Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen.

TitleSingle domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen.
Publication TypeJournal Article
Year of Publication2004
AuthorsSaerens, D., J. Kinne, E. Bosmans, U. Wernery, S. Muyldermans, and K. Conrath
JournalJ Biol Chem
Volume279
Issue50
Pagination51965-72
Date Published2004 Dec 10
ISSN0021-9258
KeywordsAmino Acid Sequence, Animals, Antibodies, Monoclonal, Camels, Female, Humans, Hybridomas, Immunoglobulin Heavy Chains, Immunoglobulin Variable Region, Lymph Nodes, Lymphocytes, Male, Molecular Sequence Data, Prostate-Specific Antigen, Protein Conformation, Protein Isoforms, Sequence Homology, Amino Acid
Abstract

The importance of the lymphocyte source to generate hybridomas or to construct antibody gene libraries from which to identify potent monoclonal antibodies is understudied. However, the few comparative studies that exist seem to favor the lymph node tissue as a B-cell source. Here the peripheral blood and lymph node lymphocytes of a dromedary immunized with prostate-specific antigen (PSA) have been employed to clone two independent gene banks of the variable domains of heavy-chain antibodies (i.e. the VHHs). Several PSA-specific VHHs were retrieved after panning of these phage-displayed VHH libraries. Some of them were derived from the same B-cell lineage, possibly reflecting the restricted primary repertoire of heavy-chain antibodies. Other binders originated from different B-cell lineages and apparently converged toward a striking homologous amino acid sequence motif in their CDR3. This illustrates the strong somatic hypermutation and stringent antigen-driven selection ongoing in these animals. Although the various antigen binders exhibit a broad range of kinetic rate constants for their interaction with the PSA, leading to equilibrium constants from 70 pM to 100 nM, no significant difference existed between the binders from the two B-cell sources. The VHHs of both libraries were categorized in three groups based on nonoverlapping epitopes. Some of these VHHs could inhibit and others could enhance the proteolytic activity of the antigen. Remarkably, VHHs seem to sense or induce conformational changes on different PSA isoforms, a feature that might be exploited to study the PSA conformational flexibility and to discriminate the stages of prostate cancer.

DOI10.1074/jbc.M409292200
Alternate JournalJ. Biol. Chem.
PubMed ID15459193