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EXHIBIT 99.4
Abstract
Analyses of single amino acid substitution mutants of adenovirus type 5 E1B-55K
protein
Y. Shen, G. Kitzes, J. Nye, A. Fattaey and T. Hermiston
The E1B-55K protein plays an important role during the human adenovirus type
5 (Ad5) productive infection. E1B-55K binds specifically to the p53 tumor
suppressor protein, blocking p53-mediated transcription activation. In
addition, E1B-55K collaborates with another adenovirus protein, E4orf6, to
target p53 for active degradation in the cytoplasm. During the late phase of
the viral infection, a protein complex that includes E1B-55K and E4orf6
modulates mRNA trafficking, facilitating efficient nuclear export of late
viral mRNAs while inhibiting the nucleocytoplasmic transport of cellular
mRNAs. In CI-1042 (ONYX-015), the E1B-55K gene is deleted, attenuating the
viral infection in normal cells and restricting the productive infection to
tumor cells lacking p53 function. Restoring the late functions of the E1B-55K
protein to the exclusion of p53 interaction may enhance the replication
efficiency and the anti-cancer efficacy. In an effort to separate the p53
binding/inactivation function and the late functions of the E1B-55K protein,
we have generated 26 single amino acid mutations in the E1B-55K protein.
These mutants were characterized for their ability to modulate p53 level,
interact with the E4orf6 protein, mediate viral late gene expression, and
support virus replication in human cancer cells.
Summary
The E1B-55K protein plays an important role both during the early phase and
the late phase of the human adenovirus type 5 (Ad5) productive infection. In
CI-1042 (ONYX-015), the E1B-55K gene is deleted, attenuating the viral
infection in normal cells and restricting the productive infection to tumor
cells lacking p53 function. In this study, we have generated twenty-six
single amino acid mutations in the E1B-55K protein and characterized their
ability to modulate p53 level, interact with the E4orf6 protein, mediate
viral late gene expression, and support virus replication in human cancer
cells. Two mutants appeared to have lost their ability to inactivate p53 but
have retained, at least partially, the late functions of the wild-type
protein. The ability to separate the p53-inactivation activity and the late
functions of E1B-55K raises the possibility to generate adenovirus variants
that retain the tumor selectivity of CI-1042 (ONYX-015), but can replicate
more efficiently than CI-1042 (ONYX-015) in a wide spectrum of cell types. We
believe this study may provide insights for new therapeutic viruses with
increased efficacy for the treatment of human cancers.
Conclusion
In this study, we have constructed a series of single amino acid substitution
mutations in the p53-binding domain and the transcriptional repression domain of
the E1B-55K protein. These mutations were recombined into an infectious virus
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(dl309) background and characterized for their abilities to modulate p53
level, interact with the E4orf6 protein, mediate viral late functions, and
support virus replication in human cancer cells. Two E1B-55K mutants, R240A
and H260A, appeared to have lost the ability to inactivate p53 but have
retained, at least partially, the late functions of the wild-type protein.
R240A fully restored the wild-type replication capacity of CI-1042 (ONYX-015)
in human cancer cells, while H260A did so only partially. The ability to
separate the p53-inactivation activity and the late functions of E1B-55K
raises the possibility to generate adenovirus variants that retain the tumor
selectivity of CI-1042 (ONYX-015), but can replicate more efficiently than
CI-1042 (ONYX-015) in a wide spectrum of cell types.
FORWARD-LOOKING STATEMENT
This abstract contains certain forward-looking statements regarding the
development of potential human therapeutic products that involve a number of
risks and uncertainties. Actual events may differ from Onyx's expectations.
