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

To create a human therapeutic vaccine able to circumvent self-tolerance against tumor necrosis factor (TNF) α, foreign T helper epitopes were inserted into human TNFα, with minimal effect on the native three-dimensional structure. TNFα variants were screened for solubility, structural stability, biological activity, and after immunization, for eliciting inhibitory antibodies. The longest and most flexible loop in TNFα, 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 TNFα molecule. Therefore, two cysteines were introduced to form a stabilizing disulfide bond between loops 2 and 3. In a second design approach, three TNFα monomers were linked by two T cell epitopes and expressed as a single chain TNFα trimer. TNFα variants that were expressed as soluble proteins also had a conserved tertiary structure, as determined by circular dichroism. The biological activity of the TNFα variants was of the same magnitude as human TNFα 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 TNFα. Antisera from mice immunized with the different TNFα variants were able to cross-react with native human TNFα and to inhibit TNFα signaling via the TNF receptors in vitro , suggesting that the structural binding epitopes of native human TNFα and thus the native conformation were conserved in the constructed vaccine candidates.