Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping.
Journal
Communications biology
ISSN: 2399-3642
Titre abrégé: Commun Biol
Pays: England
ID NLM: 101719179
Informations de publication
Date de publication:
03 Jul 2024
03 Jul 2024
Historique:
received:
29
11
2023
accepted:
25
06
2024
medline:
4
7
2024
pubmed:
4
7
2024
entrez:
3
7
2024
Statut:
epublish
Résumé
Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.
Identifiants
pubmed: 38961245
doi: 10.1038/s42003-024-06508-8
pii: 10.1038/s42003-024-06508-8
doi:
Substances chimiques
Epitopes
0
Spike Glycoprotein, Coronavirus
0
Peptide Library
0
spike protein, SARS-CoV-2
0
Antibodies, Viral
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
805Subventions
Organisme : VINNOVA (Swedish Governmental Agency for Innovation Systems)
ID : 2016-05181
Organisme : VINNOVA (Swedish Governmental Agency for Innovation Systems)
ID : 2021-02640
Organisme : VINNOVA (Swedish Governmental Agency for Innovation Systems)
ID : 2019-00103
Organisme : VINNOVA (Swedish Governmental Agency for Innovation Systems)
ID : 2017-02105
Organisme : Stiftelsen för Strategisk Forskning (Swedish Foundation for Strategic Research)
ID : SB16-0017
Informations de copyright
© 2024. The Author(s).
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