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J. Biol. Chem., Vol. 279, Issue 50, 51965-51972, December 10, 2004

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Single Domain Antibodies Derived from Dromedary Lymph Node and Peripheral Blood Lymphocytes Sensing Conformational Variants of Prostate-specific Antigen^*

Dirk Saerens, Jörg Kinne¶, Eugène Bosmans||**, Ulrich Wernery¶, Serge Muyldermans, and Katja Conrath

From the Laboratory of Cellular Immunology, Department of Cellular and Molecular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium, ^¶Central Veterinary Research Laboratory, Dubai, United Arab Emirates, and ^||DiaMed Eurogen NV, Hertoginstraat 82, 2300 Turnhout, Belgium

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.

Received for publication, August 13, 2004 , and in revised form, September 23, 2004.

^* This work was supported by a predoctoral grant from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Flanders) (to D. S.), by the European Union Project PAMELA IST-1999-13478, by the Flanders Interuniversity Institute for Biotechnology (Vlaams Interuniversitair Instituut voor Biotechnologie), and by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Present address: AML Lab, Desguinlei 88 bus 1, 2018 Antwerpen, Belgium.

To whom correspondence should be addressed. Tel.: 32-2-629-19-77; Fax: 32-2-629-19-81; E-mail: dsaerens{at}vub.ac.be.

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