CA2435967A1 - Reovirus for the treatment of lymphoid malignancies - Google Patents

Abstract

A method of treating a lymphoid malignancy in an animal, comprising administering to cells of the lymphoid malignancy an amount of reovirus sufficient to cause substantial lysis of the cells, is provided. The method is useful for treating a variety of lymphoid malignancies, including but not limited to Burkitt's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, and non-Hodgkin's lymphomas such as diffuse large B-cell lymphoma, follicular lymphoma, and mantle-cell lymphoma. The invention is further useful for purging tissues of cells of lymphoid malignancies prior to transplantation or reintroduction to an animal.

Description

REOVIRUS FOR THE TREATMENT OF LYMPHOID MALIGNANCIES
FIELD OF THE INVENTION
The present invention relates to methods of treating lymphoid malignancies using reovirus.
REFERENCES
The following publications, patent applications, and patents are cited in this application:
U.S. Patent No. 6,136,307.
U.S. Patent No. 6,596,268.
Ahuja, H.G., Foti, A., Bar-Eli, M., Cline, M.J. (1990) The pattern of mutational involvement of RAS genes in human hematologic malignancies determined by DNA amplification and direct sequencing.
Blood 75:1684-90.
Bannerji, R., Byrd, J.C. (2000) Update on the biology of chronic lymphocytic leukemia. Curr. Opin. Oncol. 12:22-29.
Barbacid, M. (1987) Ras genes. Annu. Rev. Biochem. 56:779-827.
Bischoff, J.R., Kirn, D.H., Williams, A., Heise, C., Horn, S., Muna, M., Ng, L., Nye, J.A., Sampson-Johannes, A., Fattaey, A., McCormick, F.
(1996) An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science. 274:373-76.
Bos, J. (1989) Ras oncogenes in human cancer: a review. Cancer Res.
49:4682-89.
Brooks, G.F. et al., eds. (1998) Jawetz, Melnick & Adelberg's Medical Microbiology. New York: McGraw-Hill.
Chang, H.W., Jacobs, B.L. (1993) Identification of a conserved motif that is necessary for binding of the vaccinia virus E3L gene products to double-stranded RNA. Virology 194:537-47.

Chang, H.W., Uribe, L.H., Jacobs, B.L. (1995) Rescue of vaccinia virus lacking the E3L gene by mutants of E3L. J. Virol. 69:6605-08.
Chang, H.W., Watson, J.C., Jacobs, B.L. (1992) The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase. Proc. Natl Acad. Sci. U.S.A. 89:4825-29.
Chaubert, P., Benhattar, J., Saraga, E., Costa, J. (1994) K-ras mutations and p53 alterations in neoplastic and nonneoplastic lesions associated with longstanding ulcerative colitis. Am. J. Path. 144:767-775.
Clark, H.M., Yano, T., Sander, C., Jaffe, E.S., Raffeld, M. (1996) Mutation of the ras genes is a rare genetic event in the histologic transformation of follicular lymphoma. Leukemia 10:844-47.
Dohner, H., Stilgenbauer, S., Dohner, K., Bentz, M., Lichter, P. (1999) Chromosome aberrations in B-cell chronic lymphocytic leukemia:
reassessment based on molecular cytogenetic analysis. J. Mol. Med.
77:266-8I .
Drach, J., Kaufinann, H., Urbauer, E., Schreiber, S., Ackermann, J., Huber, H. (2000) The biology of multiple myelorna. J. Cancer Res. Clin.
Oncol. 126:441-47.
Duncan, R., Horne, D., Strong, J.E., Leone, G., Pon, R.T., Yeung, M.C., Lee, P.W. (1991) Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein. Virology 182:810-19.
Gaidano, G., Pastore, C., Volpe, G. (1995) Molecular pathogenesis of non-Hodgkin lymphoma: a clinical perspective. Haematologica 80:454-72.
Haig, D.M., McInnes, C.J., Thomson, J., Wood, A., Bunyan, K., Mercer, A. ( 1998) The orf virus OVZO.OL gene product is involved in interferon resistance and inhibits an interferon-inducible, double-stranded RNA-dependent kinase. Immunology 93:335-40.
Hankin, R.C., Hunter, S.V. (1999) Mantle cell lymphoma. Arch. Pathol.
Lab. Med. 123:1182-88.
He, B., Gross, M., Roizman, B. (1997) The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1 alpha to dephosphorylate the alphasubunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc. Natl Acad. Sci.
U. S.A. 94:843-48.

2 Fueyo, J., Gomez-Manzano, C., Alemany, R., Lee, P.S., McDonnell, T.J., Mitlianga, P., Shi, Y.X., Levin, V.A., Yung, W.K., Kyritsis, A.P.
(2000) A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene. 19:2-12.
Hecht, J.L., Aster, J.C. (2000) Molecular biology of Burkitt's lymphoma.
J. Clin. Oncol. 18:3?07-21.
Hulkkonen, J., Vilpo, L., Hurme, M., Vilpo, J. (2002) Surface antigen expression in chronic lymphocytic leukemia: clustering analysis, interrelationships and effects of chromosomal abnormalities. Leukemia 16:178-85.
Jaffe, E.S. et al. eds. World Health Organization Classification of Tumours Pathology cfc Genetics. (2001) Tumours ofHaematopoietic and Lymphoid Tissues. Geneva: World Health Organization).
Kawagishi-Kobayashi, M., Silverman, J.B., Ung, T.L., Dever, T.E. (1997) Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha. Mol. Cell.
Biol. 17:4146-58.
Kuppers, R. (2002) Molecular biology of Hodgkin's lymphoma. Adv.
Cancer Res. 2002:277-3 I2.
Levitzki, A. (1994) Signal-transduction therapy. A novel approach to disease management. Eur. J. Biochem. 226:1-13.
Mah, D.C. et al. (1990) The N-terminal quarter of reovirus cell attachment protein sigma 1 possesses intrinsic virion-anchoring function. Virology 179:95-103.
Mahmoudi, M., Motoo, Y., Vela, G.R., Bollon, A.P., Osther, K. (1989) Expression of c-myc and c-Ha-ras oncogenes in human lymphobIastoid cells (Namalva). Cell. Mol. Biol. 35:75-80.
Mills, N.E., Fishman, C.L., Rom, W.N., Dubin, N., Jacobson, D.R. (1995) Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma. Cancer Res. 55:1444-47.
Motoo, Y., Mahmoudi, M., Osther, K., Bolton, A.P. (1986) Oncogene expression in human hepatoma cells PLClPRF/5. Biochem. Biophys.
Res. Commun. 135:262-68.
Nakamura, N., Nakamine, H., Tamaru, J., Nakamura, S., Yoshino, T., Ohshima, K., Abe, M. (2002) The distinction between Burkitt lymphoma and diffuse large B-Cell lymphoma with c-myc rearrangement. Mod. Pathol. 15:771-76.

