WO2012005763A1 - Use of myeloid-like progenitor cell populations to treat tumors - Google Patents

Abstract

This invention provides methods for treating tumors. The methods involve injecting or implanting a population of myeloid-like progenitor cells (e.g., human CD14⁺ cells or CD33^HI monocytes) to a subject afflicted with a tumor. The isolated cells can be further engineered to express an anti-tumor agent, e.g., an anti-angiogenic agent or a cytotoxic agent.

Description

USE OF MYELOID-LIKE PROGENITOR CELL POPULATIONS TO

TREAT TUMORS

COPYRIGHT NOTIFICATION

(0001] Unless defined Pursuant to 37 C.F.R. § 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application No. 61/399,085 (filed July 6, 2010). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

STATEMENT CONCERNING GOVERNMENT SUPPORT

[0003] This invention was made by government support under EY01 1254 awarded by the National Institutes of Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0004] The present invention generally relates to methods and compositions for inhibiting tumor growth and treating cancer.

BACKGROUND OF THE INVENTION

[0005] A tumor is an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, also called a neoplasm. Tumors may be either benign (not cancerous) or malignant (i.e., cancer). Various types of solid tumors can become cancerous which often lead to death. For example, brain tumors are an especially deadly form of cancer. Brain tumors can be present either in the brain itself (neurons, glial cells (astrocytes, oligodendrocytes, ependymal cells), lymphatic tissue, blood vessels), in the cranial nerves (myelin-producing Schwann cells), in the brain envelopes (meninges), skull, pituitary and pineal gland, or spread from cancers primarily located in other organs (metastatic tumors). The most common type of brain tumor is glioma. Gliomas are neuroectodermal tumors of neuroglial origin, and include astrocytoma, oligodendroglioma and ependymoma which are derived from astrocytes, oligodendrocytes and ependymal cells respectively.

[0006] There are a number of methods for treating tumors, e.g., surgical procedures, chemotherapy and radiation therapy. However, various problems or side effects are associated with these treatments. For example, following surgical operation to remove a primary tumor, tumor metastasis often results in recurrence of cancer in the form of the formation and growth of at least one additional or second tumor. Radiotherapeutic and chemotherapeutic agents can be toxic to normal tissues at the dose levels administered, and often create life-threatening side effects in the patient. These cancer therapies can often have high failure/remission rates which can result in death of the patient. Further, there are many solid tumors for which there is still no effective treatment.

[0007] Thus, there is an unfulfilled need in the art for alternative or more effective treatments for tumors. The present invention addresses this and other needs in the art.

SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides methods of treating a tumor in a subject. The methods entail administering to the subject suffering from a tumor an effective amount of a population of myeloid-like progenitor cells. Preferably, the myeloid-like progenitor cells are isolated from the subject to be treated. In some preferred embodiments, the subject to be treated is a human. For example, a human subject afflicted with a solid tumor can be treated with a population of human CD14⁺ cells or human monocytes. The human subject can be treated with human CD14⁺ cells isolated from a human blood sample of the subject or CD33^HI monocytes isolated from bone marrow of the subject. Preferably, at least 75% of cells in the employed population of human cells express the indicated antigen (e.g., CD14 for the blood derived cells and/or CD33 for the bone marrow derived cells). [0009] In some embodiments, the therapeutic cell population is administered via central injection to the subject. In some embodiments, the cells are stimulated with

lipopolysaccharides (LPS) or other TLR4 ligands prior to being administered to the subject. Some preferred methods of the invention are directed to treating brain tumors including, e.g., glioma, glioblastoma, ocular melanoma or a metastatic brain tumor. Some of the methods further comprise treating the tumor with sublethal radiation therapy prior to administration of the therapeutic cells.

[0010] In a related aspect, the invention provides other methods for treating a solid tumor in a subject. The methods involve administering to the subject afflicted with a solid tumor an effective amount of a population of myeloid-like progenitor cells which have been transfected or transduced with a gene that operably encodes an anti-tumor agent. In some preferred embodiments, the myeloid-like progenitor cells are human CD14⁺ cells or rodent CD44^HI cells. In some other embodiments, the administered myeloid-like progenitor cells are monocytes (e.g., human CD33^HI monocytes isolated from bone marrow).

