Constitutive expression and anticancer potency of a novel immunotoxin onconase-DV3

Onconase is an RNase of the ribonuclease A super family that is purified from the Northern leopard frog (Rana pipiens). It targets several types of malignant tumors, digests cytoplasmic transfer RNA (tRNA), and causes tumor cell apoptosis. Onconase has been employed in clinical trials as an antitumor drug, and has revealed its valuable clinical activity in several types of tumors, particularly pleural mesothelioma. However, its inefficiency in targeting tumor cells and its non-specific toxicity in normal tissues have diminished its clinical benefits. Furthermore, cyclization of the N-terminal glutamine residue (Gin), possesses more RNase activity than the structure of Met ahead of Glu in the N-terminal (99:1), which is more difficult for producing onconase by Pichia pastoris. Under the guidance of alpha-mating factor-pre (alpha-MF-pre) secretion signal, the secretion of the recombinant protein can reach a high level. In the present study, we constructed a constitutive expression vector for onconase-(DV3)(2), (Onc-DV3) production in yeast Pichia pastoris with the GAP promoter, in which the Onc-DV3 gene is inserted downstream of the truncated Saccharomyces cerevisiae alpha-mating factor-pre (alpha-MF-pre) secretion signal. The immuno-RNase Onc-DV3 expressed a high level of production and bioactivity and possessed enhanced capability to deliver the Onc molecule to tumor cell monomeric counterparts. Notably, Onc-DV3 showed strong cytotoxicity to highly metastatic tumor cells, weak cytotoxicity to lowly metastatic tumor cells and no toxicity to normal cells. These results demonstrate that the specific toxicity to highly metastatic tumor cells has made Onc-DV3 a promising antitumor drug by using two copies of DV3 for the targeted delivery of onconase.