3 Nedergaard, T., Guldberg, P., Ralfkiaer, E., Zeuthen, J. (1997) A one-step DGGE scanning method for detection of mutations in the K-, N-, and H-ras oncogenes: mutations at codons 12, 13 and 61 are rare in B-cell non-Hodgkin's lymphoma. Int. J. Cancer 71:364-69.
Neri, A., Knowles, D.M., Greco, A., McCormick, F., Dalla-Favera, R.
(1988) Analysis of RAS oncogene mutations in human Lymphoid malignancies. Proc. Nat'1 Acad. Sci. U.S.A. 85:9268-72.
Neri, A., Murphy, J.P., Cro, L., Ferrero, D., Tarella, C., Baldini, L., Dalla-Favera, R. (1989) Ras oncogene mutation in multiple myeloma. J. Exp.
Med. 170:1715-25.
Nibert, M.L., Schiff, L.A., and Fields, B.N., Reoviruses and their replication in Fields Virology, 3rd Edition, Lippencott-Raven Press, 1995, pp. 1557-96.
Ong, S.T. and Le Beau, M.M. (1998) Chromosomal abnormalities and molecular genetics of non-Hodgkin's lymphoma. Semin. Oncol.
25:447-60.
Pasqualucci, L., Neumeister, P., Goossens, T., Nanjangud, G., Chaganti, R.S., Kuppers, R., Dalla-Favera, R. (2001) Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 412:341-46.
Potter, M. (1990) Neoplastic development in B-lymphocytes.
Carcinogenesis 11:1-13.
Romano, P.R., Zhang, F., Tan, S.L., Garcia-Barno, M.T., Katze, M.G., Dever, T.E., Hinnebusch, A.G. (1998) Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain. Mol. Cell. Biol.
18:7304-16.
Sharp, T.V., Moonan, F., Romashko, A., Joshi, B., Barber, G.N., Jagus, R.
(1998) The vaccinia virus E3L gene product interacts with both the regulatory and the substrate binding regions of PKR: implications for PKR autoregulation. Virology 250:302-15.
Steenvoorden, A.C., Janssen, J.W., Drexler, H.G., Lyons, J., Tesch, H., Binder, T., Jones, D.B., Bartram, C.R. (1988) Ras mutations in Hodgkin's disease. Leukemia 2:325-26.
Turner, D.L. et al. (1992) Site directed mutagenesis of the C-terminal portion of reovirus protein sigmal :evidence for a conformation-dependent receptor binding domain. Virology 186:219-27.
TweeddaLe, M.E., Lim, B., Jamal, N., Robinson, J., Zalcberg, J., Lockwood, G., Minder, M.D., Messner, H.A. (1987) The presence of

4 clonogenic cells in high-grade malignant lymphoma: a prognostic factor. Blood 69:1307-14.
Verwer, B.J., Terstappen, L.W. (1993) Automatic lineage assignment of acute leukemias by flow cytometry. Cytometry 14:862-75.
Wiessmuller, L. and Wittinghofer, F. (1994) Signal transduction pathways involving Ras. Cellular Signaling 6:247-267.
All of the publications, patent applications, and patents, cited above or elsewhere in this application, are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
STATE OF THE ART
Lymphoid malignancies encompass a heterogeneous group of cell proliferative disorders resulting from the inappropriate proliferation of lymphoid or lymphoid precursor cells. These disorders include, but are not limited to, Burkitt's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, and non-Hodgkin's lymphomas such as diffuse large B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma and small lymphocytic lymphoma. Classification of these disorders is based on clinical presentation, cell morphology, and chromosomal abnormalities. An authoritative classification scheme is published by the World Health Organization (Jaffe et al. 2001).
Burkitt's lymphoma (BL) is a B-cell lymphoma, usually caused by Epstein-Barr virus (EBV). BL is frequently characterized by chromosomal translocations involving Myc and the ~, or K light-chain immunoglobulin genes (reviewed in Hecht et al. 2000).
Hodgkin's lymphoma (HL), or Hodgkin's disease, is a lymphoma characterized by the presence of mononucleated Hodgkin and multinucleated Reed-Sternberg cells (HRS), which occur at low frequency in tumor tissues. Hodgkin and HRS cells harbor somatically mutated, clonally rearranged immunoglobulin genes, appearing to originate from germinal center B cells. Mutation in p53, IkappaBalpha, NFkappaB, and CD95lFas and deletions at lp, 6q, and 7q have been associated with HL (reviewed in, e.g., Kuppers, 2002).
Non-Hodgkin's lymphomas (NHL) are a heterogeneous group of lymphoid malignancies arising from mature lymphoid cells. NHL is often associated with activation of proto-oncogenes such as BCL-1, BCL-2, BCL-6, and cMyc, and/or disruption of tumor suppressor genes such as p53. Other causes of NHL include virus infection (e.g., EBV), chronic antigenic stimulation, and dysregulation of cytokine networks. Documented chromosomal abnormalities include breaks at 3q27 and/or 9p13 and deletions at 6q25-q27 and 6q21-q23 (Gaidano et al. 1995; Ong et al.
1998).
Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy of mature B-lymphocytes accounting for about 40-50% of non-Hodgkin's lymphomas.
DLBCL are heterogeneous with respect to chromosomal abnormalities but are often associated with translocations affecting 3q27, to which locus maps the BCL6 gene encoding a putative zinc-finger transcription factor. Dysregulation of BCL-2 and Myc have also been associated with DLBCL (reviewed in Nakamura et al. 2002).
Follicular lymphoma (FL) is a malignancy of follicular center B-cells accounting for about 25-40% of non-Hodgkin's lymphomas. FL is usually associated with the reciprocal chromosomal translocation, t(14;18)(q32;q21), which causes deregulated expression of the anti-apoptotic gene BCL2, located on chromosome 18.
About 25-60% of FL transform to more aggressive large cell lymphomas. Other mutations associated with FL include but are not limited to BCL-2 mutations, p53 mutations, mutations in the 5' non-coding region (NCR) of BCL-6, and cMyc rearrangements (Clark et al. 1996).
Mantle-cell lymphoma (MCL) primarily affects seniors and accounts for 2%
to 8% of non-Hodgkin lymphomas. MCL arises from naive pre-germinal center cells of either the primary follicle or the mantle regions of secondary follicles.
The disease is often associated with gastrointestinal tract lymphomatous polyposis or leukemia.
MCL is characterized by a t(11;14) translocation affecting the BCL-1 locus and/or a t(I1;I4)(q13;q32) translocation that affects the Cyclin D1 gene. In both cases, proto-oncogenes are overexpressed by being placed under the control of an immunoglobulin heavy-chain gene enhancer element (Hankin and Hunter, 1999).