[0011] The anti-tumor agent can be an antiangiogenic agent, a cytotoxic agent or a tumor suppressor. In some methods, the gene encoding the anti-tumor agent is transfected into the cells via an expression vector. In some of these methods, the therapeutic cells are transduced with a lentiviral expression vector. In some embodiments, the engineered cells are injected centrally to the subject. In some preferred embodiments, the subject to be treated is a human, and the myeloid-like progenitor cells are human CD14⁺ cells isolated from the subject. Some of the methods are directed to treating a brain tumor such as glioma, glioblastoma, ocular melanoma or a metastatic brain tumor.

[0012] In another aspect, the invention provides methods for delivering an anti-tumor agent to a solid tumor in a subject. These methods entail constructing a vector expressing the anti-tumor agent, transfecting or transducing the vector into a population of myeloid-like progenitor cells, and admimstering the transfected or transduced cells to the subject. Some of these methods employ myeloid-like progenitor cells which are human CD14⁺ cells or rodent CD44^HI cells. Some other methods employ myeloid-like progenitor cells which are human monocytes (e.g., CD33^HI monocytes isolated from bone marrow). Preferably, the cells are isolated from the subject afflicted with the solid tumor. The anti-tumor agent to be delivered with the methods can be an antiangiogenic agent, a cytotoxic agent or a tumor suppressor. In some preferred embodiments, the vector for expressing the anti-tumor agent is a lentiviral vector. In some of the methods, the transfected or transduced cells are injected centrally to the tumor in the subject. Some preferred methods are used for delivering an anti-tumor agent to a human subject via a population of CD14⁺ cells isolated from the subject.

[0013] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows that rodent Lin^" myeloid-like cells migrate to tumor.

[0015] Figure 2 shows that mouse CD44^HI in combination with CD44^LO cells selectively kill sublethally irradiated tumor cells in culture.

[0016] Figure 3 shows presence of lentivirus infected Lin^" cells in 21 day tumor. The image was taken 16 days post-transduction of the cells with the vector and 15 days post- injection.

[0017] Figures 4A-4C show that human cord blood CD14⁺ cells transduced with a GFP- expressing adenoviral vector (Ad5 F16 eGFP) colocalized to 9L tumor cells in culture (4 A), reduced growth of tumor cells (4B), and down-regulated expression of proliferating cell nuclear antigen (PCNA) (4C). Shown in Fig. 4C is a reduction in PCNA expression (by implication, a decrease in DNA synthesis) resulted from addition of CD 14 cells in tumor group exposed to lOgy, but not 7.5gy.

[0018] Figure 5 shows that Ad5 F16 eGFP transduced human CD14⁺ cells, when pre- stimulated with LPS, killed lOgy irradiated 9L tumor cells in vitro.

[0019] Figure 6 shows that Ad5 F16 eGFP transduced human CD14⁺ cells, when pre- stimulated with LPS, secreted high levels of pro inflammatory cytokines.

[0020] Figure 7 shows presence in SCID mice of Ad5 F16 eGFP transduced human CD14⁺ cells co-injected with tumor cells. Shown in the figure are eGFP cells 7 days after injections.

[0021] Figure 8 shows anti-tumor activities of different cell populations from bone marrow. [0022] Figure 9 shows that different ratio of tumor cells to monocytes led to differing antitumor activities.

[0023] Figure 10 shows that both CD54 positive and CD54 negative lymphocytes, as well as the granulocyte population, are negative effector cells.

[0024] Figure 11 shows results of treatment of implanted gliosarcoma in rats with un- fractionated bone marrow cells.

[0025] Figure 12 shows in vivo anti-tumor activities of CD44 hi and CD44 low cell populations against melanoma in mice.

[0026] Figure 13 shows FACS profile of cell populations isolated from human bone marrow.

