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Biotechnology and Applied Biochemistry Mar 2020DAB IL-2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a...
DAB IL-2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a cutaneous form of T-cell lymphoma. This fusion protein has two disulfide bonds in its structure that play an essential role in toxicity and functionality of the immunotoxin. Escherichia coli (E. coli) strain BL21 (DE3) is not capable of making disulfide bonds in its reductive cytoplasm, but the E. coli strain Rosetta-gami (DE3) is a proper strain for the correct expression of the protein due to mutations in glutaredoxin reductase and thioredoxin reductase. In this study, a pET21a vector with the His6-tag fused at the N-terminus of DAB IL-2 was used to express the soluble immunotoxin in E. coli Rosetta-gami (DE3). After the purification of the soluble protein by two-step column chromatographies, the structure of DAB IL-2 was analyzed using the Native-PAGE and circular dichroism methods. In the following, the nuclease activity of soluble DAB IL-2 and its cytotoxicity activity were determined. It is concluded that the soluble recombinant protein expressed in the E. coli Rosetta-gami (DE3) has an intact structure and also functional; hence, this form of immunotoxin could be competitive with its commercial counterparts.
Topics: Antineoplastic Agents; Diphtheria Toxin; Escherichia coli; Interleukin-2; Protein Domains; Recombinant Fusion Proteins; Solubility
PubMed: 31600001
DOI: 10.1002/bab.1833 -
Immunotherapy Sep 2019T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The... (Review)
Review
T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and exotoxin A-both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.
Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Clinical Trials as Topic; Diphtheria Toxin; Drug Evaluation, Preclinical; Exotoxins; Humans; Immunity, Cellular; Immunotherapy; Lymphocyte Depletion; Neoplasms; T-Lymphocytes, Regulatory; Tumor Microenvironment; Virulence Factors; Pseudomonas aeruginosa Exotoxin A
PubMed: 31361167
DOI: 10.2217/imt-2019-0060