Chronic lymphocytic leukemia (CLL) is a proliferative disorder resulting from the accumulation of mature lymphocytes in the blood and/or bone marrow.
Approximately 95% of all CLL involves mature B-cells, hence the designation B-cell chronic lymphocytic leukemia (B-cell CLL). CLL cells typically overexpress BCL-2, e.g., from a t(14:18) translocation that causes the expression of BCL-2 (normally on chromosome 18) to be controlled by an immunoglobulin enhancer element on chromosome 14. Other molecular genetic abnormalities include deletions at 13q14 andlor 11 q, deletions and/or translocations involving 17p, trisomy 12, p53 aberrations, and ectopic expression of cell surface markers (Bannerji and Byrd, 2000;
Dohner et al. 1999).
Ras, a proto-oncogene associated with a variety of cell proliferative disorders, is a monomeric guanine nucleotide binding protein that is activated in response to upstream mitogenic signals mediated by tyrosine receptor kinases (RTKs). Ras is known to interact with a number of downstream effectors, including members of the Raf family (e.g., Rafl, B-raf, and A-raf), members of the RaIGEF family (e.g., RaIGDS, RGL1, RGLZ, and Rlf), and phosphotidylinositol-3-kinases (PI-3 kinases) (e.g., isoforms of protein kinase C, AKT kinase/protein kinase B, p70-S6 kinase, and RacGEFs). (Ward et al. 2001; de Ruiter et al. 2000; Bos 1998; and references within).
Activating Ras mutations are present in approximately 30% of all human tumors (Wiessmuller and Wittinghofer 1994; Barbacid 1987); however, such mutations occur at different frequencies in different tumor cell types. Fox example, Ras mutations are common in pancreatic cancers (80%), thyroid cancers (50%), and sporadic colorectal carcinomas (40-50%) (Millis et al. 1995; Chaubert et al.
1994;
Bos 1989) but are less common in certain hematopoietic malignancies.
While some hematopoietic malignancies are associated with Ras mutations (e.g., 30% of myeloid leukemias and 18% of acute lymphoblastic leukemias), Ras mutations are virtually undetectable in lymphoid malignancies, for example, NHL and CLL (Neri et al. 1988; Millis et al. 1995; Chaubert et al. 1994; Bos 1989).
This latter observation has been confirmed by a number of independent studies. For example, Neri et al. (1988) .screened I78 lymphomas and failed to identify N-Ras mutations (codons 12 and 13) in NHL or CLL samples. Similarly, using a PCR-based assay that simultaneously scanned six mutational "hot-spots" in codons 12, 13, and 61 of K-, N-, and H-Ras, Nedergaard et al. ( 1997) reported only three Ras mutations in 123 NHL
samples. Abuja et al. (1990) and Steenvoorden et al. (1988) reported the presence of no N-Ras mutations in six and 25 HL samples, respectively. And Clark et al.
(1996) reported the presence of only one N-Ras mutation (codon I2) in 16 samples of FL.
Accordingly, while Ras mutations appear in some acute myeloid and lymphoid malignancies, they are rare in chronic lymphoid malignancies (Abuja et al.
1990).
As described in U.S. Patent No. 6,136,307, herein incorporated by reference in its entirety, reovirus selectively replicates in cells with an activated Ras pathway.
While reovirus can infect "resistant" cells, virus gene transcription is associated with activation (via phosphorylation) of double-stranded RNA-activated protein kinase (PKR). Activated PKR subsequently affects phosphorylation of translation initiation factor eIF-2a , resulting in the inhibition of viral gene translation.
However, PKR
phosphorylation is reduced or reversed in Ras-transformed cells, relieving the translation block and allowing virus proteins to be translated. Reovirus therefore infects and productively replicates only in "susceptible" cells. Because reovirus replication causes cytopathic effects (CPE) resulting in cell death, reovirus may be used to selectively kill tumor cells in an animal, wherein the tumor cells have activated Ras pathways.
SUMMARY OF THE INVENTION
The present invention pertains to a method of treating a lymphoid malignancy in an animal, the method comprising administering to cells of the lymphoid malignancy an amount of reovirus sufficient to cause substantial lysis of the cells.
In one embodiment of the invention, the lymphoid malignancy is selected from the group consisting of Burkitt's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. In a particular embodiment of the invention, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, or small lymphocytic lymphoma.

In another embodiment of the invention, the cells of the lymphoid malignancy comprise an activated Ras pathway. In a particular embodiment of the invention, the cells of the lymphoid malignancy comprise a normal Ras gene, with the Ras pathway being activated in a manner that does not require an activating Ras mutation.
In one embodiment of the invention, reovirus is administered to hematopoietic cells in vivo. In preferred embodiments of the invention, reovirus is administered by a route selected from the group consisting of intramedullar, intravascular, intrathecal, intravenous, intramuscular, subcutaneous, intraperitoneal, topical, oral, rectal, vaginal, nasal, and intratumoral routes. In another embodiment of the invention, reovirus is administered to a cellular composition ex vivo. Reovirus may also be administered by any combination of in vivo routes, alone or in addition to ex vivo administration.
In one embodiment of the invention, more than one type of reovirus is administered. In another embodiment of the invention, more than one strain of reovirus is administered. In yet another embodiment of the invention, one or more recombinant reoviruses are administered.
In one embodiment of the invention, the reovirus is administered in a single dose. In another embodiment of the invention, the reovirus is administered in more than one dose.
In one embodiment of the invention, the animal to which the reovirus is administered is a mammal. In a preferred embodiment of the invention, the mammal is selected from the group consisting of dogs, cats, sheep, goats, cattle, horses, pigs, humans, and non-human primates.
The invention also provides a method for selectively killing cells of a lymphoid malignancy in a cellular composition suspected of containing such cells.
The method includes administering reovirus to cells of a cellular composition under conditions that result in substantial lysis of the cells of the lymphoid malignancy.
In one embodiment of the invention, the cellular composition to which reovirus is administered or provided is selected from the group consisting of hematopoietic tissue and transplant tissue. In a preferred embodiment of the invention, the hematopoietic tissue is selected from the group consisting of bone marrow, spleen, mucosa-associated lymphoid tissues (MALT), lymph nodes, Peyer's patches, thymus, tonsils, and fetal liver.
In one embodiment of the invention reovirus is administered to cells of the cellular composition by a route selected from the group consisting of intramedullar, intravascular, intrathecal, intravenous, intramuscular, subcutaneous, intraperitoneal, topical, oral, rectal, vaginal, nasal, and intratumoral routes. In another embodiment of the invention cells of a cellular composition are removed from an animal, contacted with reovirus ex vivo, then returned to an animal.
Another embodiment of the invention, comprises the additional step of inactivating the reovirus after contacting the cellular composition with reovirus under conditions which results in substantial lysis of the cells of the lymphoid malignancy.
In one embodiment of the invention, reovirus is removed by subjecting the reovirus-treated cellular composition to anti-reovirus antibodies or a combination of anti-reovirus antibodies and complement to inactivate the reovirus. Alternatively or additionally, immobilized anti-reovirus antibodies specific for reovirus surface epitopes are used to remove the reovirus particles from cellular composition.
In one embodiment of the invention, anti-reovirus antibodies are bound to a column over which the treated cellular composition is passed. In another embodiment, anti-reovirus antibodies are bound to beads or other physical structures that can be mixed with the cellular composition then recovered by filtration or centrifugation.
The reovirus-depleted cellular composition is then returned to the donor animal or provided to another animal.
Alternatively, antireovirus antibodies are administered in vivo to hematopoietic tissues treated with reovirus. In one embodiment of the invention, the anti-reovirus antibodies are provided to the tissue or tissues that were treated with reovirus. In another embodiment of the invention, the anti-reovirus antibodies are provided or administered systemically.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Bar graph showing the % viability of Raji, CA46, Daudi, Ramos, ST486, and four DLBCL cells (OCY-LY1, OCY-LY2, OCY-LY8, and OCY-LY10}
following challenge with reovirus type 3 at a MOI of 20. % viability is based on trypan blue exclusion.
Figure 2: Bar graph showing the ability of reovirus to replicate in Raji, CA46, Daudi, Ramos, ST486, and four DLBCL cells (OCY-LY1, OCY-LY2, OCY-LY8, and OCY-LY10). Growth is reported in PFUs at 0 hour (open squares) and 96 hours (filled squares) post infection.
Figure 3: Bar graphs showing the size of lymphoma-derived tumors implanted in mice following infection with either live (filled symbols) or UV-inactivated (open circles) reovirus. (A) Raji tumors, intratumoral virus administration. (B) Daudi tumors, intratumoral virus administration. (C) Raji tumors, intravenous virus injection.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
Cellular composition: Any mixture of cells obtained from an animal. Cellular compositions include but are not limited to bone marrow, tissue from the spleen, lymph nodes, Peyer's patches, thymus, tonsils, fetal liver, and bursa of Fabricus (in Avian species), mucosa-associated lymphoid tissues (MALT), whole blood, and fractions thereof.
Contacting cells with reovirus: Providing reovirus to cells of an animal such that the virus and cells are in sufficient proximity to allow virus adsorption to the cell surface.
Hematopoietic stem/progenitor cells: Partially differentiated cells cable of differentiating into a variety of different hematopoietic lineages, including lymphoid cells. The presence of CD34 is often used as a marker for hematopoietic stemlprogenitor cells (i.e., CD34+ cells), although more primitive, less differentiated, hematopoietic precursor cells may actually lack CD34 (i.e., CD34- cells).

