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

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Onconase, a ribonuclease A superfamily member from Rana pipiens, shows antitumor activity by digesting tRNA and inducing apoptosis.
• Clinical use of onconase is limited by inefficient tumor cell targeting and non-specific toxicity to normal tissues.
• Recombinant onconase production in Pichia pastoris is challenging due to N-terminal glutamine cyclization and requires optimized secretion strategies.

Purpose of the Study:

• To develop a constitutively expressed, highly secreted, and targeted onconase variant (Onc-DV3) in Pichia pastoris.
• To enhance the delivery of onconase to tumor cells and improve its therapeutic index.
• To evaluate the efficacy and specificity of Onc-DV3 against various metastatic tumor cells.

Main Methods:

• Constructed a constitutive expression vector using the GAP promoter in Pichia pastoris.
• Inserted the onconase-(DV3)2 (Onc-DV3) gene downstream of the truncated Saccharomyces cerevisiae α-mating factor-pre (α-MF-pre) secretion signal.
• Assessed recombinant protein production, bioactivity, tumor cell delivery, and cytotoxicity in vitro.

Main Results:

• Achieved high-level production and bioactivity of the immuno-RNase Onc-DV3.
• Demonstrated enhanced capability of Onc-DV3 to deliver the onconase molecule to tumor cell counterparts.
• Exhibited strong cytotoxicity to highly metastatic tumor cells, weak cytotoxicity to lowly metastatic tumor cells, and no toxicity to normal cells.

Conclusions:

• Onc-DV3, engineered with two copies of DV3 for targeted delivery, shows specific toxicity towards highly metastatic tumor cells.
• This targeted approach significantly improves the therapeutic potential of onconase.
• Onc-DV3 represents a promising antitumor drug candidate with an improved safety and efficacy profile.