CA2436196A1 - Oncolytic virus for purging cellular compositions of cells of lymphoid malignancies - Google Patents

Abstract

The present invention relates to a method for removing cells of a lymphoid malignancy from a mixed cellular composition using a virus that selectively replicates in cells of the lymphoid malignancy. A variety of viruses can be used to practice the invention, including but not limited to reovirus, adenovirus, and herpesvirus.
The invention is particularly useful for purging mixed cellular compositions comprising hematopoietic stem of cells of lymphoid malignancies prior to reintroduction or transplantation into an animal.

Description

ONCOLYTIC VIRUS FOR PURGING CELLULAR COMPOSITIONS OF CELLS
OF LYMPHOID MALIGNANCIES
FIELD OF THE INVENTION
The present invention relates to a method of selectively removing neoplastic cells from a mixed cellular composition. Also provided are compositions prepared according to this method, and kits comprising viruses useful for practicing the invention.
REFERENCES
The following publications, patent applications, and patents are cited in this application:
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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.
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(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.
Spyridonidis, A., Bernhardt, W., Fetscher, S., Behringer, D., Mertelsmann, R., Henschler, R. (1998) Minimal residual disease in autologous hematopoietic harvests from breast cancer patients. Ann. Oncol. 9:821-26.
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.
Stewart, D.A., et al. (1999) Superior autologous blood stem cell mobilization from dose-intensive cyclophosphamide, etoposide, cisplatin plus G-CSF than from less intensive chemotherapy regimens. Bone Marrow Transplant. 23: 111-117.
Stojdl, D.F., Lichty, B., Knowles, S., Marius, R., Atkins, H., Sonenberg, N., Bell, J.C. (2000) Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat. Med. 6:821-25.
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Winter, J.N. (1999) High-dose therapy with stem-cell transplantation in the malignant lymphomas. Oncology (Huntingt.) 13:1635-45.
Yoon, S.S., Nakamura, H., Carroll, N.M., Bode, B.P., Chiocca, E.A., Tanabe, K.K. (2000) An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma. FASEB J.
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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.
BACKGROUND OF THE INVENTION
Cell proliferation is regulated by a combination of growth-promoting and growth-inhibiting signals. Cells that fails to respond to growth-inhibiting signals, or over-respond to growth-promoting signals, may proliferate abnormally rapidly (referred to as neoplastic development) and may eventually develop into cancer (a malignant neoplasm).
Chemotherapy and radiation therapy are current methods of treating cancer, generally based on the rapid proliferation of cancer cells compared to most other cells in the body. Rapidly proliferating cells are more sensitive to drugs that interfere with cell proliferation, such as drugs that interfere with DNA synthesis. In theory, by carefully choosing the dosage of chemotherapeutic drugs or ionizing radiation delivered to the body, one can inhibit cancer cell proliferation without seriously damaging normal cells. However, some normal cells, such as hematopoietic stem cells, also proliferate rapidly. Therefore, any dosage that is harmful to cancer cells is likely harmful to hematopoietic stem cells.
Because it is difficult to find a chemotherapeutic drug or radiation treatment regimen that selectively kills cancer cells without causing unacceptable damage to normal cells, high-dose chemotherapy or radiation therapy, followed by autologous hematopoietic progenitor stem cell transplantation, has gained acceptance as a therapeutic approach for treating many cancers (see, e.g., Winter 1999; Nieto and Shpall 1999). According to this approach, a portion of a cancer patient's hematopoietic stem cells are removed prior to high-dose chemotherapy or radiation therapy, which will be lethal to most all rapid-proliferating cells, including cancer cells and hematopoietic stem cells. Following chemotherapy or radiation therapy, the patient's own hematopoietic stem cells (i.e., an autograft) are returned to hematopoietic centers in the body, e.g., the bone marrow, in the hope that they will repopulate the patient's immune system (these processes may be referred to herein as "reintroduction" and "repopulation," respectively).
A serious drawback of this type of therapy is that when the hematopoietic progenitor stem cells are removed from the patients, they are often contaminated with cancer cells, which will be returned to the body along with the hematopoietic cells.
This complication is especially problematic when the patient has a cancer of hematopoietic origin, although patients with solid tumors may still harbor cancer cell-contaminated hematopoietic stem cells, particularly in the case of solid tumors that have metastasized. It is therefore desirable to purge autografts of cancer cells prior to reintroduction to the patient.
Several methods have been employed to purge autografts of cancer cells (Spyridonidis et al. 1998; Bensinger 1998). The autograft can be treated ex vivo with chemotherapeutic drugs (or radiation) to kill the contaminating neoplastic cells.
However, as discussed above, it is difficult to find a therapeutic dose that selectively kills cancer cells without causing an unacceptable level of damage to hematopoietic stem cells. Autografts can also be treated with a toxin conjugated to one or more antibodies that recognize antigens specific for cancer cells. However, tumor-specific antigens are not always available, depending on the specific form of cancer involved.
It is also possible to isolate stem cells from other cells in the autograft, including cancer cells, based on stem cell-specific surface markers (e.g., CD34) using flow cytometry, affinity chromatography, or magnetic bead selection, along with appropriate antibodies. However, by selecting only certain hematopoietic cells (e.g., CD34+ cells), other hematopoietic stem cells, including less differentiated CD34- cells and more differentiated but still immature precursor cells, are also eliminated, thereby delaying or preventing the full recovery of the patient's immune system (Bensinger 1998). In addition, this procedure may result in the loss of about half the CD34+ cells while failing to remove all contaminating cancer cells (Spyridonidis et al.
1998).
Therefore, there remains a need for a highly selective and efficient method for purging autografts of neoplastic cells.
SUMMARY OF THE INVENTION
The present invention relates to a method for removing cells of a lymphoid malignancy from a mixed cellular composition using a virus that selectively replicates in cells of the lymphoid malignancy. The invention is particularly useful for purging mixed cellular compositions comprising hematopoietic stem-cells of cells of lymphoid malignancies. The purged, virus-treated hematopoietic cell compositions may then be reintroduced to the same animal or transplanted into a second animal, most likely following chemotherapy or radiation therapy.
Accordingly, the invention provides a method of selectively removing cells of a lymphoid malignancy from a mixed cellular composition, comprising the steps of a) obtaining a mixed cellular population suspected of containing cells of a lymphoid malignancy from an animal, b) contacting the mixed cellular composition with at least one virus under conditions that result in substantial killing of cells of the lymphoid malignancy, thereby producing a treated cellular composition; and c) collecting the treated cellular composition so produced.