Intramedullary: Within the bone marrow.
Lymphoid cell lines: Cell lines derived from lymphoid cells or their precursors.
Lymphoid malignancies: As used herein, this term refers broadly to a heterogeneous group of diseases, disorders, or conditions resulting from the rapid proliferation of lymphoid or lymphoid precursor cells. The current classification is based on numerous factors, including clinical presentation, cell morphology, and chromosomal abnormalities. As used herein, "lymphoid malignancies" is synonymous with "lymphoid neoplasms" and "lymphoid neoplasias" based on the classification scheme published by the World Health Organization (Jaffe et al.
2001).
Normal hematopoietic stem/progenitor cells: Hematopoietic stemlprogenitor cells not associated with a transformed or malignant growth phenotypes and not believed to harbor chromosomal abnormalities that would cause such growth phenotypes.
Normal lymphocytes: Lymphocytes not associated with a transformed or malignant growth phenotypes and not believed to harbor chromosomal abnormalities that would cause such growth phenotypes.
Normal Ras gene: A gene encoding a normal, i.e., non-transforming form, of Ras.
Activated Ras pathway: A Ras pathway that has become activated (i.e., the constitutive level of signaling through the pathway has increased compared to that of equivalent normal cells) by way of Ras gene structural mutation, elevated level of Ras gene expression, increased stability of the Ras gene message, or any mutation or other mechanism which leads to the activation of Ras or a factor or factors downstream or upstream from Ras in the Ras pathway.
Reovirus: Any virus in the family Reoviridae. The name reovirus (respiratory and enteric orphan virus) is a descriptive acronym suggesting that these viruses, although not associated with any known disease state in humans, can be isolated from both the respiratory and enteric tracts (Sabin, 1959).

SCID/NOD mice: Nonobese diabetic (NOD) mice with severe combined immunodeficiency (SCID). SCID/NOD mice lack functional T and B-lymphocytes.
SCID/NOD mice are useful for growing palpable tumor masses, derived from implantated exogenous tumor cells, for subsequent challenge with therapeutic agents.
Solid lymphoma: A lymphoid malignancy characterized by the formation of a discrete mass of predominantly malignant cells (i. e., cells of the lymphoid malignancy) at a particular location in an animal. Solid lymphomas may remain localized in an animal or may metastasize, resulting in the formation of additional malignant cell masses or resulting in a circulating lymphoid malignancy.
Substantial lysis: As used herein, substantial lysis refers to a decrease in viability, e.g., through lysis, of cells of a lymphoid malignancy. Lysis can be determined by a viable cell count of the treated cells, and the extent of decrease can be determined by comparing the number of viable cells in the treated cells to that in the untreated cells, or by comparing the viable cell count before and after reovirus treatment. Lysis can also be inferred from a reduction in the size of a solid lymphoma in terms of either (or both) mass or volume. The decrease in viability is at least about 10%, preferably at least SO%, and most preferably at least 75% of the proliferating cells. The percentage of lysis can be determined for tumor cells by measuring the reduction in the size of the tumor in the mammal or the lysis of the tumor cells in vitro. Substantial lysis also includes the complete elimination of cells of a lymphoid malignancy from an animal.
The Invention:
The instant invention is based, in part, on Applicant's discovery that reovirus is capable of replicating in tumor cells of lymphoid malignancies despite the relative infrequency of activating Ras mutations in lymphoid malignancies, compared to malignancies derived from other cell types (Neri et al. 1988; Steenvoorden et al.
1998; Ahuja et al. 1990; Clark et al. 1996; and Nedergaard et al. 1997).
The conclusion that reovirus is able to replicate in lymphoid tumor cells is based on in vitro cell culture data as well as in vivo animal model data. In a first set of experiments, a panel of nine lymphoid cell lines was assembled for challenge with reovirus. The panel comprised two BL cell lines (Raji and Daudi) in which the Epstein-Barr virus (EBV) was detected (i.e., EBV+ cell lines), three BL cell lines (CA46, Ramos, and ST486) in which EBV was not detected (i.e., EBV- cell lines), and four DLBCL cell lines (OCY-LY1, OCY-LY2, OCY-LYB, and OCY-LY10).
Reovirus replicated in six of the nine lymphoma-derived cell lines, specifically Raji, CA46, and all four DLBCL cells. Reovirus was unable to replicate in only three of these cell lines (Daudi, Ramos, and ST486).
In a second experiment, cell suspensions were prepared from 27 lymphoid tumor biopsy specimens for challenge with reovirus. Of the 27 specimens, 1 S
were associated with a clinical diagnosis of CLL, and 12 with a clinical diagnosis of NHL.
The NHL specimens could be further divided into BL (1); DLBCL (2); small lymphocytic leukemia (SLL) (2); FL, grade I (1); FL, grade II (4); FL, grade III (1);
and MCL (1). Three suspensions each of normal primary blood mononuclear cells (PBMC) and normal bone marrow, CD34+-hematopoietic stem/progenitor cells were included as negative controls.
Reovirus was able to replicate in all 15 CLL cell suspensions, the BL cell suspension, both DLBCL cell suspensions, one of the SLL cell suspensions, the MCL
cell suspension, and the FL, grade I, cell suspension. Reovirus did not replicate in 5 of the 6 FL cells suspensions, one of the SLL cell suspensions, or, as expected, any of the six negative control cell suspensions comprising normal PBMC or hematopoietic stem/progenitor cells. In total, reovirus was able to replicate in 21 of the 27 cell suspensions prepared from lymphoid tumor biopsy specimens (see, Example 2 and Table 1, below).
In vivo data using a SCID/NOD mouse xenograft model provided direct evidence that reovirus was effective in reducing the growth of a BL tumor in an animal. In this experiment, mice were injected with either the reovirus-susceptible Raji cells or the reovirus-resistant, Daudi cells, from above (see also, Example 3, below).
Following the establishment of palpable tumor masses, mice were treated with either live reovirus or UV-inactivated virus. The administration of live reovirus to mice with Raji tumors resulted in an approximately ten-fold reduction in tumor size compared to mice receiving LTV-inactivated reovirus. These results showed that reovirus could be used to treat tumors arising from lymphoid malignancies in an animal. Consistent with the results of the above in vitro experiment, reovirus was not effective in treating the mice with Daudi tumors.
While the Ras genotypes of all nine lymphoid tumor cell lines and all 27 lymphoma biopsy specimens used in the above experiments have not been formally characterized, the low frequency of Ras mutations in lymphoid cells (Neri et al. 1988;
Mills et al. 1995; Chaubert et al. 1994; Bos 1989; Ahuja et al. 1990) forecloses the possibility that 27 out of 36 (75%) lymphoma cell lines tested could harbor Ras mutations. Accordingly, the replication of reovirus in the above lymphoid tumor cell lines and biopsy specimens cannot be explained by the presence of Ras mutations in these cells. Rather, the ability of reovirus to replicate in 27 out of 36 lymphoid malignancy cell types in which Ras mutations are rare indicates that reovirus susceptibility in lymphoid cells is not merely a function of whether Ras mutations are present in the lymphoid cell type.
These findings suggest that reovirus could be used to treat lymphoid malignancies in an animal even when the cells of the lymphoid malignancies do not harbor Ras mutations. Accordingly, the present invention provides the treatment of a lymphoid malignancy in an animal, comprising the step of administering to the animal an amount of reovirus sufficient to kill the cells of the lymphoid malignancy.
In one embodiment of the invention, treatment results in a reduction in the size of a solid lymphoma or a reduction in the number of cells of a circulating lymphoma (e.g., a leukemia).
As used herein, the size of a solid lymphoma refers to the mass or volume of a solid lymphoma. In one embodiment of the invention, reduction in the size of a lymphoma is achieved when the lymphoma is less than about 50% of its original volume following reovirus treatment. In a preferred embodiment of the invention reduction in the size of a lymphoma is achieved when the lymphoma is less than about 25% of its original volume following reovirus treatment. In a most preferred embodiment, reduction in the size of a lymphoma is achieved when the lymphoma is less than about 10% of its original volume following reovirus treatment. In another embodiment of the invention, treatment results in the complete elimination of a solid lymphoma from an animal.

