Galloway Memorial Lecture. Protein engineering of tumor necrosis factor-beta and its applications in cancer, septicaemia and cachexia

The tumour necrosis factors (TNFs) are cytokines, small proteins produced by cells as part of the intercellular signalling network. The exact physiological role of TNFs is unknown, but interest in them focused on three of their capabilities: as potential anti-tumour agents, as humoral mediators of an organism's response to injury and as effector molecules in cachexia. The first TNF to be described, now known as TNF-alpha, has been relatively well studied because the recombinant protein was easily produced since it was cloned in 1984. Studies on TNF-beta or lymphotoxin were hampered by the inability of most groups to express the recombinant protein. This paper describes the expression and purification of recombinant TNF-beta in Escherichia coli, followed by studies to localise the receptor binding site of the molecule through site-directed mutagenesis. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid-50 or tyrosine-108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. This provides evidence for a new hypothesis that both the TNFs bind to their receptors as trimers, each of which is capable of binding simultaneously to three receptors. This leads further to the intriguing possibility of a new mechanism of receptor clustering through simultaneous binding to a single ligand.