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

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

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

Harvard

Sheff, J, Kelly, J, Foss, M, Brunette, E, Kemmerich, K, van Faassen, H, Raphael, S, Hussack, G, Comamala, G, Rand, K & Stanimirovic, DB 2023, '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', Journal of Biochemistry, vol. 173, no. 2, pp. 95-105. https://doi.org/10.1093/jb/mvac088

APA

Sheff, J., Kelly, J., Foss, M., Brunette, E., Kemmerich, K., van Faassen, H., Raphael, S., Hussack, G., Comamala, G., Rand, K., & Stanimirovic, D. B. (2023). 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. Journal of Biochemistry, 173(2), 95-105. https://doi.org/10.1093/jb/mvac088

Vancouver

Sheff J, Kelly J, Foss M, Brunette E, Kemmerich K, van Faassen H et al. 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. Journal of Biochemistry. 2023;173(2):95-105. https://doi.org/10.1093/jb/mvac088

Author

Sheff, Joey ; Kelly, John ; Foss, Mary ; Brunette, Eric ; Kemmerich, Kristin ; van Faassen, Henk ; Raphael, Shalini ; Hussack, Greg ; Comamala, Gerard ; Rand, Kasper ; Stanimirovic, Danica B. / 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. In: Journal of Biochemistry. 2023 ; Vol. 173, No. 2. pp. 95-105.

Bibtex

@article{450f906d823146fb965dd23906bd8b2e,

title = "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",

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.",

keywords = "blood–brain barrier, epitope mapping, hydrogen–deuterium exchange mass spectrometry, IGF1R, single-domain antibody",

author = "Joey Sheff and John Kelly and Mary Foss and Eric Brunette and Kristin Kemmerich and {van Faassen}, Henk and Shalini Raphael and Greg Hussack and Gerard Comamala and Kasper Rand and Stanimirovic, {Danica B.}",

year = "2023",

doi = "10.1093/jb/mvac088",

language = "English",

volume = "173",

pages = "95--105",

journal = "Journal of Biochemistry",

issn = "0021-924X",

publisher = "Oxford University Press",

number = "2",

}

RIS

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.

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

Department of Pharmacy