In the case of a circulating lymphoma, or circulating cells from a solid lymphoma that has metastasized, treatment results in a reduction in the number of circulating lymphoma cells. In one embodiment of the invention, treatment results in at least a 50% decrease in the number of circulating lymphoma cells. In a preferred embodiment of the invention, treatment results in at least a 75% decrease in the number of circulating lymphoma cells. In a most preferred embodiment, treatment results in at least a 90% decrease in the number of circulating lymphoma cells. In another embodiment of the invention, treatment results in the complete elimination of circulating lymphoma cells from an animal.
The amount of reovirus required for treatment of a lymphoid malignancy depends on the body mass, age, gender, and physical condition of the animal;
the type or strain of reovirus administered; the route or combination of routes of administration; the severity and characteristics of the lymphoid malignancy, the seventy of the patient's symptoms; the rate of virus replication in susceptible cells, and other factors. In addition, because reovirus replicates selectively in cells of the lymphoid malignancy, releasing progeny virus with the same specificity, the initial amount of reovirus that is administered to an animal may encompass a wide range.
Administration of an excessive number of virus particles is unlikely to cause toxic effects because of the blockage of reovirus translation in non-permissive cells.
Administration of a less than optimal number of virus particles is likely to increase the time required to kill the cells of the lymphoid malignancy because additional rounds of virus replication will be required to generate sufficient virus particles to infect the cells of the lymphoid malignancy.
Accordingly, a feature of the invention is the wide range of virus particle dosages effective in treating lymphoid malignancies. In one embodiment of the invention, an effective amount of reovirus is from about 1.0 plaque forming unit (PFU)lkilogram (kg) body weight to about 1015 PFUIkg body weight, more preferably from about 102 PFU/kg body weight to about 10'3 PFU/kg body weight. The treatment can be administered to a variety of animals, including but not limited to dogs, cats, sheep, goats, cattle, horses, pigs, humans, and non-human primates.

The reovirus may be administered in a single dose or in multiple doses. In one embodiment of the invention, the reovirus is administered to the lymphoid malignancy by an intratumoral route. In another embodiment, the reovirus is administered to the bone marrow by an intramedullary route. In another embodiment, the reovirus is administered intravenously. In yet another embodiment, the reovirus is administered by an intravascular, intrathecal, intramuscular, subcutaneous, intraperitoneal, topical, oral, rectal, vaginal, nasal, or intratumoral route, alone or in any combination with other routes discussed herein.
The reovirus used to practice the invention may be any type or strain of reovirus with a tropism for the target lymphoid malignancies in the animal to be treated. For example, to treat a human patient, a human reovirus, including but not limited to serotype 1 reovirus (Lang) , serotype 2 reovirus (Jones), or serotype 3 reovirus (bearing or Abney) is most preferable. The reovirus may also be a field isolate, laboratory strain, or engineered reovirus.
The reovirus may be a recombinant reovirus resulting from the recombination/reassortment of genomic segments from two or more genetically distinct reoviruses. RecombinationJreassortment of reovirus genomic segments may occur in nature following infection of a host organism with at least two genetically distinct reoviruses. Recombinant virions can also be generated in cell culture, for example, by co-infection of permissive host cells with genetically distinct reoviruses (Nibert et al. 1995).
Accordingly, the invention contemplates the use of recombinant reovirus resulting from reassortment of genome segments from two or more genetically distinct reoviruses, including but not limited to, human reovirus, such as type 1 (e.g., strain Lang), type 2 (e.g., strain Jones), and type 3 (e.g., strain bearing or strain Abney), non-human mammalian reoviruses, or avian reovirus. The invention further contemplates the use of recombinant reoviruses resulting from reassorhnent of genome segments from two or more genetically distinct reoviruses wherein at least one parental virus is genetically engineered, comprises one or more chemically synthesized genomic segment, has been treated with chemical or physical mutagens, or is itself the result of a recombination event. The invention further contemplates the use of recombinant reovirus that have undergone recombination in the presence of chemical mutagens, including but not limited to dimethyl sulfate and ethidium bromide, or physical mutagens, including but not limited to ultraviolet light and other forms of radiation.
The invention further contemplates the use of recombinant viruses that comprise deletions or duplications in one or more genome segments, that comprise additional genetic information as a result of recombination with a host cell genome, or that comprise synthetic genes.
The reovirus may be modified by incorporation of mutated surface proteins, for example, capsid proteins, and, where applicable, membrane proteins. The proteins may be mutated by substitution, insertion or deletion. Replacement includes the insertion of different amino acids in place of the native amino acids.
Insertions include the insertion of additional amino acid residues into the protein at one or more locations. Deletions include deletions of one or more amino acid residues in the protein. Such mutations may be generated by methods known in the art. For example, oligonucleotide site directed mutagenesis of the gene encoding for one of the coat proteins could result in the generation of the desired mutant coat protein.
Expression of the mutated protein in reovirus infected mammalian cells in vitro such as COS 1 cells will result in the incorporation of the mutated protein into the reovirus virion particle (Turner et ad. 1992; Duncan et al. 1991; Mah et al. 1990).
The reovirus may comprise more than one reovirus, including but not limited to, any combination of the reoviruses identified herein. Different reovirus may be administered simultaneously or at different times.
While reovirus is discussed as an embodiment of the invention, the invention is by no means limited to the use of reovirus to kill the cells of lymphoid malignancies. The use of other modified viruses to selectively kill cells with activated Ras pathways has been described in U.S. Patent No. 6,596,268. Representative types of modified virus included adenovirus, herpes simplex virus (HSV), parapoxvirus orf virus, or vaccinia virus. For reasons that will become apparent, these viruses may also be useful for killing cells of lymphoid malignancies One virus that was particularly useful for selectively killing cells with an activated Ras pathway was adenovirus. Adenoviruses encode several gene products that counter antiviral host defense mechanisms. For example, virus-associated RNA
(VAI RNA or VA RNAI) refers to small, structured RNAs that accumulate in the cytoplasm of infected cells late in the adenovirus replication cycle. VAI RNA
binds to the to the double stranded RNA (dsRNA) binding motifs of PKR blocking activation by phosphorylation. With PKR unable to function, adenovirus replicates in the cell, causing lysis.
Some attenuated or modified adenoviruses lack or fail to transcribe VAI RNA.
As a consequence, these viruses are unable to replicate in cells that express PKR.
However, attenuated or modified adenovirus can replicate in cells with activated Ras-pathways, which have reduced PKR activity.
In addition to VAI RNA, a 55 kDa cellular p53 inhibitor is encoded by the E1B region of the adenovirus genome. p55 allows adenovirus to overcome the replication-inhibitory effect of p53. The ONYX-015 adenovirus is deficient for p55 (Bischoff et al. 1996; WO 94/18992), limiting virus replication to cells that express mutated p53. Since p53 mutations often accompany Ras mutations, particularly in the later stages of certain cancers, the ONYX-015 adenovirus will replicate in at least a subpopulation of cells that harbor activating Ras mutations.
Similarly, the Delta24 adenovirus harbors a 24 base-pair deletion in the ElA-coding region (Fueyo et al., 2000), responsible for binding to and inhibiting the function of the cellular tumor suppressor Rb. Accordingly, Delta 24 replication is limited to cells in which Rb is inactivated, as is the case in at least a subset of cancer cells.
Based on the discovery that reovirus, known to replicate in cells with activated Ras pathways, also replicates in the cells of lymphoid malignancies, it follows that at least some attenuated or modified adenovirusese will also replicate in the cells of lymphoid malignancies. Accordingly, attenuated or modified adenoviruses may be used to practice the instant invention.

