Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and AlphaFold

• Published in: mAbs Vol. 15; no. 1; p. 2175311
• Main Authors: Pruvost, Tiphanie, Mathieu, Magali, Dubois, Steven, Maillère, Bernard, Vigne, Emmanuelle, Nozach, Hervé
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 2023; Taylor & Francis Group
• Subjects: Animals; Antibodies, Monoclonal; Antigens - metabolism; cross-species reactivity; deep mutational scanning; epitope mapping; Epitope Mapping - methods; Epitopes; LAMP-1; Life Sciences; Mice; monoclonal antibodies; Mutation; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; yeast surface display; LAMP-1; deep mutational scanning; yeast surface display; cross-species reactivity; epitope mapping; monoclonal antibodies; LAMP-1 DMS: Deep Mutational Scanning YSD: Yeast Surface Display NGS: next-generation sequencing cryo-EM: cryogenic electron microscopy BLI: biolayer interferometry FACS: Fluorescence-activated cell sorting CDRs: complementarity-determining regions HDX-MS: Hydrogen deuterium exchange mass spectrometry RMSD: root-mean-square deviation; Monoclonal antibodies; Monoclonal antibodies deep mutational scanning yeast surface display epitope mapping cross-species reactivity LAMP-1 DMS: Deep Mutational Scanning YSD: Yeast Surface Display NGS: next-generation sequencing cryo-EM: cryogenic electron microscopy BLI: biolayer interferometry FACS: Fluorescence-activated cell sorting CDRs: complementarity-determining regions HDX-MS: Hydrogen deuterium exchange mass spectrometry RMSD: root-mean-square deviation
• ISSN: 1942-0862; 1942-0870; 1942-0870
• DOI: 10.1080/19420862.2023.2175311

Delineating the precise regions on an antigen that are targeted by antibodies has become a key step for the development of antibody therapeutics. X-ray crystallography and cryogenic electron microscopy are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, they are labor-intensive and a successful outcome is not guaranteed. We used deep mutational scanning (DMS) of the human LAMP-1 antigen displayed on yeast surface and leveraged next-generation sequencing to observe the effect of individual mutants on the binding of two LAMP-1 antibodies and to determine their functional epitopes on LAMP-1. Fine-tuned epitope mapping by DMS approaches is augmented by knowledge of experimental antigen structure. As human LAMP-1 structure has not yet been solved, we used the AlphaFold predicted structure of the full-length protein to combine with DMS data and ultimately finely map antibody epitopes. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one of the two antibodies with a LAMP-1 luminal domain. Finally, we used AlphaFold models of non-human LAMP-1 to understand the lack of mAb cross-reactivity. While both epitopes in the murine form exhibit multiple mutations in comparison to human LAMP-1, only one and two mutations in the Macaca form suffice to hinder the recognition by mAb B and A, respectively. Altogether, this study promotes a new application of AlphaFold to speed up precision mapping of antibody-antigen interactions and consequently accelerate antibody engineering for optimization.