In one embodiment of the invention, the mixed cellular composition comprises hematopoietic stem cells. In a preferred embodiment, the mixed cellular composition comprises CD34+ cells. In one embodiment of the invention, the mixed cellular composition has been harvested from bone marrow. In another embodiment, the mixed cellular composition has been harvested from blood. In yet another embodiment, the mixed cellular composition is selected from the group consisting of a tissue, an organ, or a portion of a tissue or organ, wherein the corresponding treated cellular composition is useful for transplantation. In another embodiment, the mixed cellular composition comprises cultured cells, semen, or eggs. In a preferred embodiment of the invention, steps (a) and (b), above, are performed ex vivo.
In one embodiment of the invention, the method is useful for treating a mixed cellular population comprising (or suspected of comprising) cells of at least one lymphoid malignancy. In a preferred embodiment of the invention, the lymphoid malignancy is a B-cell lymphoid malignancy. Examples of B-cell malignancies that can be treated by the instant invention include but are not limited to Burkitt's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, and mantle-cell lymphoma. The cells of the lymphoid malignancy may comprise normal ras genes.
Numerous different viruses can be used to practice the invention. In one embodiment, the virus or viruses are replication competent. In a preferred embodiment of the invention, at least one reovirus is used. The reovirus may be a human reovirus, a non-human reovirus, a modified reovirus, a recombinant reovirus, combinations thereof, or chimeras thereof. In another embodiment of the invention, at least one virus is an adenovirus, herpesvirus, vaccinia virus, parapoxvirus orf virus, Newcastle disease virus, or vesicular stomatitis virus.
In the case of adenovirus, the E 1 A-coding region may be mutated such that the resulting ElA gene product does not bind to Rb. Alternatively or additionally, the adenovirus may be mutated in the E 1 B-coding region such that the resulting E

gene product does not bind to p53. The adenovirus may also be capable of expressing a wild type p53 protein. Examples of adenovirus useful for practicing the instant invention include but are not limited to Delta24 and ONYX-O1 S.

In another embodiment of the invention, at least one virus is mutated or modified such that the virus does not produce a gene product that inhibits double stranded RNA kinase (PKR). In another embodiment of the invention, the virus is an interferon sensitive virus and the method further comprises a step of adding interferon to the mixed cellular composition.
In another embodiment of the invention, virus is removed from the mixed cellular composition or inactivated in the mixed cellular composition following treatment.
In one embodiment of the invention, the mixed cellular composition is stored either before or after treatment with virus. In a preferred embodiment, the treated cellular composition is stored. In a most preferred embodiment, the treated cellular composition is stored in liquid nitrogen in a solution comprising DMSO.
In a preferred embodiment of the invention, the method further comprises the step of reintroducing the treated cellular composition to the animal from which it was obtained. In a most preferred embodiment of the invention, such an animal is treated with chemotherapeutic drugs or radiation therapy following the harvesting of the mixed cellular composition but prior to the reintroduction of the treated cellular composition. In one embodiment of the invention, the treated cellular composition is reintroduced into the bone marrow of the animal. In another embodiment of the invention, the treated cellular composition is reintroduced into the blood of the animal.
In a variation of this preferred embodiment of the invention, virus is administered to an animal or patient following the harvesting of a mixed cellular composition from the animal or patient but prior to treatment of the animal or patient with chemotherapy or radiation therapy. The animal or patient is then allowed to produce neutralizing antibodies to the virus before being treated with chemotherapy or radiation therapy. The mixed cellular composition is treated with the same virus as administered to the animal or patient prior to chemotherapy or radiation therapy, or a variant thereof, providing that both viruses are recognized by at least some of the same antibodies produced in the animal or patient. When the treated cellular composition is returned to the animal or patient, the animal or patient will already possess circulating antibodies that will recognize and inactivate virus present in the treated cellular composition. In one particular embodiment of this invention, virus present in the treated cellular composition is inactivated primarily by these circulating antibodies. In another particular embodiment, circulating antibodies remove only residual virus that was not completely inactivated or removed from the treated cellular composition by other means, prior to reintroduction to the animal or patient.
In another embodiment of the invention, a composition comprising anti-virus antibodies is administered to the animal or patient prior to, simultaneously with, or soon after, the treated cellular composition is reintroduced to the animal or patient.
These antibodies recognize and inactivate virus present in the treated cellular composition though passive immunity.
In yet another embodiment of the invention, the treated cellular composition is administered to at least one second animal, wherein the second animal is not the animal from which the mixed cellular composition was harvested. In a preferred embodiment of the invention, the second animal is genetically compatible with the animal from which the mixed cellular composition was harvested. In one embodiment of the invention, immunosuppresants are administered to the second animal to prevent or minimize rejection of the transplanted cellular composition.
The invention may be administered to a variety of different animals. In a preferred embodiment of the invention, the animal is selected from the group consisting of dogs, cats, sheep, goats, cattle, horses, pigs, humans, and non-human primates.
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-LYB, and OCY-LY10) following challenge with reovirus at an 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-LYB, and OCY-LY10). Growth is reported in PFUs at 0 hour (open squares) and 96 hours (filled squares) post infection.
Figure 3: Graphs showing the sizes of lymphoma-derived tumors implanted in mice following infection with either live (filled symbols) or UV-inactivated (open circles) reovirus. Panel A: Raji tumors, intratumoral virus administration.
Panel B:
Daudi tumors, intratumoral virus administration. Panel C: Raji tumors, intravenous virus injection.
Figure 4: Bar graphs showing cell viability following reovirus infection.
Panels A-C show the purging effects of reovirus on mixed cellular compositions comprising apheresis product and tumor cells selected from the group consisting of MCF7, MDA MB 468 and SKBR3 cells. Panel D shows a control experiment in which CD34+ cells were infected with reovirus.
Figure 5: Bar graph showing the number of colonies of different cell types originating from CD34+ cells at various times following reovirus infection.
NV: no virus. LV: live virus. G: granulocytes. E: erythroids. GEMM: granulocyte erythroid macrophage megakaryocyte.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of the following description, the following terms have been given the following meanings unless otherwise indicated.
Definitions 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.
Adenovirus: Double-stranded DNA (dsDNA) viruses of the family Adenoviridae. In humans, adenoviruses can replicate and cause disease in the eye and in the respiratory, gastrointestinal, and urinary tracts. About one-third of the 47 known human serotypes are responsible for most human diseases associated with adenovirus (Brooks et al. 1998).
Apheresis: A method of isolating specific fractions of whole blood wherein whole blood is withdrawn from an animal or donor and separated into various components. The desired components are then retained while the remainder is returned to the animal or donor.
Burkitt's lymphoma (BL): A B-cell lymphoma, usually caused by Epstein-Barr virus (EBV).
Cellular composition suspected of containing neoplastic cells: A cellular composition that may contain neoplastic cells. For example, any autograft obtained from a patient, clinical test subject, or experimental animal known to have or suspected of having a neoplastic disorder. Cells that have been growing in culture for a considerable amount of time may also give rise to neoplastic cells as a result of spontaneous mutations.
Cellular composition: Any cells or 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, liver, bursa of Fabricus (in Avian species), mucosa-associated lymphoid tissues (MALT), spleen, whole blood, and other mixtures of animals cells from various tissues or organs, as well as fractions and/or combinations thereof.
Chronic lymphocytic leukemia (CLL): A proliferative disorder resulting from the accumulation of mature lymphocytes in the blood andlor bone marrow.
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.
Diffuse large B-cell lymphoma (DLBCL): An aggressive malignancy of mature B-lymphocytes accounting for about 40-50% of non-Hodgkin's lymphomas.