DETAILED DESCRIPTION

I. Overview

[0027] The present invention relates to methods of killing tumor cells, inhibiting tumor growth and treating cancers with a population of isolated or engineered myeloid-like progenitor cells. The invention is predicated in part on the discovery of the present inventors that myeloid-like progenitor cell populations such as human CD14⁺ cells or rodent Lin- myeloid like cells are capable of both tumor-homing and/or tumor-killing. As detailed in the Examples below, it was found that bone marrow derived rodent Lin- progenitor cells, upon injection into mice brains, can localize and proliferate within brain tumors in the mice. In addition, it was found that the observed tumor-homing activity of the Lin- cells is primarily attributable to a CD44^HI subset of cells. In contrast, CD44^LO cells generally do not localize and home to the tumors. Further, the CD44^HI cells under appropriate conditions can exhibit anti-tumor activity. It was found that they were able to mediate phagocytosis or killing of various tumors that have been sublethally irradiated. Similarly to rodent myeloid-like progenitor cells, a population of blood derived human CD14⁺ cells was shown to be able to target and kill tumor cells in vitro. In vivo, the human cells were similarly able to localize to injected tumor cells.

[0028] The inventors further determined that monocytes, not lymphocytes or

granulocytes, from rodent bone marrow are the effector cell population with anti-tumor activities, and were able to isolate pure populations of the effector cells. The inventors observed profound in vivo anti-glioma activity of isolated rat monocytes against syngeneic 9L glioma cells. Also, anti-melanoma activity was observed with mouse bone marrow cell populations. Similarly fractionated cell populations were also obtained from human bone marrow. In addition, the inventors observed in vivo activity of whole bone marrow in reducing tumor size and improving survival. Moreover, it was determined that sublethal tumor cell irradiation is not necessary to elicit anti-tumor activity of the effector cells. The sublethal irradiation was used to promote the activation of the immune cells. The effector cells exhibited potent anti-tumor activity without sublethal irradiation of the tumor cells.

[0029] In accordance with these discoveries, the present invention provides methods of using myeloid-like progenitor cell populations (e.g., human CD14⁺ cells from blood or human CD33^HI monocytes from bone marrow) to treat various types of tumors, e.g., brain tumors. The cells can be used directly to kill tumor cells, e.g., tumors that have been subject to sub-lethal UV or radiation treatment or other types of sub-lethal stressors. The isolated cells can also be engineered to express an anti-tumor agent such as an antiangiogenic agent or a cytotoxic polypeptide (e.g., by using an adenoviral or adeno-associated viral vector). The engineered myeloid-like progenitor cells can be used to specifically deliver therapeutic agents to tumors and to treat tumors in subjects in need of treatment. Cells engineered to stably express an anti-tumor agent (e.g., via a lentivirus vector that integrates into the genome of the cells) allow continual expression of anti-tumor agents in daughter cells.

[0030] The isolated or engineered myeloid-like progenitor cells are highly migratory. They can target tumors at the site of administration (e.g., site of local injection) as well as at distant tumor satellites. The cells can be introduced centrally or locally into the subject, e.g., at tumor resection cavity or through an implanted catheter. Because the cells specifically target to tumors, toxicity is greatly reduced since normal cells are spared. In addition, when the cells are autologous to the subject to be treated, there is also reduced risk of any undesirable immune response.

[0031] The following sections provide more detailed guidance for practicing the methods of the invention.

II. Definitions [0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and

Technology, Morris (Ed.), Academic Press (1^st ed., 1992); Illustrated Dictionary of

Immunology, Cruse (Ed.), CRC Pr I Lie (2^nd ed., 2002); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1^st ed., 1999);

Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer- Verlag Telos (1994);

Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0033] Hematopoietic stem cells are stem cells that are capable of developing into various blood cell types e.g., B cells, T cells, granulocytes, platelets, and erythrocytes. The lineage surface antigens (surface markers) are a group of cell-surface proteins that are markers of mature blood cell lineages, including CD2, CD3, CD1 1, CD1 la, Mac-1

(CDl lb:CD18), CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, CD45RA, murine Ly-6G, murine TER-119, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leukocyte antigen DR (HLA-DR), and CD235a (Glycophorin A). Hematopoietic stem cells that do not express significant levels of these antigens are commonly referred to as lineage negative (Lin .). Human hematopoietic stem cells commonly express other surface antigens such as CD31 , CD34, CD117 (c-kit) and/or CD133. Murine hematopoietic stem cells commonly express other surface antigens such as CD34, CD117 (c-kit), Thy-1, and/or Sca-1.

