Developing novel oncolytic adenoviruses through bioselection.
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.
Yan
Wen
W
ONYX Pharmaceuticals, Inc., 3031 Research Drive, Richmond, CA 94806, USA.
Kitzes
Galila
G
Dormishian
Farid
F
Hawkins
Lynda
L
Sampson-Johannes
Adam
A
Watanabe
Josh
J
Holt
Jenny
J
Lee
Vivian
V
Dubensky
Thomas
T
Fattaey
Ali
A
Hermiston
Terry
T
Balmain
Allan
A
Shen
Yuqiao
Y
D000260:Adenoviruses, Human / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D003588:Cytopathogenic Effect, Viral; D006801:Humans; D016296:Mutagenesis; D012641:Selection, Genetic; D012692:Serial Passage; D014407:Tumor Cells, Cultured; D014773:Virology / Q000379:methods; D014779:Virus Replication