Ex vivo: Outside the body. As use herein, ex vivo refers to a process that is performed under cell culture conditions using a cellular composition obtained from an animal. Following manipulation of the mixed cellular composition (e.g., treatment with one or more viruses), the treated cellular composition may be reintroduced to the same animal or administered to at least one second animal.
Follicular lymphoma (FL): A malignancy of follicular center B-cells accounting for about 25-40% of non-Hodgkin's lymphomas.
Genetically compatible: As used herein, "genetically compatible" refers to animals that express substantially identical cell surface antigens (or major histocompatability markers (MHC markers)) on their cell surfaces.
Hematopoietic stem/progenitor cells: Undifferentiated and/or partially differentiated cells cable of differentiating into a variety of different hematopoietic lineages, including myeloid and lymphoid cells. The presence of CD34 is often used as a marker for hematopoietic stem/progenitor cells (i.e., CD34+ cells), although more primitive, less differentiated, hematopoietic precursor cells may actually lack CD34 (i.e., CD34- cells).
Herpesvirus: Members of the family Herpesviridae, including but not limited to herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), Kaposi's sarcoma-associated virus (KSHV), and other numbered herpesviruses (e.g., HHV-6, 7, etc.) of humans, pigs, cattle, horses, chickens, turkeys, and other animals. HSV-1 and are the best studied herpesviruses. HSV gene Y134.5 encodes the gene product infected-cell protein 34.5 (ICP34.5) that can prevent the antiviral effects exerted by PKR. ICP34.5 has a unique mechanism of preventing PKR activity by interacting with protein phosphatase 1 and redirecting its activity to dephosphorylate eIF-2a (He et al. 1997). In cells infected with either wild-type or the genetically engineered virus from which the y,34.5 genes were deleted, eIF-2oc is phosphorylated and protein synthesis is turned off in cells infected with y~ 34.5-minus virus. It would be expected that the Y134.5 minus virus would be replication competent in cells with an activated Ras pathway in which the activity of ICP34.5 would be redundant.

Hodgkin's lymphoma (HL) (or Hodgkin's disease): A lymphoma characterized by the presence of mononucleated Hodgkin and multinucleated Reed-Sternberg cells (HRS), which occur at low frequency in tumor tissues.
Interferon sensitive virus: A virus that does not replicate in or kill normal cells in the presence of interferon. As defined below, a normal cell is a cell that is not neoplastic. To test whether a virus is interferon sensitive, a culture of normal cells may be incubated with the virus in the presence of varying concentrations of interferon, and the survival rate of the cells is determined according to well-known methods in the art. A virus is interferon sensitive if less than 20%, preferably less than 10%, of the normal cells is killed at a high concentration of interferon (e.g., 100 units per ml).
Intramedullary: Within or pertaining to the bone marrow of an animal.
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. Examples of lymphoid malignancies 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. 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 ).
Mantle-cell lymphoma (MCL): A lymphoma arising 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, primarily affects seniors, and accounts for 2% to 8% of non-Hodgkin lymphomas.

