Ribonucleases and immunoRNases as anticancer drugs.

Ribonucleases degrade RNA, now considered an important drug target. The parent member of this protein superfamily is bovine pancreatic RNase A that functions as a digestive enzyme. Other physiological roles and activities have been ascribed to more recently discovered members of this superfamily. Angiogenin was isolated by following angiogenic activity from cell culture media conditioned by colon cancer cells. ONCONASE kills tumor cells in vitro and in vivo and has advanced to a phase IIIb confirmatory clinical trial for the treatment of unresectable malignant mesothelioma. All three of these RNA degrading enzymes have been used to generate immunoRNases; chemical conjugates and ligand-RNase fusion proteins, for cancer therapy. The properties of each of these RNases are described along with the increasingly sophisticated construction of recombinant immunoRNases. The advantages of using RNase as an antibody payload is compared to using plant or bacterial toxins in the construction of immunotoxins, a related strategy for specifically killing malignant cells.

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme.

Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V(H)-71, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 37 degrees C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUC1(+) tumour cells with an IC(50) of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1(+) malignancies.