title: Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.
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.
authors: From the Department of Chemical Engineering and Materials Science.
authors: Caitlin A
authors: CA
authors: Kowalsky
authors: Department of Biochemistry and Molecular Biology.
authors: Matthew S
authors: MS
authors: Faber
authors: Genetics Graduate Program.
authors: Aritro
authors: A
authors: Nath
authors: From the Department of Chemical Engineering and Materials Science.
authors: Hailey E
authors: HE
authors: Dann
authors: From the Department of Chemical Engineering and Materials Science.
authors: Vince W
authors: VW
authors: Kelly
authors: Department of Microbiology and Molecular Genetics, and.
authors: Li
authors: L
authors: Liu
authors: From the Department of Chemical Engineering and Materials Science.
authors: Purva
authors: P
authors: Shanker
authors: the Department of Chemical Engineering, University of Texas, Austin, Texas 78712.
authors: Ellen K
authors: EK
authors: Wagner
authors: the Department of Chemical Engineering, University of Texas, Austin, Texas 78712.
authors: Jennifer A
authors: JA
authors: Maynard
authors: From the Department of Chemical Engineering and Materials Science, Department of Biochemistry and Molecular Biology, Genetics Graduate Program.
authors: Christina
authors: C
authors: Chan
authors: From the Department of Chemical Engineering and Materials Science, Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, Michigan 48824 and taw@egr.msu.edu.
authors: Timothy A
authors: TA
authors: Whitehead
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016296:Mutagenesis; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000939:Epitopes; C499380:TACSTD2 protein, human; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha
keywords: Bordetella pertussis; TROP2; antibody; antibody engineering; conformational epitope mapping; epitope mapping; protein-protein interaction; tumor necrosis factor (TNF)