Mixed cellular (or cell) composition: A cellular composition comprising at least two kinds of cells. Typically, the mixed cellular composition comprises both normal and neoplastic cells. It is preferable that most of the cells in the cellular composition are dividing cells and a virus selectively replicates in the neoplastic cells.
Mutated (or modified) adenovirus: As used herein, mutated adenoviruses are those in which the gene product (or products) that prevent the activation of PKR are lacking, inhibited, or mutated such that PKR activation is not blocked.
Adenovirus encodes several gene products that counter antiviral host defense mechanisms.
The virus-associated RNA (VAI RNA or VA RNAI) of the adenovirus are small, structured RNAs that accumulate in high concentrations in the cytoplasm at late time after adenovirus infection. These VAI RNA bind to the double stranded RNA
(dsRNA) binding motifs of PKR and block the dsRNA-dependent activation of PKR
by autophosphorylation. Thus, PKR is not able to function and the virus can replicate within the cell. The overproduction of virons eventually leads to cell death.
In a mutated or modified adenovirus, the VAI RNA's are preferably not transcribed.
Such mutated or modified adenovirus would not be able to replicate in normal cells that do not have an activated Ras-pathway; however, it would be able to infect and replicate in cells having an activated Ras-pathway.
Mutated (or modified) HSV: As used herein, mutated or modified HSV are those herpesviruses in which the gene product (or products) which prevent the activation of PKR are lacking, inhibited or mutated such that PKR activation is not blocked. Preferably, the HSV gene y134.5 is not transcribed. Such mutated or modified HSV would not be able to replicate in normal cells that do not have an activated Ras-pathway, however, it would be able to infect and replicate in cells having an activated Ras-pathway.
Mutated (or modified) parapoxvirus orf: A parapoxvirus orf virus in which the gene product or products that prevent activation of PKR are lacking, inhibited, or mutated such that PKR activation is not blocked. Preferably, the gene OV20.OL
is not transcribed. Such mutated or modified parapoxvirus orf viruses would not be able to replicate in normal cells that do not have an activated Ras-pathway; however, they would be able to infect and replicate in cells having an activated Ras-pathway.

Mutated (or modified) vaccinia virus: As used herein, these terms refer to vaccinia virus variants in which the gene product or products that prevent the activation of PKR are lacking, inhibited or mutated, such that PKR activation is not blocked. Preferably, the E3L gene and/or the K3L gene is not transcribed. Such mutated or modified vaccinia viruses are unable to replicate in normal cells that do not have activated ras pathways; however, they are able to infect and replicate in cells having activated ras pathways.
Neoplasm: As used herein, neoplasm is used to describe benign or malignant tumors. As used herein, malignant tumors may include various forms of cancer.
Malignant neoplasms/tumors can be broadly classified into three major types.
Malignant neoplasms arising from epithelial structures are called carcinomas;
malignant neoplasms that originate from connective tissues such as muscle, cartilage, fat or bone are called sarcomas; and malignant tumors arising from hematopoietic cells, are called leukemias and lymphomas. Other neoplasms include but are not limited to neurofibromatosis.
Neoplastic cells: Cells that proliferate aberrantly with respect to their normal counterparts and typically do not respond to growth inhibition signals. A new growth comprising neoplastic cells is a neoplasm or tumor. A neoplasm is an abnormal tissue growth, generally forming a distinct mass, which grows more rapidly than normal cells. Neoplasms may show partial or total lack of structural organization and functional coordination with normal tissue. As used herein, a neoplasm is intended to encompass hematopoietic neoplasms as well as solid neoplasms. Also as used herein, the term "neoplastic cells," or substantially similar terms, refer to cells with proliferative disorders.
Non-Hodgkin's lymphomas (NHL): A heterogeneous group of lymphoid malignancies arising from mature lymphoid cells.
Normal cell: A cell that that is not neoplastic, responds to growth stimulatory and inhibitory signals in a manner typical for the particular cell type, does not cause tumors in animals, and is not believed to harbor chromosomal abnormalities that would result in a neoplastic phenotype.

Normal hematopoietic stem/progenitor cells: Hematopoietic stem/progenitor cells that are not associated with a cell proliferative disorder or neoplasm and are not believed to harbor chromosomal abnormalities that would cause such growth phenotypes.
Normal lymphocytes: Lymphocytes that are 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.
Parapoxvirus orf A poxvirus that induces acute cutaneous lesions in different mammalian species, including humans. Parapoxvirus orf viruses naturally infect sheep, goats, and humans through broken or damaged skin, replicates in regenerating epidermal cells, and induces pustular lesions that turn to scabs. The parapoxvirus orf viruses encode the gene OV20.OL that is involved in blocking PKR activity (Haig et al. 1998).
Pluripotency: Potential to differentiate into different cell types. As used herein, pluripotency refers to cells that have broad "differentiation potential" with respect to the type of cells that may arise from the undifferentiated cells.
Ras-activated neoplastic cells or ras-mediated neoplastic cells: Cells that proliferate at an abnormally high rate due to, at least in part, activation of the ras pathway. The ras pathway may be activated by way of ras gene structural mutation, elevated level of ras gene expression, elevated 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, thereby increasing the ras pathway activity. For example, activation of EGF receptor, PDGF receptor or Sos results in activation of the ras pathway. Ras-mediated neoplastic cells include, but are not limited to, ras-mediated cancer cells, which are cells proliferating in a malignant manner due to activation of 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).
Replication competent virus: A virus that is capable of replicating in at least one cell type.
Resistance of cells to viral infection: Infection of the cells with the particular virus or viruses does not result in significant viral production or yield.
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 one or more loci 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 or from a mixed cellular composition.

Transplant recipient: An animal, including but not limited to dogs, cats, sheep, goats, cattle, horses, pigs, humans, and non-human primates, that receives a transplant of one or more cellular compositions. Preferably the recipient is a human, and more preferably the recipient is a human who is receiving a transplant in the treatment of cancer.
Vaccinia virus: Viruses of the orthopoxvirus genus that infect humans and produce localized lesions (Brooks et al. 1998). Vaccinia virus encodes two genes that play a role in the down regulation of PKR activity through two entirely different mechanisms. E3L gene encodes two proteins of 20 and 25 kDa that are expressed early in infection and have dsRNA binding activity that can inhibit PKR
activity.
Deletion or disruption of the E3L gene creates permissive viral replication in cells having an activated Ras pathway. The K3L gene of vaccinia virus encodes pK3, a pseudosubstrate of PKR.
Viral oncolysate: A composition prepared by treating tumor cells with an oncolytic virus in vitro, which composition is subsequently administered to a tumor patient with the same kind of tumor in order to induce immunity in the tumor patient against this tumor. As such, viral oncolysates are essentially virus-modified cancer cell membranes.
The Invention:
The present invention is directed to a method for selectively removing neoplastic cells from a mixed cellular composition, for example an autograft, by using a virus that selectively replicates in neoplastic cells. The instant invention is based, in part, on Applicant's discovery that reovirus is capable of replicating in 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 cells of lymphoid malignancies 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-LY8, and OCY-LY10). Reovirus replicated in six of the nine lymphoma-derived cell lines, specifically Raji, CA46, and all four DLBCL cells (Figure 1). Reovirus was unable to replicate in only three of these cell lines (Daudi, Ramos, and ST486). This experiment is described in more detail in Example 1.
In a second experiment, cell suspensions were prepared from 27 lymphoid tumor biopsy specimens for challenge with reovirus. Of the 27 specimens, 15 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 S
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 (Figure 2). In total, reovirus was able to replicate in 21 of the 27 cell suspensions prepared from lymphoid tumor biopsy specimens. This experiment is described in more detail in Example 2.
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.

