Generation and Characterization of Native and Sialic Acid-Deficient IgE

Autor:	Alex J. McCraw, Richard A. Gardner, Anna M. Davies, Daniel I. R. Spencer, Melanie Grandits, Gerd K. Wagner, James M. McDonnell, Sophia N. Karagiannis, Alicia Chenoweth, Silvia Crescioli
Rok vydání:	2022
Předmět:	AllergoOncology Receptors IgE - metabolism neuraminidase Receptors Fc Catalysis Inorganic Chemistry Antigens Neoplasm Animals antibodies Humans Physical and Theoretical Chemistry Molecular Biology IgE glycosylation Spectroscopy Receptors IgE Organic Chemistry General Medicine Immunoglobulin E allergy N-Acetylneuraminic Acid Rats Computer Science Applications glyco-engineered IgE immunoglobulin E IgE IgE purification sialic acid Chromatography Gel
Zdroj:	International Journal of Molecular Sciences; Volume 23; Issue 21; Pages: 13455 McCraw, A J, Gardner, R A, Davies, A M, Spencer, D I R, Grandits, M, Wagner, G K, McDonnell, J M, Karagiannis, S N, Chenoweth, A & Crescioli, S 2022, ' Generation and characterization of native and sialic acid-deficient IgE ', International Journal of Molecular Sciences, vol. 23, no. 21, 13455 . https://doi.org/10.3390/ijms232113455
ISSN:	1422-0067
DOI:	10.3390/ijms232113455
Popis:	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.
Databáze:	OpenAIRE
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