Chimaeric toxins have considerable therapeutic potential to treat various malignancies. We have previously used the fungal ribonucleolytic toxin restrictocin to make chimaeric toxins in which the ligand was fused at either the N-terminus or the C-terminus of the toxin. Chimaeric toxins containing ligand at the C-terminus of restrictocin were shown to be more active than those having ligand at the N-terminus of the toxin. Here we describe the further engineering of restrictocin-based chimaeric toxins, anti-TFR(scFv)-restrictocin and restrictocin-anti-TFR(scFv), containing restrictocin and a single chain fragment variable (scFv) of a monoclonal antibody directed at the human transferrin receptor (TFR), to enhance their cell-killing activity. To promote the independent folding of the two proteins in the chimaeric toxin, a linear flexible peptide, Gly-Gly-Gly-Gly-Ser, was inserted between the toxin and the ligand to generate restrictocin-linker-anti-TFR(scFv) and anti-TFR(scFv)-linker-restrictocin. A 12-residue spacer, Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu, containing the recognition site for the protease furin, was incorporated between the toxin and the ligand to generate restrictocin-spacer-anti-TFR(scFv) and anti-TFR(scFv)-spacer-restrictocin. The incorporation of the proteolytically cleavable spacer enhanced the cell-killing activity of both constructs by 2-30-fold depending on the target cell line. However, the introduction of linker improved the cytotoxic activity only for anti-TFR(scFv)-linker-restrictocin. The proteolytically cleavable spacer-containing chimaeric toxins had similar cytotoxic activities irrespective of the location of the ligand on the toxin and they were found to release the restrictocin fragment efficiently on proteolysis in vitro.
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January 2000
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Research Article|
January 10 2000
Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins
Anita GOYAL;
Anita GOYAL
1Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi-110067, India
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Janendra K. BATRA
Janendra K. BATRA
1
1To whom correspondence should be addressed (e-mail janendra@;nii.res.in or jkbatra@;yahoo.com).
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Publisher: Portland Press Ltd
Received:
July 12 1999
Revision Received:
September 30 1999
Accepted:
October 27 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 345 (2): 247–254.
Article history
Received:
July 12 1999
Revision Received:
September 30 1999
Accepted:
October 27 1999
Citation
Anita GOYAL, Janendra K. BATRA; Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins. Biochem J 15 January 2000; 345 (2): 247–254. doi: https://doi.org/10.1042/bj3450247
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