[0034] The cells that circulate in the bloodstream are generally divided into three types: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets or thrombocytes. Leukocytes include granulocytes (polymorphonuclear leukocytes) and agranulocytes (mononuclear leucocytes). Granulocytes are leukocytes characterized by the presence of differently staining granules in their cytoplasm when viewed under light microscopy. There are three types of granulocytes: neutrophils, basophils, and eosinophils. Agranulocytes (mononuclear leucocytes) are leukocytes characterized by the apparent absence of granules in their cytoplasm. Although the name implies a lack of granules, these cells do contain non-specific azurophilic granules, which are lysosomes. Agranulocytes include

lymphocytes, monocytes, and macrophages.

[0035] Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. They constitute between three to eight percent of the leukocytes in the blood. In the tissues monocytes mature into different types of macrophages at different anatomical locations. Monocytes have two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (aprox. 8-12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Monocytes are usually identified in stained smears by their large bilobate nucleus.

[0036] The term "myeloid-like progenitor cells" refers to a population of cells which is isolated from rodent (e.g., mouse or rat) bone marrow or blood and contains a majority of cells that express CD44 antigen (CD44^HI cells). It also encompasses human analogue to rodent CD44^HI cells, e.g., human CD14⁺ cells which can be isolated from human blood (e.g., cord blood or peripheral blood) or bone marrow. The term also includes monocyte populations isolated from bone marrow (e.g., human CD33^HI monocytes). Typically, the rodent CD44^HI cell population contains at least about 50 percent of cells that express CD44. Preferably, at least 75% or even 90% of the cells in the population express CD44. When the employed myeloid-like progenitor cells are human CD14⁺ cells or CD33^HI cells, the cell population typically contains at least about 50% cells that express CD 14 or CD33, with preferably at least about 75%, 85%, 90%, 95% or 99% of the cells expressing the antigen.

[0037] In some embodiments, the CD44^HI rodent myeloid-like progenitor cells of the invention are also defined by being lineage negative (Lin ) in addition to expressing CD44. Depending on the level of CD44 expression, lineage negative bone marrow cells can be divided into two subpopulations: CD44^H1 cells and CD44^LO cells. The CD44^HI cells are primarily myeloid in origin while the CD44^LO cells are largely lymphoid in origin (see, e.g., Ritter et al., J. Clin. Invest. 116:3266-76, 2006). Unless otherwise specified, the rodent myeloid-like progenitor cells used in the practice of the invention is a population of CD44^HI cells. Unless otherwise noted, the Lin^" CD44⁺ myeloid-like cells is used herein

interchangeably with "lineage negative CD44⁺ hematopoietic stem cells." When the myeloid-like progenitor cells are isolated from bone marrow, they are also termed myeloid- like bone marrow (MLBM) cells. Specifically, the bone marrow derived myeloid-like progenitor cells include monocytes.

[0038] The myeloid-like progenitor cells to be used in the practice of the invention can be isolated in accordance with methods that have been known in the art (Ritter et al., J. Clin. Invest. 116:3266-76, 2006) or the procedures described in the Examples below for isolating human CD14⁺ cells. For example, cells from a sample of bone marrow, peripheral blood or umbilical cord blood can be first selected to isolate lineage negative cells. This can be accomplished by negative selection with antibodies against one or more of the lineage markers described above. The isolated Lin^" cells can then be further subject to positive selection for CD44⁺ cells. This can be performed with an antibody specific for CD44 (hyaluronic acid receptor). If desired, the Lin^" CD44⁺ cells may be further selected for expression of other surface markers that are indicative of myeloid origin. Other than selection via a specific antibody, the various selection steps may also be performed by other means commonly practiced in the art, e.g., flow cytometry.

