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.

Combining deep mutational scanning to heatmap of HLA class II binding of immunogenic sequences to preserve functionality and mitigate predicted immunogenicity

“…This makes it possible to evaluate very quickly and systematically the impact of all possible substitutions in a protein-protein interface. The applications of DMS are very diverse, ranging from epitope mapping (45), through optimization of the affinity of monoclonal antibodies (7), to the evaluation of antigen mutations leading to immune escape, for example for viruses (46,47). Here, we are searching for affinityneutral mutations, similar to the concept of neutral drift in protein evolution, i.e.…”