Tumor necrosis factor analogs: identification of functional domains

Truncated tumor necrosis factor analogs were produced by making in vitro deletions of the coding sequence of the human TNF gene. One of these analogs, TNF-desA7, lacked seven amino terminal residues of the mature TNF protein and the other two analogs, TNF-desC7 and TNF-desC2, had deletions of seven and two carboxy terminal amino acids, respectively. While the deletion of the first seven amino acid residues did not affect the biological activity of the protein, the deletions of carboxy terminal residues in both analogs resulted in the complete loss of biological activity. A direct correlation of the biological activity of the TNF protein and its binding to neutralizing monoclonal antibodies was observed. The carboxy terminal-deleted TNF analogs, with no biological activity, did not bind to neutralizing monoclonal antibodies while the amino terminal-deleted analog, which retained complete biological activity, did bind. These results indicate that the carboxy terminal amino acids of the TNF protein are essential for TNF biological activity and are part of an epitope recognized by neutralizing monoclonal antibodies.