[0039] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant (i.e., cancer) or benign, and all pre-cancerous and cancerous cells and tissues. Solid tumors are tumors which have a physical swelling or lesion formed by an abnormal growth of cells. Examples of solid tumors include brain tumor, melanoma, breast cancer, lung cancer, liver cancer, colon cancer, renal cancer, pancreatic cancer, esophageal cancer, bladder cancer, prostate cancer, head and neck cancer, gastric cancer, and ovarian cancer.

[0040] A brain tumor refers to any tumor that grows in the brain, which can be cancerous or non-cancerous (benign). It is defined as any intracranial tumor created by abnormal and uncontrolled cell division that normally presents in the brain itself (neurons, glial cells (astrocytes, oligodendrocytes, ependymal cells), lymphatic tissue, blood vessels), in the cranial nerves (myelin-producing Schwann cells), in the brain envelopes (meninges), skull, pituitary and pineal gland, or that spreads from cancers primarily located in other organs (metastatic tumors). Primary (true) brain tumors are commonly located in the posterior cranial fossa in children and in the anterior two-thirds of the cerebral hemispheres in adults, although they can affect any part of the brain. Examples of brain tumors include, but are not limited to, glioma, glioblastoma, medulloblastoma, astrocytoma, and other primitive neuroectoderma.

[0041] Retroviruses are enveloped viruses that belong to the viral family Retroviridae. The virus itself stores its nucleic acid, in the form of a +mRNA (including the 5 '-cap and 3'- PolyA inside the virion) genome and serves as a means of delivery of that genome into host cells it targets as an obligate parasite, and constitutes the infection. Once in a host's cell, the virus replicates by using a viral reverse transcriptase enzyme to transcribe its RNA into DNA. The DNA is then integrated into the host's genome by an integrase enzyme. The retroviral DNA replicates as part of the host genome, and is referred to as a provirus.

Retroviruses include the genus of Alpharetrovirus (e.g., avian leukosis virus), the genus of Betaretrovirus; (e.g., mouse mammary tumor virus), the genus of Gammaretrovirus (e.g., murine leukemia virus or MLV), the genus of Deltaretrovirus (e.g., bovine leukemia virus and human T-lymphotropic virus), the genus of Epsilonretrovirus (e.g., Walleye dermal sarcoma virus), and the genus of Lentivirus.

[0042] Lentivirus is a genus of viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. Examples of lentiviruses include human immunodeficiency viruses (HIV- 1 and HIV-2), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Additional examples include BLV, EIAV and CEV.

[0043] The term "operably linked" when referring to a polynucleotide, means a linkage of polynucleotide elements in a functional relationship. A polynucleotide element is "operably linked" to another polynucleotide element when the two are placed into a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it controls or affects the transcription of the coding sequence. Operably linked means that the linked polynucleotide elements are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

[0044] The terms "subject" and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as dogs, cats, sheeps, cows, pigs, rabbits, chickens, and etc.

Preferred subjects for practicing the therapeutic methods of the present invention are human. Subjects in need of treatment include patients already suffering from a cancer or a tumor, as well as those who are at risk of or prone to developing such a condition or disease.

[0045) Sublethal irradiation refers to radiation at a dose level that, when applied to a cell (e.g., a tumor cell), does not cause the cell to die but instead allows the cell to repair damages itself. General guidance for appropriate radiation dose in order to achieve sublethal or lethal effect is well known in the art, e.g., as provided by the International Commission on Radiological Protection. For example, a dose range from 0.25 to 2 sievert may be considered sublethal. Other than the sievert (or Sv) unit, a sublethal dose may also be expressed as an absorbed dose measured in gray (Gy). For example, an absorbed dose of less than 8 Gy, less than 6 Gy, or less than 5 Gy may be considered sublethal. The exact dose level for a specific subject should be determined based on many other factors, like age, sex, health etc.

