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

Tiphanie Pruvost (1) , Magali Mathieu (2) , Steven Dubois (1) , Bernard Maillère (1) , Emmanuelle Vigne (2) , Hervé Nozach (1, 3)

1 SIMoS - Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO)
2 Sanofi [Vitry-sur-Seine]
3 MTS - Médicaments et Technologies pour la Santé

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.

Mots clés: 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; 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

Domaines: Sciences du Vivant [q-bio]

Tiphanie Pruvost, Magali Mathieu, Steven Dubois, Bernard Maillère, Emmanuelle Vigne, et al.. Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and AlphaFold. mAbs, 2023, 15 (1), pp.2175311. ⟨10.1080/19420862.2023.2175311⟩. ⟨cea-03993824⟩