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
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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. / Sheff, Joey; Kelly, John; Foss, Mary; Brunette, Eric; Kemmerich, Kristin; van Faassen, Henk; Raphael, Shalini; Hussack, Greg; Comamala, Gerard; Rand, Kasper; Stanimirovic, Danica B.
In: Journal of Biochemistry, Vol. 173, No. 2, 2023, p. 95-105.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - 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
AU - Sheff, Joey
AU - Kelly, John
AU - Foss, Mary
AU - Brunette, Eric
AU - Kemmerich, Kristin
AU - van Faassen, Henk
AU - Raphael, Shalini
AU - Hussack, Greg
AU - Comamala, Gerard
AU - Rand, Kasper
AU - Stanimirovic, Danica B.
N1 - Publisher Copyright: © The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - blood–brain barrier
KW - epitope mapping
KW - hydrogen–deuterium exchange mass spectrometry
KW - IGF1R
KW - single-domain antibody
U2 - 10.1093/jb/mvac088
DO - 10.1093/jb/mvac088
M3 - Journal article
C2 - 36346120
AN - SCOPUS:85147389824
VL - 173
SP - 95
EP - 105
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 2
ER -
ID: 336124894