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Published on: April 3, 2018
Developing novel oncolytic adenoviruses through bioselection
Wen Yan1, Galila Kitzes, Farid Dormishian
1ONYX Pharmaceuticals, Inc., 3031 Research Drive, Richmond, CA 94806, USA.
Abstract:
Mutants of human adenovirus 5 (Ad5) with enhanced oncolytic activity were isolated by using a procedure termed bioselection. Two mutants, ONYX-201 and ONYX-203, were plaque purified from a pool of randomly mutagenized Ad5 that was repeatedly passaged in the human colorectal cancer cell line HT29, and they were subsequently characterized. ONYX-201 and ONYX-203 replicated more rapidly in HT29 cells than wild-type Ad5, and they lysed HT29 cells up to 1,000-fold more efficiently. The difference was most profound when cells were infected at a relatively low multiplicity of infection, presumably due to the compounding effects of multiple rounds of infection. This enhanced cytolytic activity was observed not only in HT29 cells but also in many other human cancer cell lines tested. In contrast, the cytotoxicity of the bioselected mutants in a number of normal primary human cells was similar to that of wild-type Ad5, thus enhancing the therapeutic index (cytotoxicity in tumor cells versus that in normal cells) of these oncolytic agents. Both ONYX-201 and -203 contain seven single-base-pair mutations when compared with Ad5, four of which were common between ONYX-201 and -203. The mutation at nucleotide 8350, shared by both mutant viruses, was shown to be essential for the observed phenotypes. This mutation was mapped to the i-leader region of the major late transcription unit, resulting in the truncation of 21 amino acids from the C terminus of the i-leader protein. This work demonstrates that bioselection is a powerful tool for developing novel tumor-selective oncolytic viruses. Other potential applications of this technology are discussed.
Insights
Bioselection created novel human adenovirus 5 (Ad5) mutants, ONYX-201 and ONYX-203, with significantly enhanced oncolytic activity against cancer cells but not normal cells. A specific mutation in the i-leader region proved crucial for this tumor selectivity.
Area of Science:
- Virology
- Oncology
- Molecular Biology
Background:
- Human adenovirus 5 (Ad5) is a potential oncolytic virus for cancer therapy.
- Developing tumor-selective oncolytic viruses with enhanced efficacy is crucial for improving cancer treatment outcomes.
Purpose of the Study:
- To isolate and characterize novel Ad5 mutants with improved oncolytic activity and tumor selectivity using bioselection.
- To identify the genetic basis for the enhanced oncolytic properties of the selected mutants.
Main Methods:
- Bioselection of Ad5 mutants by serial passaging in human colorectal cancer cells (HT29).
- Plaque purification and characterization of ONYX-201 and ONYX-203 mutants.
- Comparative analysis of viral replication, cell lysis, and cytotoxicity in tumor and normal cells.
- Genetic sequencing to identify mutations and mapping of critical mutations.
Main Results:
- Two Ad5 mutants, ONYX-201 and ONYX-203, exhibited significantly enhanced replication and cytolytic activity (up to 1,000-fold) in HT29 and other cancer cell lines compared to wild-type Ad5.
- The enhanced oncolytic activity was observed at low multiplicities of infection, suggesting efficient viral spread.
- Cytotoxicity in normal primary human cells remained similar to wild-type Ad5, leading to an improved therapeutic index.
- Both mutants shared seven single-base-pair mutations, with a key mutation at nucleotide 8350 in the i-leader region responsible for truncating the i-leader protein.
Conclusions:
- Bioselection is an effective strategy for generating tumor-selective oncolytic Ad5 viruses.
- The mutation at nucleotide 8350 is essential for the enhanced oncolytic phenotype and tumor selectivity of ONYX-201 and ONYX-203.
- These findings support the potential of bioselected Ad5 mutants as novel oncolytic agents for cancer therapy.