Following the establishment of palpable tumor masses, mice were treated with either live reovirus or LTV-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 UV-inactivated reovirus (Figure 3). 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.
This experiment is described in more detail in Example 3.
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.
It is also known that reovirus can selectively purge neoplastic cells from a mixed population of cells comprising CD34+ cells, with minimal effect on the viability and pluripotency of the CD34+ cells. Experiments supporting these conclusions are described in Examples 4 and 5.
In the experiment described in Example 4, cells of one of three neoplastic cell lines (i.e., MCF7, SKBR3, and MDA MB 468), known to be susceptible to reovirus infection (data not shown), were mixed with an apheresis product comprising CD34+
cells. The mixed cellular populations were then infected by reovirus and counted daily to determine the extent of cell death and the types of cells killed by reovirus. As shown in Figures 4A-4D, reovirus infection resulted in the selective killing of the neoplastic cells with minimal effect on CD34+ cells. These data indicate that reovirus may be effective in purging a cell population of neoplastic cells without affecting the viability of the normal cells.

To determine whether reovirus infection affected the pluripotency of the CD34+ cells, CD34+ cells were allowed to differentiate in culture after being exposed to reovirus. These cell cultures were compared to control cell cultures comprising control cells that had not been exposed to virus). As shown Figure 5, CD34+
cells gave rise to similar numbers of granulocutes (G), erythrocytes (E), or granulocyte erythroid macrophage megakaryocytes (GEMM) regardless of whether they were exposed to reovirus. Therefore, reovirus treatment did not change the differentiation potential of CD34+ cells.
These findings suggest that reovirus could be used to purge mixed cellular compositions of cells of lymphoid malignancies even when the cells of the lymphoid malignancies do not harbor ras mutations. Accordingly, the present invention provides the treatment of mixed cellular compositions comprising or suspected of comprising cells of a lymphoid malignancy in an animal, comprising the step of administering to the mixed cellular compositions animal an amount of reovirus sufficient to kill the cells of the lymphoid malignancy.
The amount of reovirus required for killing cells of a lymphoid malignancy in a mixed cellular composition depends on numerous factors, including but not limited to the type or strain of virus administered; the volume and cell density of the mixed cellular composition; the sensitivity of the cells of the lymphoid malignancy to virus infection, and the number of cells of the lymphoid malignancy present in the sample.
However, because the virus will replicate selectively in cells of the lymphoid malignancy, releasing progeny virus with the same specificity, the initial amount of reovirus that should be administered to a mixed cellular composition may encompass a wide range. Administration of an excessive number of virus particles is unlikely to cause toxic effects because of the blockage of virus 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 purging mixed cellular compositions of cells of lymphoid malignancies. In one embodiment of the invention, an effective amount of reovirus is from about 1.0 plaque forming unit (PFU)/kilogram (kg) sample weight (i.e., the sample weight of the mixed cellular composition) to about 1015 PFU/kg sample weight, more preferably from about 102 PFU/kg sample weight to about 1013 PFU/kg sample 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.
In one embodiment of the invention, the mixed cellular population is a hematopoietic stem cell-comprising autograft that is obtained from an animal or patient prior to the administration of a chemotherapeutic or radiotherapeutic regimen.
The autograft is treated with virus prior to transplantation to remove contaminating neoplastic cells. This "cleaned up" or "purged" autograft is returned to the animal or patient following high-dose chemotherapy/radiation therapy.
In a preferred embodiment of the invention, the mixed cellular composition is obtained from a hematopoietic center in the animal known to have or suspect of having a lymphoid malignancy. In a most preferred embodiment of the invention, the mixed cellular composition is obtained from the bone marrow of such an animal.
In another preferred embodiment of the invention, the mixed cellular composition is obtained from the blood of the animal. In another embodiment, the mixed cellular composition is an apheresis product.
The invention may be used to purge a mixed cellular composition of cells of a variety of lymphoid malignancies. In one embodiment, the invention is used to purge cells of a B-cell lymphoid malignancy. In another embodiment, the invention is used to purge cells of a T-cell lymphoid malignancy. In yet another embodiment of the invention, reovirus is used to purge cells of a lymphoid malignancy comprising both B-cells and T-cells, in any proportion. The invention may also be used to purge cells of 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 purge cells of a B-cell lymphoid malignancy such as Burkitt's lymphoma (BL). In another embodiment of the invention, reovirus is used to purge cells of 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 purge cells of small lymphocytic lymphoma (SLL) or mantle-cell lymphoma (MCL).
In a preferred embodiment of the invention, hematopoietic progenitor stem cells can be obtained from the bone marrow of an animal or patient.
Alternatively, hematopoietic stem cells may be obtained from the peripheral blood of an animal, with or without the use of colony stimulating factor priming. In the latter case, hematopoietic progenitor stem cells can be obtained from blood as an apheresis product, which may be stored for a long time before being transplanted. The present invention can be applied to stem cell-containing autografts that are harvested from any tissue source, including bone marrow and blood.
In addition to hematopoietic stem cells, the present invention can be broadly applied to remove neoplastic cells from many other cellular compositions. For example, reovirus can be used as a routine practice to "clean-up" any tissue or organ transplant prior to implantation.
Of particular interest will be the use of the claimed methods to clean-up whole blood or any portion thereof for a subsequent transfusion. Similarly, since tissue and organ transplantation has become increasingly common, it will be beneficial to clean-up the transplant tissues or organs to remove cells of lymphoid malignancies prior to transplantation. Liver, kidney, heart, cornea, skin graft, pancreatic islet cells, bone marrow, or any portions thereof are just a few examples of the tissues or organs to which this invention can be applied.
The tissue or organ can be autologous, allogeneic, or xenogeneic. The tissue or organ may also be derived from a transgenic animal, may be a tissue/organ that is developed in vitro from stem cells, or may be expanded ex vivo. The tissue or organ to be treated with reovirus can be from an embryonic or adult origin. For example, embryonic neuronal cells can be treated before being transplanted into an Alzheimer's patient. Similarly, the invention can be used to treat semen or donor eggs ex vivo.
In all instances of transplantation, a transplant recipient may be given treatments to stimulate the immune system and/or to reduce the risk of virus infection.

