R
  • single-domain antibody

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