[0046] The term "substantially pure" or "substantial purity" when referring to an isolated cell population means the percentage of a given cell (target cell) in the population is significantly higher than that found in a natural environment (e.g., in a tissue or a blood stream of a subject). Typically, percentage of the target cell (e.g., Lin^" and/or CD44⁺ cells) in a substantially pure cell population is at least about 50%, preferably at least about 60%, 70%, 75%, and more preferably at least about 80%, 85%, 90% or 95% of total cells in the cell population.

[0047] As used herein, "treating" or "ameliorating" includes (i) preventing a pathologic condition (e.g., tumor) from occurring (e.g. prophylaxis); (ii) inhibiting the pathologic condition (e.g., tumor growth) or arresting its development; and (iii) relieving symptoms associated with the pathologic condition. Thus, "treatment" includes the administration of a cell population of the invention and/or other therapeutic compositions or agents to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease described herein, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. "Treatment" further refers to any indicia of success in the treatment or amelioration or prevention of tumor growth or cancer development, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient. Detailed procedures for the treatment or amelioration of a tumor or symptoms thereof can be based on objective or subjective parameters, including the results of an examination by a physician.

[0048] The term "transfection" refers to the introduction of foreign DNA into cells. A cell has been "transformed" or "transfected" by exogenous or heterologous polynucleotide when such polynucleotide has been introduced inside the cell. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co- precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment) and the like. When a viral vector is introduced into a bacterial host cell, the process is termed "transduction."

[0049] The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into the genomic DNA.

[0050] The term "transient transfection" or "transiently transfected" refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell. The foreign DNA persists in the nucleus of the transfected cell for a period of time (e.g., several days). The term "transient transfectant" refers to cells that have taken up foreign DNA but have failed to integrate this DNA.

[0051] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as "expression vectors". A retrovirus (e.g., a lentivirus) based vector or a vector derived from the virus means that genome of the vector comprises components from the virus as a backbone. The viral particle generated from the vector as a whole contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Usually these will include the gag and pol proteins derived from the virus. If the vector is derived from a lentivirus, the viral particles are capable of infecting and transducing non-dividing cells. Recombinant retroviral particles are able to deliver a selected exogenous gene or polynucleotide sequence such as therapeutically active genes, to the genome of a target cell.

Mveloid-like progenitor cell populations [0052] The present invention provides methods of employing myeloid-like (ML) progenitor cell populations to treat tumors. Examples of such cell populations include human CD14⁺ cell populations and rodent CD44^HI cell populations. In some embodiments, human myeloid-like progenitor cells are used. The human myeloid-like cell population can be isolated from a blood sample (e.g., cord blood or peripheral blood) or a bone marrow sample from a human subject. In some other embodiments, a population of rodent myeloid- like progenitor cells is used. Typically, the rodent myeloid-like cell population contains a majority of cells that express CD44 (CD44^HI). In addition, the rodent cell population can also be characterized by the lack of significant levels of lineage surface antigens (Lin) on their cell surfaces. Thus, the cell population suitable for use in the invention should contain mammalian cells in which at least about 50% of the cells express the respective surface marker (CD44 for rodent cells and CD 14 for human cells). Preferably, the employed cell population contains at least about 60%, 70%, 80%, 90%, 95%, 99% or more of cells that express the marker.

[0053] The myeloid-like progenitor cell populations can be obtained from bone marrow or a blood sample from a mammalian subject (e.g., a rodent or a human subject) as described herein or taught in the art. Preferably, the cells are isolated from the same subject in need of treatment of a tumor. For example, human CD 14+ cell populations can be obtained in accordance with the procedures described in the Examples below. Rodent myeloid-like progenitor cell populations can be obtained from a subject in accordance with procedures disclosed herein or well known in the art. See, e.g., WO 06/104609; and Ritter et al., J. Clin. Invest. 116:3266-76, 2006. They can be isolated from a bone marrow sample, a peripheral blood sample or an umbilical cord blood sample of the subject. For example, a bone marrow can be obtained postnatally from juvenile and adult subjects. As exemplification, the lineage negative progenitor cells from bone marrow of a rodent can be isolated by the following standard procedures: (a) extracting bone marrow from an adult mammal; (b) separating a plurality of monocytes from the bone marrow; (c) labeling the monocytes with biotin- conjugated lineage panel antibodies to one or more lineage surface antigens, preferably lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD1 1 , CD1 la, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G (murine), TER-119 (murine), CD45RA, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leukocyte antigen DR (HLA-DR), and CD235a (Glycophorin A); and (d) removing monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative cells.

