A novel tumor necrosis factor‐α mutant with significantly enhanced cytotoxicity and receptor binding affinity

Abstract

A novel tumor necrosis factor-u, mutant (mutant M3), in which Ser and Tyr at positions 52 and 56 were substituted by Ile and Phe, respectively, along with deletion of 7 N-terminal amino acids, was prepared and its biological activities were investigated. The mutant exhibited a 14-to 24-fold increase in the cytotoxicity relative to the wildtype TNF on various cancer cell lines. The binding affinity of the mutant to TNF-R55 and TNF-R75 receptors was over 10-fold higher than that of the wild-type. TNF-c~ and the mutant show similar CD spectra in the far-UV region, indicating that the overall structure was not influenced by the mutations. The production of highly potent TNF-c~ mutant utilizing increase of hydrophobicity in the region 52-56 may provide a structural basis for a design of optimized TNF-ct as a therapeutic purpose.

Development of human tumor necrosis factor-α muteins with improved therapeutic potential

“…As shown previously by our laboratory (17), the most dramatic increase in cytotoxic activity arise from the deletion of 7 N-terminal amino acid residues in region 1 and mutations in region 2 (mutein R2-3). Further mutations either in regions 1 and/or 4 in addition to the mutations in region 2 resulted in the decrease of cytotoxic activities.…”

“…Acute lethal toxicities of wild-type TNF-α and selected muteins in D-galactosamine-sensitized ICR mice at positions 52 and 56 on the bottom region of the TNF-α trimer. This mutein showed a high cytotoxic activity towards various human tumor cells and exhibited anticancer activities against transplanted human tumors in nude mice (16)(17)(18). The diverse activities of TNF are mediated by binding to each of the two receptors, TNF-R55 (55 kDa) and TNF-R75 (75 kDa), on the cell surface.…”

Potential for Using Histidine Tags in Purification of Proteins at Large Scale

“…For efficient isolation of recombinant proteins histidine tags offer numerous advantages but in the case of pharmaceutically useful proteins, the authentic structure is usually compulsory, therefore, histidine tags if employed for purification purposes must be removed for final usage. Typical example is a high-level production of human growth hormone in the form of a fusion protein with a His10-tag and the enterokinase recognition site included to enable the enzymatic cleavage and thus generation of the authentic structure [51]. On the other hand, histidine-tagged antibodies as such, without tag removal, have been described as useful for diagnostic purposes [52] and for cost-effective production of biosensors, in which histidine tags serve for oriented immobilization [53].…”

Targeting immune cell types of tumor microenvironment to overcome resistance to PD-1/PD-L1 blockade in lung cancer

“…A mutated human tumor necrosis factor alpha (TNF-α) has been reported to improve the therapeutic index in the mouse fibroblast cell line L929 and mice ( Yan et al, 2006 ). Similarly, TNF-α mutant was also found to promote cytotoxicity and receptor binding affinity ( Shin et al, 1998 ). Pharmacokinetics of the recombinant mutated human TNF-α (rmhTNF-α) displayed that rmhTNF-α has a low systemic toxicity and high anticancer ability ( Li et al, 2010 ).…”