Cloning and cytotoxicity of a human pancreatic RNase immunofusion

Immunotechnology. 1997 Jun;3(2):127-36. doi: 10.1016/s1380-2933(97)00070-5.


Background: Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins.

Objectives: The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines.

Study design: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity.

Results: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate.

Conclusion: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.

MeSH terms

  • Animals
  • Cell-Free System / chemistry
  • Cloning, Molecular
  • Cytotoxicity, Immunologic*
  • Genes
  • Humans
  • Immunoglobulin Fragments / chemistry
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Variable Region / chemistry
  • Immunoglobulin Variable Region / genetics
  • Immunotoxins / genetics
  • Immunotoxins / immunology*
  • Immunotoxins / toxicity*
  • Mice
  • Plasmids / chemical synthesis
  • Polymerase Chain Reaction
  • Protein Synthesis Inhibitors / pharmacology
  • Receptors, Transferrin / genetics
  • Receptors, Transferrin / immunology
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology*
  • Recombinant Fusion Proteins / toxicity*
  • Ribonuclease, Pancreatic / genetics
  • Ribonuclease, Pancreatic / immunology*
  • Ribonuclease, Pancreatic / toxicity*
  • Tumor Cells, Cultured


  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Immunotoxins
  • Protein Synthesis Inhibitors
  • Receptors, Transferrin
  • Recombinant Fusion Proteins
  • Ribonuclease, Pancreatic