[0054] In addition or alternative to the negative selection, the bone marrow or blood cells can also be positively selected for CD44 expression (for rodent cells) or CD 14 expression (for human cells) to obtain the myeloid-like progenitor cell populations suitable for the present invention. For example, positive selection for CD44 expression can be performed with, e.g., using an antibody specific for the CD44 antigen (anti-CD44), and then selecting cells that immunoreact with the antibody. Typically, the antibody can be removed from the cells by methods that are well known in the art. In any of these selection steps, the cells can be selected by using flow cytometry, using antibodies bound to or coated on beads followed by filtration, or using other separation methods that are well known in the art.

[0055] In some preferred embodiments for treating human subjects, a population of human myeloid-like progenitor cells (CD14⁺ cells) is used. As exemplified herein, the human myeloid-like progenitor cells that express the respective surface marker can also be isolated from a blood sample from a human subject, e.g., umbilical cord blood. It can also be obtained from other samples such as peripheral blood and bone marrow. Regardless of the manner by which the cell population is obtained, at least about 25% or about 40% of the cells in the population express the surface marker CD 14. With some selection scheme, a majority of the selected cells (e.g., at least 50%, 75% or 90%) express the surface. Prior to administration to a subject, the isolated cell populations can be maintained in an appropriate culture medium such as phosphate buffered saline (PBS). The CD14⁺ human cells were isolated from human cord blood.

[0056] Methods for obtaining human myeloid-like cell population from a whole blood sample are described in detail in U.S. Provisional Application No. 61/208,173 (filed

February 20, 2009). Briefly, red blood cells in the blood sample (e.g., peripheral blood or cord blood) were lysed (e.g., with ammonium chloride) and separated from other cells via sedimentation. Erythrocytes and granulocytes present in thee blood were also removed, e.g., via centrifugation. The other cells in the sample were subject to fluorescence-activated cell sorting to separate monocytes from lymphocytes based on differences in cell size and granularity. Other than FACS based sorting, the human myeloid-like cell populations can also be obtained by density centrifugation. It was found that that the isolated human myeloid-like cell population contains CD14⁺/CD33⁺ progenitor cells with purities of 80% - 85%.

[0057] In some embodiments of the invention, a rodent myeloid like cell population containing cells with high expression of CD44 (CD44^HI) is used. Such CD44^HI myeloid cells can be prepared and obtained in accordance with methods described in the art, e.g., Ritter et al., J. Clin. Invest. 116:3266-76, 2006. The CD44^HI subpopulation of isolated Lin^" CD44⁺ myeloid like progenitor cells typically contains cells in which at least about 50% of the cells express CD44. More preferably, at least about 65%, 75% or 90% of the cells in the CD44^HI subpopulation express CD44.

[0058] In some embodiments, the CD44^HI myeloid like cell population is further selected for lack of expression of one or more of the following cell markers: Term 19, CD45RB220, and CD3e. It is known that bone marrow cells that do not express CD44 (CD44^LO cells) generally express one or more of these markers. Thus, the CD44^HI myeloid-like cell population to be used in the present invention can be isolated by a method involving further negative cell-marker selection. The method involve contacting a plurality of bone marrow cells, peripheral blood cells or umbilical cord cells with one or more antibodies specific for Terl 19, CD45RB220, and CD3e, removing cells from the plurality of bone marrow cells that immunoreact with Terl 19, CD45RB220, and CD3e antibodies, and recovering myeloid- like bone marrow cells that are depleted in Terl 19, CD45RB220, and CD3e-expressing cells. With such negative selection, a myeloid-like cell population can be recovered in which greater than 90 percent of the cells express CD44.

