Immunotoxins are targeted therapeutics designed to kill cancer cells. The targeting moiety of an immunotoxin selectively binds to a tumor cell and targets it for death via an attached toxin. Because the toxins are typically of plant or bacterial origin, their clinical use is limited by immunogenicity and nonspecific toxicity. To circumvent these problems, we have begun to engineer immunotoxins containing human pancreatic ribonuclease. Here we describe the generation of ribonuclease mutants designed to evade a ubiquitous cytosolic inhibitor that would otherwise block cytotoxicity. Two mutants retained catalytic activity and were relatively resistant to the inhibitor. To deliver them to human T leukemic cells, these ribonuclease variants were fused to a single chain Fv fragment specific for CD7. The ribonuclease-sFv fusion proteins bound CD7+ T cells and were internalized yet were not cytotoxic. Transfection of the proteins directly into the cytosol reduced cell viability, suggesting that the failure of the immunotoxins to kill cells when added externally resulted from the inability of the ribonuclease moiety to access the cytosol efficiently. Our results indicate appropriate intracellular routing, as well as resistance to inhibition, is critical to the cytotoxicity of human ribonuclease-based immunotoxins.
Bibliographical noteFunding Information:
We thank John Kersey for the SEM-K2 cell line, John Lipscomb for helpful discussions regarding the enzyme kinetic assays, and Daniel Vallera for DA7 and the G89R RNase 1 construct. This work was supported in part by an NIH training grant (T32 CA09138) to H.A.E. and an NIH grant (R01 CA82766) to C.A.P.
- Cancer therapy
- Human RNase
- Ribonuclease inhibitor