title: De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody.
abstract: Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent.
authors.affiliation: Department of Biomolecular Engineering, University of California Santa Cruz , 1156 High Street, Santa Cruz, California 95064, United States.
authors.forename: Walter A
authors.initials: WA
authors.lastname: Bogdanoff
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine , New York, New York 10016, United States.
authors.forename: David
authors.initials: D
authors.lastname: Morgenstern
authors.affiliation: Protein Metrics , San Carlos, California 94070, United States.
authors.forename: Marshall
authors.initials: M
authors.lastname: Bern
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine , New York, New York 10016, United States.
authors.forename: Beatrix M
authors.initials: BM
authors.lastname: Ueberheide
authors.affiliation: Viral Gastroenteritis Unit, Centro Nacional de Microbiología, Instituto de Salud Carlos III , Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Sanchez-Fauquier
authors.affiliation: Department of Biomolecular Engineering, University of California Santa Cruz , 1156 High Street, Santa Cruz, California 95064, United States.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: DuBois
keywords: X-ray crystallography; antibody; astrovirus; mass spectrometry; protein engineering; protein sequencing