This treatment may be performed prior to, contemporaneously with, or after the transplantation, but is preferably performed prior to the transplantation.
Administering immunostimulants to a patient is particularly desirable when the treated cellular compositions are autografts, which should not invoke a graft-versus-host reaction upon reintroduction to the patient or animal.
Virus may also be administered to a transplant recipient following the harvesting of a mixed cellular composition but prior to chemotherapy or radiation therapy. The transplant recipient is then allowed to produce neutralizing antibodies to the virus before being treated with chemotherapy or radiation therapy. The mixed cellular composition (in this case, transplant tissue) is treated with the same virus as administered to the transplant recipient prior to chemotherapy or radiation therapy, or a variant thereof, providing that both viruses are recognized by at least some of the same antibodies produced in the transplant recipient. When the transplant tissue is given to the transplant recipient, circulating anti-virus antibodies will recognize and inactivate virus present in the transplant tissue. In another embodiment of the invention, the transplant recipient is administered with a composition comprising anti-virus antibodies that will recognize and inactivate virus present in the transplant tissues through passive immunity.
Application of the present invention is not limited to autografts and transplants. Rather, any cellular compositions can be "cleaned up" with reovirus for any purpose. Thus, all the examples described above or below are applicable even if the tissue or organ is not meant for transplantation.
Cell lines may also be treated routinely to safeguard against spontaneous or contaminating neoplastic cells. Again, any cell line will be a good candidate for this treatment, providing that reovirus, or another suitable virus, selectively grows in the neoplastic cells.
Numerous viruses may be used to practice the invention. In one embodiment of the invention, the virus is a reovirus. 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, chimera, recombinant, or otherwise engineered reovirus, or a reovirus comprising any combination of these viruses.
Such a recombinant reovirus may result from the recombination/reassortment of genomic segments from two or more genetically distinct reoviruses.
Recombination/reassortment 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 reassortment 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 COS1 cells will result in the incorporation of the mutated protein into the reovirus virion particle (Turner et al. 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 is particularly useful for selectively killing cells with an activated Ras pathway is 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 SS 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-O1 S 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 adenoviruses 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 (ICP34.5) of both type 1 and type 2 herpes simplex viruses (HSV) can also prevent the antiviral effects exerted by PKR. ICP34.5 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 ICP34.5 thereby allows herpesvirus to prevent or reverse PKR activation. Herpesviruses that lack or are unable to express ICP34.5 cannot replicate in cells with activated (i.e., phosphorylated) 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 ICP34.5-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 25 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-2oc 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.
Vesicular stomatitis virus (VSV) selectively kills neoplastic cells in the presence of interferon. Interferons are circulating factors which bind to cell surface receptors which ultimately lead to both an antiviral response and an induction of growth inhibitory and/or apoptotic signals in the target cells. Although interferons can theoretically be used to inhibit proliferation of tumor cells, this attempt has not been very successful because of tumor-specific mutations of members of the interferon pathway.
However, by disrupting the interferon pathway to avoid growth inhibition exerted by interferon, tumor cells may simultaneously compromise their anti-viral response. Indeed, it has been shown that VSV, an enveloped, negative-strand RNA
virus, rapidly replicated in and killed a variety of human tumor cell lines in the presence of interferon, while normal human primary cell cultures were apparently protected by interferon. Intratumoral injection of VSV also reduced tumor burden in nude mice bearing subcutaneous human melanoma xenografts (Stojdl et al. 2000).
Accordingly, in another embodiment of the present invention, VSV is used to remove neoplastic cells from a mixed cellular composition in the presence of interferon. Moreover, it is contemplated that this aspect of the invention be applied to any other interferon-sensitive virus (WO 99/18799), i.e., a virus that does not replicate in a normal cell in the presence of interferon. Such a virus may be identified by growing a culture of normal cells, contacting the culture with the virus of interest in the presence of varying concentrations of interferons, then determining the percentage of cell killing after a period of incubation.
It is also possible to take advantage of the fact that some neoplastic cells express high levels of particular enzymes and construct a virus that is dependent on these enzymes. For example, the enzyme ribonucleotide reductase is abundant in cells associated with liver metastases but is scarce in normal liver cells. In a related experiment, an HSV-1 mutant defective in ribonucleotide reductase expression (hrR3) was shown to replicate in colon carcinoma cells but not normal liver cells (Moon et al.
2000).
In addition to the viruses discussed above, a variety of other viruses have been associated with oncolysis, although the underlying mechanisms were not always apparent. For example, Newcastle disease virus (NDV), particularly the 73-T
strain, replicates preferentially in malignant cells (Reichard et al. 1992; Zorn et al 1994; Bar-Eli et al. 1996). NDV reduction of tumor burden after intratumor inoculation was also observed in cervical, colorectal, pancreatic, gastric, and renal cancer, in addition to melanoma (WO 94/25627; Nemunaitis 1999). Therefore, NDV can be used to remove neoplastic cells from a mixed cellular composition.
Tumor regression has also been described in tumor patients infected with, e.g., varicella-zoster virus (VZV), hepatitis virus, influenza virus, and measles virus (reviewed in Nemunaitis 1999).
