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Generation and Characterization of Native and Sialic Acid-Deficient IgE

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Original languageEnglish
Article number13455
Number of pages22
JournalInternational Journal of Molecular Sciences
Volume23
Issue number21
DOIs
Accepted/In press28 Oct 2022
Published3 Nov 2022

Bibliographical note

Funding Information: S.N. Karagiannis is a founder and shareholder of Epsilogen Ltd. S.N. Karagiannis declares patents on IgE technologies. M. Grandits is funded by a grant by Epsilogen Ltd. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. D.I.R. Spencer & R.A. Gardner are employed by Ludger Ltd. a company that commercializes glycan analytics. All other authors have declared that no conflict of interest exists. Funding Information: The authors acknowledge support by Cancer Research UK (C30122/A11527; C30122/A15774); the Guy’s and St Thomas’ Foundation Trust Charity Melanoma Special Fund (SPF573); CR UK//NIHR in England/DoH for Scotland, Wales and Northern Ireland Experimental Cancer Medicine Centre (C10355/A15587); Breast Cancer Now (147; KCL-BCN-Q3); and the Medical Research Council (MR/L023091/1). A.J.M is supported by the UK Medical Research Council (MR/R015643/1) and King’s College London member of the MRC Doctoral Training Partnership in Biomedical Sciences. The research was supported by the National Institute for Health and Care Research Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London and the NIHR Clinical Research Facility (NIHR Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London, IS-BRC -1215-20006). The authors are solely responsible for the study design, data collection, analysis, decision to publish, and preparation of the manuscript. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health and Social Care. Publisher Copyright: © 2022 by the authors.

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Abstract

Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs.

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