title: De Novo MS/MS Sequencing of Native Human Antibodies.
abstract: One direct route for the discovery of therapeutic human monoclonal antibodies (mAbs) involves the isolation of peripheral B cells from survivors/sero-positive individuals after exposure to an infectious reagent or disease etiology, followed by single-cell sequencing or hybridoma generation. Peripheral B cells, however, are not always easy to obtain and represent only a small percentage of the total B-cell population across all bodily tissues. Although it has been demonstrated that tandem mass spectrometry (MS/MS) techniques can interrogate the full polyclonal antibody (pAb) response to an antigen in vivo, all current approaches identify MS/MS spectra against databases derived from genetic sequencing of B cells from the same patient. In this proof-of-concept study, we demonstrate the feasibility of a novel MS/MS antibody discovery approach in which only serum antibodies are required without the need for sequencing of genetic material. Peripheral pAbs from a cytomegalovirus-exposed individual were purified by glycoprotein B antigen affinity and de novo sequenced from MS/MS data. Purely MS-derived mAbs were then manufactured in mammalian cells to validate potency via antigen-binding ELISA. Interestingly, we found that these mAbs accounted for 1 to 2% of total donor IgG but were not detected in parallel sequencing of memory B cells from the same patient.
authors: Mapp Biopharmaceutical, Inc. , 6160 Lusk Boulevard #C105, San Diego, California 92121, United States.
authors: Adrian
authors: A
authors: Guthals
authors: Department of Proteomics & Biological Resources, Genentech, Inc. , South San Francisco, California 94080, United States.
authors: Yutian
authors: Y
authors: Gan
authors: Department of Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
authors: Laura
authors: L
authors: Murray
authors: Department of Antibody Engineering, Genentech, Inc. , South San Francisco, California 94080, United States.
authors: Yongmei
authors: Y
authors: Chen
authors: Department of Molecular Biology, Genentech, Inc. , South San Francisco, California 94080, United States.
authors: Jeremy
authors: J
authors: Stinson
authors: Department of Antibody Engineering, Genentech, Inc. , South San Francisco, California 94080, United States.
authors: Gerald
authors: G
authors: Nakamura
authors: Department of Proteomics & Biological Resources, Genentech, Inc. , South San Francisco, California 94080, United States.
authors: Jennie R
authors: JR
authors: Lill
authors: Department of Proteomics & Biological Resources, Genentech, Inc. , South San Francisco, California 94080, United States.
authors: Wendy
authors: W
authors: Sandoval
authors: Department of Computer Science and Engineering, University of California, San Diego , 9500 Gilman Drive, Mail Code 0404, La Jolla, California 92093, United States.|Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego , 9500 Gilman Drive, Mail Code 0657, La Jolla, California 92093, United States.
authors: Nuno
authors: N
authors: Bandeira
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000917:Antibody Formation; D000921:Antibody-Producing Cells / Q000166:cytology / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology* / Q000821:virology; D002846:Chromatography, Affinity / Q000379:methods; D003587:Cytomegalovirus / Q000254:growth & development; D003586:Cytomegalovirus Infections / Q000097:blood / Q000276:immunology* / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007106:Immune Sera; D007074:Immunoglobulin G; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus
keywords: B cell; antibody discovery; de novo sequencing; mass spectrometry; monoclonal antibody; polyclonal antibody; shotgun protein sequencing