Method of producing infectious reovirus

Details Method of producing infectious reovirus Method of producing infectious reovirus

2003-01-08

2004-03-09

Housel, James (Department: 1648)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

C435S239000, C435S239000, C435S325000, C435S261000, C435S006120

Reexamination Certificate

active

06703232

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method of producing infectious mammalian reovirus which is suitable for clinical administration to mammals, including human beings.
REFERENCES
U.S. Pat. No. 5,023,252.
Berry et al., Biotechnology and Bioengineering, “Production of Reovirus Type-1 and Type-3 from Vero Cells Grown on Solid and Macroporous Microcarriers”,
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Coffey, M. C., et al., “Reovirus therapy of tumors with activated Ras pathway”,
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Davis, et al.,
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Japanese Patent 63044532A, published Feb. 25, 1988.
McRae, M. A. and Joklik, W. K., “The nature of the polypeptide encoded by each of the 10 double-stranded RNA segments of reovirus type 3”,
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Strong, J. E. and P. W. Lee, “The v-erbV oncogene confers enhanced cellular susceptibility to reovirus infection”,
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WO99/08692A1, published Feb. 25, 1999.
All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Reovirus is a double-stranded RNA virus with a segmented genome. The receptor for the mammalian reovirus, sialic acid, is a ubiquitous molecule, therefore reovirus is capable of binding to a multitude of cells. However, most cells are not susceptible to reovirus infection and binding of reovirus to its cellular receptor results in no viral replication or virus particle production. This is probably the reason why reovirus is not known to be associated with any particular disease.
It was discovered recently that cells transformed with the ras oncogene become susceptible to reovirus infection, while their untransformed counterparts are not (Strong et al., 1998). For example, when reovirus-resistant NIH 3T3 cells were transformed with activated Ras or Sos, a protein which activates Ras, reovirus infection was enhanced. Similarly, mouse fibroblasts that are resistant to reovirus infection became susceptible after transfection with the EGF receptor gene or the v-erbB oncogene, both of which activate the ras pathway (Strong et al., 1993; Strong et al., 1996). Thus, reovirus can selectively infect and replicate in cells with an activated Ras pathway.
The ras oncogene accounts for a large percentage of mammalian tumors. Activating mutations of the ras gene itself occur in about 30% of all human tumors (Bos, 1989), primarily in pancreatic (90%), sporadic colorectal (50%) and lung (40%) carcinomas, as well as myeloid leukemia (30%). Activation of factors upstream or downstream of ras in the ras pathway is also associated with tumor. For example, overexpression of HER2/Neu/ErbB2 or the epidermal growth factor (EGF) receptor is common in breast cancer (25-30%), and overexpression of platelet-derived growth factor (PDGF) receptor or EGF receptor is prevalent in gliomas and glioblastomas (40-50%). EGF receptor and PDGF receptor are both known to activate ras upon binding to their respective ligand, and v-erbB encodes a constitutively activated receptor lacking the extracellular domain.
Since a large number of human tumors are accounted for by genetic alteration of the proto-oncogene ras or a high Ras activity, reovirus therapy is a new, promising therapy for such conditions (Coffey et al., 1998). Reovirus therapy is highly selective for Ras-associated tumor cells and leaves normal cells uninfected. Consequently, a simple and cost-effective method for the production of infectious reovirus suitable for clinical administration in human beings is needed.
Because reovirus does not pose a serious threat to human health, there has not been an intensive effort to produce reovirus efficiently. The mammalian reovirus is traditionally grown in mouse L-929 fibroblasts (Nibert et al., 1996). It has also been reported to grow in Chinese hamster ovary cells and Vero cells, an African green monkey kidney cell line (Taber et al., 1976; Davis et al., 1990). In addition, a primary culture of swine kidney was used to culture a swine reovirus (Japanese Patent 63044532A, published Feb. 25, 1988). In a study aiming at mass production of the reovirus, Berry et al. conducted an investigation of the optimal methods of culturing Vero cells and the subsequent reovirus infection (Berry et al., 1999). Vero cells were grown in either Cytodex-1 or Cultispher-G microcarriers, and culture parameters such as cell density, time course of viral growth and the ratio of cells to beads in the microcarrier were varied and virus yield determined. The study showed that the yield of virus varied greatly with the culture parameters, and complicated culture conditions (e.g. cell number per beads relative to multiplicity of infection) were required to obtain reasonable yield. Therefore, there remains a need for a simple, efficient method to produce clinically useful reovirus.
SUMMARY OF THE INVENTION
The present invention is directed to a simple and efficient method of producing reovirus by culturing reovirus in human embryo kidney 293 (HEK 293) cells. The yield in HEK 293 cells is unexpectedly high, particularly in comparison to previous work using L-929 cells or Vero cells. Moreover, by using the present invention, a high yield is achieved early in the course of infection, before viral particles are released from the cells. Therefore, the virus can be harvested early from intact cells, allowing the virus to be purified in the absence of the culture media. This makes the purification procedure relatively simple.
Accordingly, one aspect of the invention provides a method of producing mammalian reovirus, comprising the steps of contacting HEK 293 cells with a mammalian reovirus under conditions which result in reoviral infection of said HEK 293 cells; incubating the culture of said infected cells for a period of time sufficient to allow for viral replication; and harvesting the virus produced.
Any mammalian reovirus can be produced using this method. In particular, human reovirus for clinical administration in human beings can be produced using this method. Most particularly, reovirus serotype 3 and the Dearing strain are produced. Other mammalian reovirus can be produced efficiently by the presently claimed method as well.
Another feature of the invention is that because reovirus is produced rapidly in the HEK 293 cells, this invention provides for a fast and cost-effective method of producing reovirus. Also, since virus titer in the HEK 293 cell culture is high before the cells are completely lysed, the virus may be harvested when it is still associated with the cell, hence the purification procedure of the virus can be relatively simple, further reducing the cost of production. Therefore, this invention provides for a method wherein the virus is harvested

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