Tumor necrosis factor mutants with selective cytotoxic activity.

Tumor necrosis factor (TNF-alpha) has a cytotoxic or cytostatic effect when tested with various malignant cell lines. Clinical trials in cancer patients, however, revealed high systemic toxicity of TNF-alpha. The existence of two types of receptor may partially explain the pleiotropic activity of TNF-alpha.

[Interaction of anti-TNF-alpha monoclonal antibodies E7H2 with mutant and hybrid proteins].

A number of hybrid proteins containing amino acid sequences of human lymphotoxin and human and mouse tumor necrosis factors were obtained by means of genetic engineering. By using these proteins, an antigenic determinant in the molecule of human tumor necrosis factor for monoclonal antibodies E7H2 (MAb E7H2), previously developed against this factor was localized. It was demonstrated by Western blot analysis that the MAb E7H2-binding site is located in the sequence region 37-49 of human tumor necrosis factor and includes the sequence Val41GluLeuArg44 directly interacting with MAb E7H2.

Determination of binding site of anti-tumour necrosis factor-alpha monoclonal antibody using hybrid and mutant proteins.

In order to map the immunogenic epitope for the monoclonal antibody E7H2 on the human tumour necrosis factor (hTNF-alpha) molecule, a number of chimeric proteins were developed by in-frame joining segments of the human genes encoding TNF-alpha and lymphotoxin (TNF-beta) as well as by coupling appropriate coding regions for human and mouse TNF-alpha. High level expression of these chimeric genes was achieved in Escherichia coli by placing the coding sequences under control of either E. coli trp-promoter or a tandem of bacteriophage T7 constitutive promoters A2 and A3.

[Human tumor necrosis factor mutants: preparation and some properties].

Using polymerase chain reaction, a number of mutant genes encoding human tumor necrosis factor (TNF-alpha) with amino acid substitutions and a deletion were obtained. The mutant proteins (muteins) contained point mutations R32H, A33S, F144L, I118M, and I118A; double mutation R32H-F144L; and deletion of four amino acid residues 67-70. The mutant genes were expressed in E.