Infected-cell protein 34.5 (ICP34.S) of both type 1 and type 2 herpes simplex viruses (HSV) can also prevent the antiviral effects exerted by PKR. ICP34.S
causes cellular protein phosphatase-1 to act on eIF-2a, resulting in dephosphorylation of eIF-2a (He 1997), the same protein phosphorylated by PKR. The activity of ICP34.S
thereby allows herpesvirus to prevent or reverse PKR activation. Herpesviruses that lack or are unable to express ICP34.S cannot replicate in cells with activated PKR;
however, such attenuated or mutated viruses can replicate in cells with activated Ras pathways, in which PKR activity is reduced. Accordingly, based on the finding that reovirus can replicate in the cells of lymphoid malignancies, it is reasonable to predict that ICP34.S-deficient herpesviruses can also replicate in cells of lymphoid malignancies and therefore be used to practice the instant invention.
Parapoxvirus orf virus is a poxvirus that induces acute cutaneous lesions in different mammalian species, including humans. Parapoxvirus orf virus naturally infects sheep, goats, and humans through broken or damaged skin, replicates in regenerating epidermal cells and induces pustular lesions that turn to scabs (Haig 1998). The virus encodes gene OV20.OL, involved in blocking PKR activity (Haig 1998). Parapoxvirus orf viruses deficient in the expression of OV20.OL cannot escape the effect of PKR activation. However, such viruses can replicate in cells that are deficient in PKR activation, such as cells with activated Ras pathways.
Accordingly, OV20.OL-deficient parapoxvirus orf viruses are also predicted to replicate selectively in cells of lymphoid malignancies, and are therefore useful for practicing the instant invention.
Vaccinia virus is a member of the Orthopoxvirus genus that infects humans, producing characteristic localized lesions (Brooks 1998). The virus encodes two proteins that play a role in down-regulating PKR activity through different mechanisms.
The E3L gene encodes proteins of 20 and 2S kDa that are expressed early in infection. The amino terminal region of the E3 proteins interacts with the carboxy-terminal region of PKR, preventing function (Chang et al. 1992, 1993, and 1995;
Sharp et al. 1998; and Romano et al. 1998). Deletion or disruption of the E3L
gene precludes vaccinia virus from replicating in cells with activated PKR, limiting its replication to cells with reduced PKR activity, such as cells with an activated Ras pathway.
The vaccinia virus K3L gene encodes pK3, a protein possessing a carboxy-terminal region that is structurally analogous to residues 79-83 of eIF-2a.
pK3 acts as an eIF-2a-decoy for PKR, preventing the activation of eIF-2a and allowing vaccinia virus to replicate. Carboxy-terminal mutations or truncations in K3L protein abolish its PKR-inhibitory function (Kawagishi-Kobayashi et al. 1997), thereby limiting the replication of vaccinia virus to cells with reduced or absent PKR activity, such as cells with an activated Ras pathway.
Attenuated or modified vaccinia viruses are deficient in terms of E3L or pK3 function, and preferably both functions. Such attenuated or modified viruses are unable to replicate in normal cells in which PKR is activated. Accordingly, replication of these viruses is limited to cells having an activated Ras-pathway, or, as predicted from the findings related to the instant invention, cells of lymphoid malignancies. Accordingly, an attenuated or modified vaccinia virus should be useful for practicing the instant invention.
The invention may be used to treat a variety of lymphoid malignancies providing reovirus is able to replicate in the cells of the lymphoid malignancy. In one embodiment of the invention, reovirus is used to treat a B-cell lymphoid malignancy.
In another embodiment of the invention, reovirus is used to treat a T-cell lymphoid malignancy. In yet another embodiment of the invention, reovirus is used to treat a lymphoid malignancy comprising both B-cells and T-cells, in any proportion.
The invention may also be used to treat a lymphoid malignancy arising from lymphoblasts, prolymphocytes, or other lymphoid cells at various stages of differentiation and/or maturity.
In one embodiment of the invention, reovirus is used to treat a B-cell lymphoid malignancy such as Burkitt's lymphoma (BL). In another embodiment of the invention, reovirus is used to treat a non-Hodgkin's lymphoma (NHL), including but not limited to chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma (FL). In one embodiment of the invention, the FL is a type I FL. In another embodiment of the invention, reovirus is used to treat small lymphocytic lymphoma (SLL) or mantle-cell lymphoma (MCL).
In yet another embodiment of the invention, reovirus is used to treat precursor B-lymphoblastic leukemia (also called precursor B-cell acute lymphoblastic leukemia), prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma, extranodal marginal zone B-cell lymphoma of MALT type, nodal marginal zone B-cell lymphoma, mediastinal large B-cell lymphoma, or primary effusion lymphoma.
In another embodiment of the invention, reovirus is used to treat a B-cell lymphoma such as Hodgkin's lymphoma (HL), including but not limited to nodular lymphocyte-predominant Hodgkin's lymphoma in addition to classical Hodgkin's lymphoma, comprising nodular sclerosis Hodgkin's lymphoma (grades 1 and 2), lymphocyte-rich classical Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma, and lymphocyte depletion Hodgkin's lymphoma.
In yet another embodiment of the invention, reovirus is used to treat predominantly T-cell and natural killer (NK)-cell neoplasms, including but not limited to precursor T-cell neoplasm, precursor T-lymphoblastic lymphoma/leukemia (also called precursor T-cell acute lymphoblastic leukemia), T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell lymphoma/leukemia (e.g., caused by HTLV-1), extranodal NK/T-cell lymphoma (nasal type), enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides/Sezary syndrome, anaplastic large-cell lymphoma, T/null cell (primary cutaneous type), anaplastic large-cell lymphoma (T/null cell, primary systemic type), angioimmunoblastic T-cell lymphoma, and peripheral T-cell lymphomas that are not otherwise characterized.
In yet another embodiment of the invention, reovirus is used to treat post-transplant lymphoproliferative disorders (PTLD), including but not limited to polyclonal PTLD, monoclonal PTLD, reactive plasmacytic hyperplasia, infectious mononucleosis-like PTLD, and monomorphic PTLD.

