Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins.
Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia coli (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.
Deonarain
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MP
Imperial Cancer Research Fund Oncology Unit, Imperial College School of Medicine at the Hammersmith Hospital, London, UK.
Epenetos
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AA
D000469:Alkaline Phosphatase / Q000276:immunology; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use; D008222:Lymphokines / Q000737:chemistry* / Q000235:genetics; D010920:Placenta / Q000201:enzymology; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000235:genetics; D014407:Tumor Cells, Cultured
C089422:Fv protein, human; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D008222:Lymphokines; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; C055324:phospholipase A2-activating protein; D004722:Endoribonucleases; C032523:ribonuclease SPL; D000469:Alkaline Phosphatase