scholarly journals Construction, expression and characterization of chimaeric toxins containing the ribonucleolytic toxin restrictocin: intracellular mechanism of action

1997 ◽  
Vol 324 (3) ◽  
pp. 815-822 ◽  
Author(s):  
Dharmendar RATHORE ◽  
Janendra K. BATRA
Keyword(s):  
Transferrin Receptor ◽  
Metabolic Inhibitors ◽  
Target Cells ◽  
Cytotoxic Activities ◽  
Dna Encoding ◽  
Single Chain ◽  
Human Transferrin ◽  
Human Transferrin Receptor ◽  
A Cell

Restrictocin is a ribonucleolytic toxin produced by the fungus Aspergillus restrictus. Two chimaeric toxins containing restrictocin directed at the human transferrin receptor have been constructed. Anti-TFR(scFv)–restrictocin is encoded by a gene produced by fusing the DNA encoding a single-chain antigen-combining region (scFv) of a monoclonal antibody, directed at the human transferrin receptor, at the 5′ end of that encoding restrictocin. The other chimaeric toxin, restrictocin–anti-TFR(scFv), is encoded by a gene fusion containing the DNA encoding the single-chain antigen-combining region of antibody to human transferrin receptor at the 3′ end of the DNA encoding restrictocin. These gene fusions were expressed in Escherichia coli, and fusion proteins purified from the inclusion bodies by simple chromatography techniques to near-homogeneity. The two chimaeric toxins were found to be equally active in inhibiting protein synthesis in a cell-free in vitrotranslation assay system. The chimaeric toxins were selectively toxic to the target cells in culture with potent cytotoxic activities. However, restrictocin–anti-TFR(scFv) was more active than anti-TFR(scFv)–restrictocin on all cell lines studied. By using protease and metabolic inhibitors, it can be shown that, to manifest their cytotoxic activity, the restrictocin-containing chimaeric toxins need to be proteolytically processed intracellularly and the free toxin or a fragment thereof thus generated is translocated to the target via a route involving the Golgi apparatus.

scholarly journals Single-chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv).

1991 ◽  
Vol 11 (4) ◽  
pp. 2200-2205 ◽  
Author(s):  
J K Batra ◽  
D J Fitzgerald ◽  
V K Chaudhary ◽  
I Pastan
Keyword(s):  
Transferrin Receptor ◽  
Diphtheria Toxin ◽  
Gene Fusion ◽  
Antigen Binding ◽  
Dna Encoding ◽  
Single Chain ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A ◽  
Human Transferrin ◽  
Human Transferrin Receptor

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.

Single-chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv)

1991 ◽  
Vol 11 (4) ◽  
pp. 2200-2205
Author(s):  
J K Batra ◽  
D J Fitzgerald ◽  
V K Chaudhary ◽  
I Pastan
Keyword(s):  
Transferrin Receptor ◽  
Diphtheria Toxin ◽  
Gene Fusion ◽  
Antigen Binding ◽  
Dna Encoding ◽  
Single Chain ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A ◽  
Human Transferrin ◽  
Human Transferrin Receptor

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.

Structural, functional, and tissue distribution analysis of human transferrin receptor-2 by murine monoclonal antibodies and a polyclonal antiserum

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3782-3789 ◽  
Author(s):  
Silvia Deaglio ◽  
Andrea Capobianco ◽  
Angelita Calı̀ ◽  
Francesca Bellora ◽  
Federica Alberti ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Distribution ◽  
Transferrin Receptor ◽  
Polyclonal Antiserum ◽  
Target Cells ◽  
Distribution Analysis ◽  
Human Transferrin ◽  
Normal Tissues ◽  
Human Transferrin Receptor ◽  
Transferrin Receptor 2

Human transferrin receptor-2 (TFR-2) is a protein highly homologous to TFR-1/CD71 and is endowed with the ability to bind transferrin (TF) with low affinity. High levels of TFR-2 mRNA were found in the liver and in erythroid precursors. Mutations affecting the TFR-2gene led to hemochromatosis type 3, a form of inherited iron overload. Several issues on distribution and function of the receptor were answered by raising a panel of 9 monoclonal antibodies specific for TFR-2 by immunizing mice with murine fibroblasts transfected with the human TFR-2 cDNA. A polyclonal antiserum was also produced in mice immunized with 3 peptides derived from the TFR-2 sequence, exploiting an innovative technique. The specificity of all the reagents produced was confirmed by reactivity with TFR-2+ target cells and simultaneous negativity with TFR-1+ cells. Western blot analyses showed a dominant chain of approximately 90 kDa in TFR-2 transfectants and HepG2 cell line. Analysis of distribution in normal tissues and in representative cell lines revealed that TFR-2 displays a restricted expression pattern—it is present at high levels in hepatocytes and in the epithelial cells of the small intestine, including the duodenal crypts. Exposure of human TFR-2+cells to TF-bound iron is followed by a significant up-regulation and relocalization of membrane TFR-2. The tissue distribution pattern, the behavior following exposure to iron-loaded TF, and the features of the disease resulting from TFR-2 inactivation support the hypothesis that TFR-2 contributes to body iron sensing.

scholarly journals A DNA Aptameric Ligand of Human Transferrin Receptor Generated by Cell-SELEX

2021 ◽  
Vol 22 (16) ◽  
pp. 8923
Author(s):  
Nan Zhang ◽  
Tao Bing ◽  
Luyao Shen ◽  
Le Feng ◽  
Xiangjun Liu ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Human Tumor ◽  
Dna Aptamer ◽  
Live Cells ◽  
Human Tumor Cells ◽  
Human Transferrin ◽  
High Affinity ◽  
Human Transferrin Receptor ◽  
A Cell ◽  
Promising Ligand

General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.

scholarly journals Structural model of phospholipid-reconstituted human transferrin receptor derived by electron microscopy

Structure ◽  
1998 ◽  
Vol 6 (10) ◽  
pp. 1235-1243 ◽  
Author(s):  
Hendrik Fuchs ◽  
Uwe Lücken ◽  
Rudolf Tauber ◽  
Andreas Engel ◽  
Reinhard Geßner
Keyword(s):  
Electron Microscopy ◽  
Transferrin Receptor ◽  
Structural Model ◽  
Human Transferrin ◽  
Human Transferrin Receptor
Sign in / Sign up

Export Citation Format

Share Document