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J. Biol. Chem., Vol. 279, Issue 32, 33593-33600, August 6, 2004

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Insertion of Foreign T Cell Epitopes in Human Tumor Necrosis Factor with Minimal Effect on Protein Structure and Biological Activity^*

Finn Stausholm Nielsen, Jørgen Sauer¶, Johan Bäcklund, Bjørn Voldborg, Klaus Gregorius||, Søren Mouritsen, and Tomas Bratt

From the Pharmexa A/S, Kogle Allé 6, DK-2970 Hørsholm, Denmark, the ^¶ALK-Abelló A/S Bøge Allé 6-8, DK-2970 Hørsholm, Denmark, and ^||PreciSense A/S Dr. Neergaards Vej 3, DK-2970 Hørsholm, Denmark

To create a human therapeutic vaccine able to circumvent self-tolerance against tumor necrosis factor (TNF) , foreign T helper epitopes were inserted into human TNF, with minimal effect on the native three-dimensional structure. TNF variants were screened for solubility, structural stability, biological activity, and after immunization, for eliciting inhibitory antibodies. The longest and most flexible loop in TNF, also designated loop 3, is the only region that is not involved in intra- or intermolecular interactions and therefore constitute an attractive insertion site. However, the extension of the flexible loop by epitope insertions destabilized the TNF molecule. Therefore, two cysteines were introduced to form a stabilizing disulfide bond between loops 2 and 3. In a second design approach, three TNF monomers were linked by two T cell epitopes and expressed as a single chain TNF trimer. TNF variants that were expressed as soluble proteins also had a conserved tertiary structure, as determined by circular dichroism. The biological activity of the TNF variants was of the same magnitude as human TNF in cellular assays. Introduction of three separate single-point mutations (D143N, A145R, or Y87S) diminished the cytotoxicity of the mutated variants 50-800-fold compared with native TNF. Antisera from mice immunized with the different TNF variants were able to cross-react with native human TNF and to inhibit TNF signaling via the TNF receptors in vitro, suggesting that the structural binding epitopes of native human TNF and thus the native conformation were conserved in the constructed vaccine candidates.

Received for publication, March 19, 2004 , and in revised form, May 26, 2004.

^* 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.

To whom correspondence should be addressed. Tel.: 45-45-16-25-25; Fax: 45-45-16-25-00; E-mail: fn{at}pharmexa.com.

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