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Benchmarking glycoform-resolved affinity separation – mass spectrometry assays for studying FcγRIIIa binding: mass spectrometry assays for studying FcγRIIIa binding

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Titel: Benchmarking glycoform-resolved affinity separation – mass spectrometry assays for studying FcγRIIIa binding: mass spectrometry assays for studying FcγRIIIa binding
Autoren: Gstöttner, C., Lippold, S., Hook, M., Yang, F., Haberger, M., Wuhrer, M., Falck, D., Schlothauer, T., Domínguez-Vega, E.
Quelle: Front Immunol
Frontiers in Immunology, Vol 15 (2024)
Verlagsinformationen: Frontiers Media SA, 2024.
Publikationsjahr: 2024
Schlagwörter: 0301 basic medicine, FcγRIIIA receptor, 0303 health sciences, glycosylation, Fc gamma RIIIA receptor, Immunology, Receptors, IgG, Antibodies, Monoclonal, RC581-607, affinity capillary electrophoresis, Mass Spectrometry, affinity chromatography, 03 medical and health sciences, monoclonal antibody, Polysaccharides, affinity interaction, Immunoglobulin G, Immunologic diseases. Allergy, Mannose, mass spectrometry
Beschreibung: The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan interaction with FcγRIIIa receptor, differences in antibody glycosylation can drastically influence the binding affinity. Understanding the differential binding of mAb glycoforms is a very important, yet challenging task due to the co-existence of multiple glycoforms in a sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated with mass spectrometry (MS) can provide glycoform-resolved affinity profiles of proteins based on their differences in either dissociation (AC) or equilibrium (ACE) constants. To cross-validate the affinity ranking provided by these complementary novel approaches, both techniques were benchmarked using the same FcγRIIIa constructs. Both approaches were able to assess the mAb – FcγRIIIa interaction in a glycoform selective manner and showed a clear increase in binding for fully versus hemi-fucosylated mAbs. Also, other features, such as increasing affinity with elevated galactosylation or the binding affinity for high mannose glycoforms were consistent. We further applied these approaches to assess the binding towards the F158 allotype of FcγRIIIa, which was not reported before. The FcγRIIIa F158 allotype showed a very similar profile compared to the V158 receptor with the strongest increase in binding due to afucosylation and only a slight increase in binding with additional galactosylation. Both techniques showed a decrease of the binding affinity for high mannose glycoforms for FcγRIIIa F158 compared to the V158 variant. Overall, both approaches provided very comparable results in line with orthogonal methods proving the capabilities of separation-based affinity approaches to study FcγR binding of antibody glycoforms.
Publikationsart: Article
Other literature type
Dateibeschreibung: application/pdf
ISSN: 1664-3224
DOI: 10.3389/fimmu.2024.1347871
Zugangs-URL: https://pubmed.ncbi.nlm.nih.gov/38469305
https://doaj.org/article/17823215efd24dc59d0603caae56befe
https://hdl.handle.net/1887/4175766
Rights: CC BY
Dokumentencode: edsair.doi.dedup.....d6d5ed7f502d142a9c1e1ffa6410f4e1
Datenbank: OpenAIRE
Beschreibung
Abstract:The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan interaction with FcγRIIIa receptor, differences in antibody glycosylation can drastically influence the binding affinity. Understanding the differential binding of mAb glycoforms is a very important, yet challenging task due to the co-existence of multiple glycoforms in a sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated with mass spectrometry (MS) can provide glycoform-resolved affinity profiles of proteins based on their differences in either dissociation (AC) or equilibrium (ACE) constants. To cross-validate the affinity ranking provided by these complementary novel approaches, both techniques were benchmarked using the same FcγRIIIa constructs. Both approaches were able to assess the mAb – FcγRIIIa interaction in a glycoform selective manner and showed a clear increase in binding for fully versus hemi-fucosylated mAbs. Also, other features, such as increasing affinity with elevated galactosylation or the binding affinity for high mannose glycoforms were consistent. We further applied these approaches to assess the binding towards the F158 allotype of FcγRIIIa, which was not reported before. The FcγRIIIa F158 allotype showed a very similar profile compared to the V158 receptor with the strongest increase in binding due to afucosylation and only a slight increase in binding with additional galactosylation. Both techniques showed a decrease of the binding affinity for high mannose glycoforms for FcγRIIIa F158 compared to the V158 variant. Overall, both approaches provided very comparable results in line with orthogonal methods proving the capabilities of separation-based affinity approaches to study FcγR binding of antibody glycoforms.
ISSN:16643224
DOI:10.3389/fimmu.2024.1347871