According to the methods disclosed herein and techniques well known in the art, a skilled artisan can test the ability of any of the above viruses, or other viruses, to selectively kill cells of a lymphoid malignancy in a particular mixed cellular composition. If desired, a biopsy specimen (including but not limited to a bone marrow specimen or a blood sample) comprising cells of the lymphoid malignancies (or the suspected lymphoid malignancies) may be obtained from the patient or animal.
The biopsy specimen may be harvested in advance of chemotherapy or radiation treatment and tested with different viruses to determine which viruses efficiently kill the cells of the lymphoid malignancy. The Examples, below, describe assays useful for performing such tests, although other assays are known in the art and may be equally useful for such purposes. Based on results obtained with biopsy specimens, one or more viruses can be selected to practice the invention.
As an optional feature of many of the embodiments of the invention described herein, cellular compositions that have been treated with a virus, or are intended to be treated with a virus, are stored before reintroducing the treated cell compositions to the same animal or transplanting the treated cell composition into at least one second animal.
In a preferred embodiment of the invention, the mixed cellular composition to be treated with virus, or the treated cell composition, is frozen in a solution containing DMSO and thawed prior to reintroduction or transplantation. Freezing all or a portion of the cellular composition may allow more time to administer chemotherapy or radiation therapy or may provide a second opportunity to reintroduce or transplant a cellular population into a patient or animal if the first attempt fails to yield a suitable clinical outcome. In instances wherein the cellular compositions are frozen after virus treatment, freezing the compositions in DMSO may have the added advantage of inactivating the virus prior to transplantation.
In one embodiment of the invention, virus is administered to mixed cellular compositions in conjunction with standard techniques for purging hematopoietic mixed cellular compositions, including but not limited to chemotherapy and/or radiation therapy. Known chemotherapeutic agent include but are not limited to 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. 1n another embodiment of the invention, virus is administered mixed cellular compositions prior to or in place of a chemotherapeutic agent or radiation.
Prior to, contemporaneous with, or following reintroduction or transplantation of a treated cellular composition to an animal, immunosuppressants and/or immunoinhibitors may be given to the animal. The use of immunosuppressants and/or immunoinhibitors is particularly desirable when transplanting treated cellular compositions from one animal to another, even if such animals are genetically compatible. Immunosuppressants and immunoinhibitors known to those of skill in the art include but are not limited to such agents as cyclosporin, rapamycin, tacrolimus, mycophenolic acid, azathioprine and their analogs, and the like (see, e.g., Goodman and Gilman, 7'h Edition, page 1242, the disclosure of which is incorporated herein by reference).
Immunoinhibitors also include anti-antivirus antibodies, which are antibodies directed against anti-virus antibodies. Such anti-antivirus antibodies may be administered prior to, at the same time, or shortly after the reintroduction or transplantation of the virus. Preferably an effective amount of the anti-antivirus antibodies are administered in sufficient time to reduce or eliminate an immune response to residual live virus or residual virus antigens.
The invention includes pharmaceutical compositions that comprise, as an active ingredient, one or more of the above identified viruses associated with pharmaceutically acceptable Garners or excipients. The pharmaceutical compositions may also comprise an appropriate immunosuppresant or immunostimulant associated with pharmaceutically acceptable Garners 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 virus 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-antivirus antibodies. Such kits may comprise a number of different viruses, allowing the practitioner to determine which virus is most efficient in killing cells of a particular lymphoid malignancy, for example, using a biopsy specimen as described above.
The following Examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of the present invention.
Examples In the examples below, the following abbreviations have the following meanings. Abbreviations not defined have their generally accepted meanings:
pg microgram pl microliter pM micromolar B-cell B-cell chronic lymphocytic CLL leukemia BL Burkitt's lymphoma CLL chronic lymphocytic leukemia CPE cytopathic effects DLBCL diffuse large B-cell lymphoma DMEM Dulbecco's modified Eagle's medium DMSO dimethylsulfoxide DTT dithiothrietol EBV Epstein-Barr virus EBV- Epstein-Barn virus not detected EBV+ Epstein-Barr virus detected EGF epidermal growth factor FBS fetal bovine serum FL follicular lymphoma GCSF granulocyte colony stimulating factor HL Hodgkin's lymphoma hr hour M molar MCL mantle-cell lymphoma 2-ME 2-mercaptoethanol (also called ~3-ME) MEM -modif ed Eagle's medium mg milligram min minute ml milliliter mM millimolar mm2 square millimeters MOI multiplicity of infection n number of test subjects in a particular group NHL non-Hodgkin's lymphoma C degree Celsius PAGE polyacrylamide gel electrophoresis PBMC primary blood mononuclear cells PBS phosphate buffered saline PDGF platelet derived growth factor PFU plaque forming units PKR double-stranded RNA activated protein kinase rpm revolutions per minute SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SLL small lymphocytic leukemia UV ultraviolet Examyle 1: 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 l, 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, EBV-No 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-LY 10 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 1. Reovirus failed to replicate in the control PBMC and CD34+ cells but appeared to replicate in 15/15 CLL samples and 6/12 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 specimensspecimens specimens CLL Chronic lymphocytic 1 S 0 leukemia 15 NHL Burkitt's lymphoma 1 1 0 Diffuse largeB-cell lymphoma NHL Small lymphocytic lymphoma2 1 1 NHL Follicular lymphoma, 1 1 0 grade I

