Abstract
Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day.
Highlights
A new method using comprehensive mutagenesis libraries, yeast display, and deep sequencing is proposed to determine fine conformational epitopes for three antibody-antigen interactions
Three distinct populations are collected: an unselected population of cells that passed through a cell-size gate, a displayed population of cells passing through the previous gate as well as a gate confirming display of the C-terminal c-myc epitope tag, and a binding population of cells satisfying these two previous gates as well as a gate on the fluorescence channel associated with antibody binding
Mutation of Gln-67 to aromatic residue increased binding affinity for the infliximab interaction (Fig. 2a). Based on this comprehensive mutagenesis dataset enabled by deep sequencing, we conclude that this improved yeast display-deep sequencing pipeline is effective in identifying fine conformational epitopes for antibody-antigen interactions
Summary
A new method using comprehensive mutagenesis libraries, yeast display, and deep sequencing is proposed to determine fine conformational epitopes for three antibody-antigen interactions. Full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions We show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. Yeast surface display [22] coupled to deep mutational scanning [23] was used to understand the sequence effects of binding for nearly every single point mutant for two computationally designed proteins targeting a conserved epitope on influenza hemagglutinin [7] This method was used to confirm the paratope for both small proteins, as validated by crystal structures. Because additional inefficiencies exist at several stages in the deep sequencing and analysis workflow, we asked whether we could simplify the yeast display-deep sequencing pipeline to increase the method throughput, reduce cost, and improve the ability to resolve complete conformational epitopes for full-length proteins
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