Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins.
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.
Goyal
A
A
Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi-110067, India.
Batra
J K
JK
D000485:Allergens; D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D052179:Antigens, Plant; D002460:Cell Line; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D045683:Furin; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000494:pharmacology*; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011990:Receptors, Transferrin / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013381:Subtilisins / Q000494:pharmacology*; D018675:Toxicity Tests; D014357:Trypsin / Q000494:pharmacology
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D000903:Antibiotics, Antineoplastic; D052179:Antigens, Plant; D005656:Fungal Proteins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D009842:Oligopeptides; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; C036196:MITF protein, Aspergillus restrictus; D012260:Ribonucleases; D013381:Subtilisins; D014357:Trypsin; D045683:Furin