NHL Follicular lymphoma, 4 0 4 grade II

NHL Follicular lymphoma, 0 0 1 grade III

NHL Mantle-cell lymphoma 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 15 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 CD10+/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.
Example 3: Efficacy Of Reovirus Treatment On Lymphoid Tumors In A
Xenograft Model.
A marine xenograft model was used to evaluate the ability of reovirus to treat lymphoma-derived tumors in vivo. About 10' 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 10' PFU of live (n=8) or UV-inactivated (n=7) reovirus in 50 p,l 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 10' (n=7) or Sx 10' (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 administration.
Example 4: Reovirus selectively removed cancer cells from a mixed cellular composition MCF7 (ATCC number HTB-22), SKBR3 (ATCC number HTB-30), and MDA MB 468 (ATCC number HTB 132) are known to be susceptible to reovirus infection (i.e., these cells are killed by reovirus). Cells of each type were mixed with apheresis product and subjected to reovirus infection to investigate if reovirus can selectively remove neoplastic cells from the mixed cellular composition.
Apheresis product was prepared according to a procedure previously described (Stewart et al.
1999; Duggan et al. 2000).
When admixtures of apheresis product (90%) and, e.g., MCF7 (10%) were treated with reovirus and tested daily for cell count and viability, there was a 100-fold depletion in the numbers of cytokeratin-positive MCF7 cells while the CD34+
stem cells remained intact and viable. Figures 4A-4C show the purging effect of reovirus to mixtures of apheresis product with MCF7, SKBR3 or MDA MB 468 cells.
However, in a control experiment, infection of CD34+ cells with reovirus did not significantly alter the number of viable cells in culture (Figure 4D). Taken together, these results suggest that reovirus selectively kills cancer cells while not significantly affecting the viability of CD34+ stem cells.
Example 5: Reovirus treatment neither inhibited cell proliferation nor altered differentiation potential of CD34+ cells While the number of CD34+ cells was unaffected by reovirus infection (data not shown), there remained the question whether reovirus changed the potential of CD34+ stem cells to differentiate into all the hematopoietic lineages in the appropriate proportion. To investigate this possibility, CD34+ cells were incubated with reovirus for 2, 24, 48 or 72 hours, respectively. The reovirus was then removed and the cells were diluted and cultured in fresh media for 14 days to allow colonies to form. Each colony was examined to determine if it belongs to the granulocyte, erythroid, or granulocyte erythroid macrophage megakaryocyte lineage. As shown in Figure S, stem cells treated with live virus (LV) yielded similar numbers of granulocutes (G), erythrocytes (E) or granulocyte erythroid macrophage megakaryocytes (GEMM) as the no virus (NV) control. Therefore, reovirus treatment did not change the differentiation potential of CD34+ cells.
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 (30)

1. A method of selectively removing cells of a lymphoid malignancy from a mixed cellular composition suspected of containing cells of a lymphoid malignancy, comprising the steps of:
d) obtaining a mixed cellular population from an animal having the lymphoid malignancy, e) contacting the mixed cellular composition with at least one virus under conditions that result in substantial killing of cells of the lymphoid malignancy, thereby producing a treated cellular composition; and f) collecting the treated cellular composition so produced.

2. The method of claim 1 wherein the mixed cellular composition comprises hematopoietic stem cells.

3. The method of claim 2 wherein the mixed cellular composition comprises CD34+ cells.

4. The method of claim 1 wherein the mixed cellular composition has been harvested from bone marrow.

5. The method of claim 1 wherein the mixed cellular composition has been harvested from blood.

6. The method of claim 1 wherein the mixed cellular composition is selected from the group consisting of a tissue, an organ, or a portion of a tissue or organ, and wherein the mixed cellular composition is useful for transplantation.

7. The method of claim 1 wherein the mixed cellular composition comprises cultured cells, semen, or eggs.

8. The method of claim 1 wherein the mixed cellular population comprises or is suspected of comprising cells of at least one lymphoid malignancy and wherein at least one lymphoid malignancy is a B-cell lymphoid malignancy.

9. The method of claim 8 wherein the B-cell malignancy is selected from the group consisting of Burkitt's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, and mantle-cell lymphoma.

10. The method of claim 1 wherein the cells of the lymphoid malignancy comprise normal ras.

11. The method of claim 1 wherein the virus is a replication competent virus.

12. The method of claim 1 wherein at least one virus is a reovirus.

13. The method of claim 12 wherein the reovirus is selected from the group consisting of a human reovirus, a non-human reovirus, a modified reovirus, and a recombinant reovirus.

14. The method of claim 1 wherein at least one virus is selected from the group consisting of adenovirus, herpesvirus, vaccinia virus, parapoxvirus orf virus, Newcastle disease virus, and vesicular stomatitis virus.

15. The method of claim 14 wherein the at least one virus is mutated or modified such that the virus does not produce a gene product that inhibits double stranded RNA kinase (PKR).

16. The method of claim 14 wherein the adenovirus has been mutated in E1A
region such that the resulting E1A gene product does not bind to Rb.

17. The method of claim 14 wherein the adenovirus has been mutated in E1B
region such that the resulting E1B gene product does not bind to p53.

18. The method of claim 14 wherein the adenovirus is capable of expressing a wild type p53 protein.

19. The method of claim 14 wherein the adenovirus is selected from the group consisting of Delta24 and ONYX-015.

20. The method of claim 1 further comprising the step of removing the virus from the treated cellular composition.

21. The method of claim 1 further comprising the step of storing the virus treated cellular composition.

22. The method of claim 21 wherein the cellular composition is stored in a solution comprising DMSO.

23. The method of claim 14 wherein the virus is an interferon sensitive virus and the method further comprises a step of adding interferon to the mixed cellular composition.

24. The method of claim 1 wherein steps (a) and (b) are performed ex vivo.

25. The method of claim 1 further comprising the step of reintroducing the treated cellular composition to said animal.

26. The method of claim 25 wherein the treated cellular composition is reintroduced into the bone marrow of said animal.

27. The method of claim 24 wherein the treated cellular composition is reintroduced into the blood of said animal.

28. The method of claim 1 further comprising the step of administeing the treated cellular composition to at least one second animal, wherein the second animal is genetically compatible with the animal from which the mixed cellular composition was harvested.

29. The method of claim 28 further comprising administering immunosuppressants to said second animal.

30. The method of claim 1, wherein the animal is selected from the group consisting of dogs, cats, sheep, goats, cattle, horses, pigs, humans, and non-human primates.