Reovirus can be used to treat any lymphoid malignancy in which cells it is able to replicate. Moreover, the lymphoid malignancy need not be clinically, morphologically, or genetically characterized prior to or in order to be treated with reovirus. That reovirus is able to replicate in the cells of the malignancy is sufficient to practice the invention. One method of determining whether reovirus is able to replicate in cells of a lymphoid malignancy is provided in Example 2, below.
Accordingly, lymphoma cells from a biopsy specimen obtained from an animal with a lymphoid malignancy, or suspected of having a lymphoid malignancy, can be grown in culture and infected with reovirus. Reovirus replication can be measured using the assays described in the Examples, below, or by other methods known in the art, including but not limited to plaque assays and labeled nucleoside or nucleotide monophosphate incorporation. The ability of reovirus to replicate in cells derived from the biopsy specimen is evidence that administration of reovirus to an animal will be effective in treating the malignancy or suspected malignancy.
In another embodiment of the invention, cells of hematopoietic tissues are removed from an animal contacted with reovirus ex vivo. The treated hematopoietic cells are then returned to the animal, preferably to the site from which they were harvested.
In another embodiment of the invention, reovirus is used to remove cells of a lymphoid malignancy from a tissue or organ prior to transplantation. Because this method can be applied without regard to the type or age of the transplant or the specific nature of the lymphoid malignancy, and because the reovirus is essentially harmless to normal cells and tissues, this method can be used as a routine practice to "clean up" any transplant before transplantation. Transplanted tissues may be autologous, allogeneic, or even xenogeneic. Preferably the transplanted tissues are autologous.
Reovirus may optionally be removed following treatment of a cellular composition, such as hematopoietic tissues treated ex vivo or transplant tissue, by subjecting the mixture to anti-reovirus antibodies or a combination of anti-reovirus antibodies and complement to inactivate the reovirus (e.g., by lysis or capsid disruption). Alternatively or additionally, anti-reovirus antibodies that recognize epitopes on the surface of reovirus may be used to remove the reovirus particles by immobilizing the antibodies, applying the cellular composition to the immobilized antibodies, and collecting the part of the composition that does not bind to the antibodies. The cellular composition is then transplanted into an animal or returned to the animal from which it was derived.
Alternatively, antireovirus antibodies are administered to an animal in vivo, following administration of reovirus and, preferably, after substantial lysis of cells of a lymphoid malignancy has occurred. According to this embodiment of the invention, the antibodies are administered by the same route as the reovirus they are administered to inactivate, by a separate route of administration, or any combination thereof.
Reovirus may also be used to treat more than one lymphoid malignancy in an animal, including but not limited to any combination of the diseases, disorders, and conditions identified herein, or any combination of lymphoid malignancies that have not been characterized but in which reovirus is able to replicate.
In one embodiment of the invention, reovirus is administered in conjunction with surgery or removal of at least a portion of the lymphoid malignancy. The reovirus may be administered in conjunction with or in addition to radiation therapy and/or known anticancer compounds or chemotherapeutic agents. Such agents, include, but are not limited to, 5-fluorouracil, mitomycin C, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracyclins (Epirubicin and Doxurubicin), antibodies to receptors, such as herceptin, etopside, pregnasome, platinum compounds such as carboplatin and cisplatin, taxanes such as taxol and taxotere, hormone therapies such as tamoxifen and anti-estrogens, interferons, aromatase inhibitors, progestational agents and LHRH analogs. In another embodiment of the invention, reovirus is administered prior to or in place of radiation therapy and/or chemotherapeutic agent.
In another embodiment of the invention, a method is provided for reducing the growth of metastastic tumors in a mammal comprising administering an effective amount of a reovirus to the mammal.

The reovirus may be administered to immunocompetent mammals in conjunction with the administration of immunosuppressants and/or immunoinhibitors.
Such immunosuppressants and immunoinhibitors are known to those of skill in the art and include such agents as cyclosporin, rapamycin, tacrolimus, mycophenolic acid, azathioprine and their analogs, and the like. Other agents are known to have immunosuppressant properties as well (see, e.g., Goodman and Gilman, 7~' Edition, page 1242, the disclosure of which is incorporated herein by reference).
Immunoinhibitors include anti-antireovirus antibodies, which are antibodies directed against anti-reovirus antibodies. Such anti-antireovirus antibodies may be administered prior to, at the same time, or shortly after the administration of the reovirus. Preferably an effective amount of the anti-antireovirus antibodies are administered in sufficient time to reduce or eliminate an immune response by the mammal to the administered reovirus.
The invention includes pharmaceutical compositions that comprise, as an active ingredient, one or more of the reoviruses associated with pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions may also comprise an appropriate immunosuppresant, associated with pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions may be solid, semi-solid, or liquid, in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, gelatin capsules, suppositories, sterile injectable solutions, transdermal patches, and sterile packaged powders, where appropriate.
Examples of suitable excipients include but are not limited to lactose, dextrose (glucose), sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose and sterile water.
Pharmaceutical compositions may additionally comprise lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates;
sweetening agents; and flavoring agents. The pharmaceutical compositions may be formulated to provide quick, sustained or delayed release of the active ingredients) following administration to the patient. Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences.
The reovirus or the pharmaceutical composition comprising the reovirus may be packaged into convenient kits providing the necessary materials packaged into suitable containers. It is contemplated the kits may also include chemotherapeutic agents and/or anti-antireovirus antibody.
In order to further illustrate the present invention and advantages thereof, the following specific examples are given but are not meant to limit the scope of the claims in any way.
EXAMPLES
In the examples below, the following abbreviations have the following meanings. Abbreviations not defined have their generally accepted meanings:
~1 microliter B-cell CLL B-cell chronic lymphocytic leukemia BL Burkitt's lymphoma CLL chronic lymphocytic leukemia CPE cytopathic effects DLBCL diffuse large B-cell lymphoma EBV Epstein-Barr virus EBV- Epstein-Barr virus not detected EBV+ Epstein-Barr virus detected FL follicular lymphoma HL Hodgkin's lymphoma MCL mantle-cell lymphoma mm2 square millimeters MOI multiplicity of infection n number of test subjects in a particular group NHL non-Hodgkin's lymphoma PBMC primary blood mononuclear cells PBS phosphate-buffered saline PFU plaque forming units SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SLL small lymphocytic leukemia UV ultraviolet Example l: Susceptibility Of Various Lymphoid Cell Lines To Reovirus Infection.
To determine the susceptibility of lymphoid cells to reovirus infection, a panel of lymphoma-derived cells was assembled for challenge with reovirus. The panel included EBV+ BL cells (Raji and Daudi), EBV- BL cells (CA46, Ramos, and ST486), and DLBCL cells (OCY-LY1, OCY-LY2, OCY-LYB, and OCY-LY10).
About 106 cells of each type were challenged with reovirus type 3 at a MOI of 20. While no morphological changes were detected in Daudi, Ramos, or ST486 cells 96 hours post-infection, Raji, CA46, and all four lines of DLBCL cells exhibited CPE.
Cells with CPE were determined to have 40-70% reduced viability,.based on trypan blue exclusion straining (Figure 1).
Virus replication in infected cells was also assayed by pulse labeling infected cultures with [35S]-methionine for six hours, followed by immunoprecipitating the labeled extracts with a rabbit polyclonal antireovirus type 3 antibody. The immune complexes were then analyzed by SDS-PAGE, and the results visualized by autoradiography. Reovirus proteins were observed in Raji, CA46, and all DLBCL
cells but not in Daudi, Ramos, or ST486 cells, consistent with the pattern of CPE
observed in the cells.
The results of the above experiments are summarized in Table 1, below.
These results demonstrate that reovirus was able to replicate in six out of nine lymphoid malignancy-derived cell lines, with CPE corresponding to the presence of virus proteins. The presence of virus protein further indicates that virus translation in Raji, CA46, and the DLBCL cells is not blocked by PKR activation.
Table 1: Susceptibility of lymphoid cell lines to reovirus Cell line Cell type Reovirus replication Raji Burkitt's lymphoma, EBV+Yes Daudi Burkitt's lymphoma, EBV+No CA46 Burkitt's lymphoma, EBV-Yes Ramos Burkitt's lymphoma, EBV-No ST486 Burkitt's lymphoma, No EBV-OCY-LY1 Diffuse large B-cell Yes lymphoma OCY-LY2 Diffuse large B-cell Yes lymphoma OCY-LY8 Diffuse large B-cell Yes lymphoma OCY-LY10 Diffuse large B-cell Yes lymphoma Example 2: Reovirus Infection Of Primary Lymphoma Cells.
A total of 27 lymphoid tumor biopsy specimens were obtained for the preparation of cell suspensions for reovirus challenge. The specimens included peripheral blood, bone marrow, lymph nodes, or other tissues. In the case of solid biopsy specimens, the tumor masses were disrupted to obtain cell suspensions.
Of the 27 biopsy specimens, 15 were associated with a clinical diagnosis of CLL. The remaining 12 samples were associated with a clinical diagnosis of NHL
and could be further divided into BL (1); DLBCL (2); SLL (2); FL, grade I (1);
FL, grade II (4); FL, grade III ( 1 ); and MCL ( 1 ). PBMC {n=3) and CD34+-hematopoietic stem/progenitor cells (n=3) from normal individuals were used as negative controls.
About 106 cells from each sample were infected with reovirus at a MOI of 20 then pulse labeled, immunoprecipitated, and resolved by SDS-PAGE, as described in Example I. Reovirus failed to replicate in the control PBMC and CD34+ cells but appeared to replicate in 15/IS CLL samples and 6/I2 NHL samples (i.e., 1/1 BL;

DLBCL; 1/2 SLL; 1/1 FL, grade I; 0/4 FL, grade II; 0/1 FL, grade III; and 1/1 MCL).
The results are shown in Figure 2 and summarized in Table 2, below.

