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

ABSTRACT

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.

KEYWORDS:

• monoclonal antibodies
• deep mutational scanning
• yeast surface display
• epitope mapping
• cross-species reactivity
• LAMP-1

Abbreviations

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

Acknowledgments

The authors wish to acknowledge Fabienne Soubrier, Cecile Capdevila, Francis Duffieux, Alain Dupuy and Alexey Rakfor the key contributions they made to this work. LAMP-1 proteins and domains as well as Fab B were cloned by FS, produced by CC, and purified by FD (Sanofi, LMR, France). The authors also thank Raphaël Sierocki (Deeptope SAS) for help with the NGS data analysis scripts and useful discussions about the epitope mapping processes.

Disclosure statement

TP, MM and EV are Sanofi employees and may hold shares and/or stock options in the company. The authors have no additional conflict of interest.

Author contribution

TP, MM and SD contributed to data collection; TP, MM and HN contributed to data analysis; data interpretation was performed by TP, HN, MM, EV and BM; and BM, EV and HN contributed to the writing and design of the study.

All authors have approved the final version of this manuscript and agreed both to be personally accountable for their contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even that in which the author was not personally involved, are appropriately investigated, and resolved and the resolution documented in the literature.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2175311

Additional information

Funding

This work was financially supported by Sanofi (Collaboration agreement Sanofi/CEA). TP was supported by a CIFRE fellowship (No. 2018/0802) funded in part by ANRT (National Association for Research and Technology) on behalf of the French Ministry of Education and Research and in part by Sanofi.