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J Proteome Res. 2017 Jan 6;16(1):45-54. doi: 10.1021/acs.jproteome.6b00608. Epub 2016 Nov 2.

De Novo MS/MS Sequencing of Native Human Antibodies.

Author information

1
Mapp Biopharmaceutical, Inc. , 6160 Lusk Boulevard #C105, San Diego, California 92121, United States.
2
Department of Proteomics & Biological Resources, Genentech, Inc. , South San Francisco, California 94080, United States.
3
Department of Protein Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.
4
Department of Antibody Engineering, Genentech, Inc. , South San Francisco, California 94080, United States.
5
Department of Molecular Biology, Genentech, Inc. , South San Francisco, California 94080, United States.
6
Department of Computer Science and Engineering, University of California, San Diego , 9500 Gilman Drive, Mail Code 0404, La Jolla, California 92093, United States.
7
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego , 9500 Gilman Drive, Mail Code 0657, La Jolla, California 92093, United States.

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.

KEYWORDS:

B cell; antibody discovery; de novo sequencing; mass spectrometry; monoclonal antibody; polyclonal antibody; shotgun protein sequencing

PMID:
27779884
PMCID:
PMC5574256
DOI:
10.1021/acs.jproteome.6b00608
[Indexed for MEDLINE]
Free PMC Article

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