Table 2: Susceptibility of lymphoid biopsy specimens to reovirus Disease Cell type Total SusceptibleResistant s ecimens s ecimenss ecimens CLL Chronic lymphocytic 15 15 0 leukemia NHL Burkitt's lym homa 1 1 0 NHL Diffuse largeB-cell 2 2 0 lymphoma NHL Small lym hoc is 1 2 1 1 homa NHL Follicular lym homa, 1 1 0 grade I

NHL Follicular lymphoma, 4 0 4 grade II

NHL Follicular lym homa, 0 0 1 ade III

NHL Mantle-celllyrnphoma 1 ~ 1 ~ 0 The CLL cells, as well as the BL cells, were also analyzed by flow cytometry, before and after infection, to identify the population of cells killed by reovirus infection. CLL cells are characterized by expression of the CDS and CD20 cell-surface markers (i.e., the cells are CDS+/CD20+) (Hulkkonen et al. 2002). BL
cells are characterized by expression of the CD10 and CD20 cell-surface markers (i.e., the cells are CD10+/CD20+) (Nakamura et al. 2002). Before infection and at approximately 96 hours post-infection, cells were washed with PBS then incubated with CD10, CDS, and CD20-specific antibodies in the presence of 7-amino-actinomycin D, for 1 S minutes at room temperature, in the dark. The cells were then washed and resuspended in PBS.
Flow cytometry of CLL cell populations before and after infection revealed significant reductions in CDS+/CD20+ cells, but not other cells, as a result of reovirus infection, indicating that CLL cells were selectively killed as a result of reovirus infection. Similarly, flow cytometry a BL cell population before and after infection revealed a significant reduction in CD 10+/CD20+ cells, but not other cells, as a result of reovirus infection, indicating that BL cells were selectively killed as a result of reovirus infection.
These results show that reovirus is able to replicate in cells of CLL and NHL
lymphoid malignancies, taken directly from biopsy specimens, and that malignant cells are selectively killed as a result of infection.

Examule 3: Efficacy Of Reovirus Treatment On Lymphoid Tumors In A
Xenograft Model.
A murine xenograft model was used to evaluate the ability of reovirus to treat lymphoma-derived tumors in vivo. About 107 Raji or Daudi cells in about 100 pl PBS
were administered by subcutaneous injection in the hind flank of 6-8-week old SCID/NOD mice. Once palpable tumor masses were established, animals received either live or UV-inactivated reovirus by either intratumoral or intravenous injection (day 0).
Animals receiving intratumoral reovirus were injected with approximately 107 PFU of live (n=8) or UV-inactivated (n=7) reovirus in 50 pl PBS, delivered to the tumor masses. Tumors size was measured every other day for 30 days or until animals showed excess tumor burden.
Animals receiving intravenous reovirus were injected with either 107 (n=7) or 5x107 (n=7) PFU reovirus, or no reovirus (n=7) in 100 p,l saline solution, delivered into the tail vain. Tumors size was measured every other day for 20 days or until animals showed excess tumor burden. The results are shown in Figure 3.
The growth of Raji-derived tumors was reduced at least 10-fold (in terms of tumor area, expressed in mm2) by intratumoral administration of live reovirus.
UV-inactivated reovirus had no effect on tumor size (Figure 3A). Daudi tumors were resistant to reovirus treatment (Figure 3B). The growth of Raji-derived tumors was reduced about a 5-fold following intravenous administration of live reovirus at either of the concentrations tested. UV-inactivated reovirus had no effect on tumor size (Figure 3C).
Hematoxylin and eosin staining of paraffin-embedded sections prepared at day 20 days following live or UV-inactivated reovirus administration confirmed the killing of Raji tumor cells in animals treated with live reovirus.
Immunohistochemical staining using an antireovirus polyclonal antibody and avidin biotin horseradish peroxidase color-development system (Vector, Burlingame, CA) confirmed the presence of reovirus proteins in residual tumor cells, confirming virus replication (data not shown).
These results show that reovirus was able to infect and kill human Burkitt's Lymphoma cells (Raji) in vivo following either intratumoral or intravenous admini stration.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (19)

What is claimed is

1. A method of treating a lymphoid malignancy in an animal, the method comprising administering to cells of the lymphoid malignancy an amount of reovirus sufficient to cause substantial lysis of the cells.

2. The method of claim 1, wherein the lymphoid malignancy is selected from the group consisting of Burkitt's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

3. The method of claim 2, wherein the non-Hodgkin's lymphoma is selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, and small lymphocytic lymphoma.

4. The method of claim 1, wherein the cells of the lymphoid malignancy comprise an activated Ras pathway.

5. The method of claim 4, wherein cells of the lymphoid malignancy comprise a normal Ras gene.

6. The method of claim 1, wherein the reovirus is administered by a route selected from the group consisting of intramedullar, intravascular, intrathecal, intravenous, intramuscular, subcutaneous, intraperitoneal, topical, oral, rectal, vaginal, nasal, and intratumoral routes.

7. The method of claim 1, wherein more than one type of reovirus is administered.

8. The method of claim 1, wherein more than one strain of reovirus is administered.

9. The method of claim 1, wherein the reovirus is one or more recombinant reoviruses.

14. The method of claim 1, wherein the reovirus is administered in a single dose.

11. The method of claim 1, wherein the reovirus is administered in more than one dose.

12. The method of claim 1, wherein the animal is a mammal.

13. The method of claim 12, wherein said mammal is selected from the group consisting of dogs, cats, sheep, goats, cattle, horses, pigs, humans, and non-human primates.

14. A method for selectively killing cells of a lymphoid malignancy in a cellular composition suspected of containing such cells, the method comprising administering reovirus to cells of the cellular composition under conditions that result in substantial lysis of the cells of the lymphoid malignancy.

15. The method of claim 14 wherein the cellular composition is selected from the group consisting of hematopoietic tissue and transplant tissue.

16. The method of claim 15 wherein the hematopoietic tissue is selected from the group consisting of bone marrow, spleen, mucosa-associated lymphoid tissues (MALT), lymph nodes, Peyer's patches, thymus, tonsils, and fetal liver.

17. The method of claim 14, wherein reovirus is administered to cells of the cellular composition by a route selected from the group consisting of intramedullar, intravascular, intrathecal, intravenous, intramuscular, subcutaneous, intraperitoneal, topical, oral, rectal, vaginal, nasal, and intratumoral routes.

18. The method of claim 14 wherein cells of a cellular composition are removed from an animal, contacted with reovirus, then returned to an animal.

19. The method of claim 14, comprising the additional step of inactivating the reovirus following the step of contacting the cellular composition with reovirus under conditions which results in substantial lysis of the cells of the lymphoid malignancy.