[0059] The various selection procedures for isolating the myeloid-like progenitor cell populations may also be performed or assisted with flow cytometry or FACS. Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus. Typically, a beam of light (usually laser light) of a single wavelength is directed onto a hydro-dynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each cell's fluorescence, scattered light, and transmitted light.

[0060] Modern flow cytometers are able to analyze several thousand particles every second in real time, and can actively separate and isolate particles having specified properties. A flow cytometer is similar to a microscope, except that instead of producing an image of the cell, flow cytometry offers high-throughput automated quantification of set parameters. A flow cytometer has 5 main components: a flow cell - liquid stream, a light source (e.g., laser), a detector and Analogue to Digital Conversion (ADC) system which generate FSC and SSC as well as fluorescence signals, an amplification system, and a computer for analysis of the signals. The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates". Specific gating protocols exist for diagnostic and clinical purposes especially in relation to haematology. The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally.

[0061] Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet. Just before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems the charge is applied directly to the stream and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.

[0062] In some embodiments, FACS can be carried out on a BD FACSAria Cell-Sorting System (BD Biosciences, San Jose, CA) using a series of gates. No antibodies or other selection agents are used in the sorting. Dead cells and debris can be first gated out by drawing a region that includes only viable white blood cells. Thereafter, doublets or aggregated cells can be removed with secondary and tertiary gates that interrogate forward scatter width (FSC-W) vs. forward scatter area (FSC-A) and side scatter width (SSC-W) vs. side scatter area (SSC-A), respectively. The procedures can be performed in accordance with standard protocols well known in the art, e.g., Flow cytometry - A practical approach, Ormerod (ed.), Oxford University Press, Oxford, UK (3^rd ed., 2000); and Handbook of Flow Cytometry Methods, Robinson et al. (eds.), Wiley-Liss, New York (1993). rV. Engineered myeloid-like progenitor cell populations

[0063] In addition to using the myeloid-like progenitor cells isolated from a subject, the invention can also employ isolated cells that have been subsequently modified. For example, the isolated cells can be engineered to express an anti-tumor agent. The anti-tumor agent can include, e.g., antiangiogenic agents, cytotoxic agents or proteins encoded by tumor suppressor genes. In these embodiments, the isolated myeloid like cell populations are stably or transiently transfected with a vector harboring a polynucleotide that encodes the therapeutically useful agent. Examples of genes encoding such agents and methods for their transfection into myeloid like progenitor cells have been described in the art. See, e.g., PCT publication WO 04/098499. In some preferred embodiments, the cells are engineered with a vector that allows the anti-tumor agent to be stably expressed.

[0064] The transfected cells can include any gene which is therapeutically useful for treatment of tumors. In some embodiments, the transfected cells from the myeloid-like cell population of the present invention harbors a gene operably encoding an antiangiogenic agent. Examples of such agents include fragments derived from the carboxyl-terminal fragment of tryptophan tRNA synthetase (T2-TrpRS), angiopoietin 2, endostatin, angiostatin, PEX, IL-12, IFN-a, prolactin and thrombospondin TSP-1 and TSP-2. In some other embodiments, the transfected cells are engineered to express a cytotoxic protein or peptide. The cytotoxic protein or peptide can be one that is naturally occurring or synthetic in nature. Examples of naturally occurring cytotoxic peptides that can be expressed by the transfected cells include venoms (e.g., bee venom, scorpion toxins, and jumper ant venom) and peptides involved in autocytotoxicity (e.g., amyloid polypeptide IAPP). Synthetic peptides that possess cytotoxic activity similar to that of the naturally occurring cytotoxic peptides are also known in the art. Examples of such peptides are disclosed in, e.g., Chen et al., FEBS Lett 236: 462-466, 1988; and Cornut et al., FEBS Lett 349: 29-33, 1994. Other than these peptide agents, the myeloid like cells can also be engineered to express a cytotoxic protein or polypeptide drug. Examples of such protein agents include, e.g., IL-2, GM-CSF, TNFa, gelonin, momordin, Diphtheria toxin, and Pseudomonas exotoxin.

[0065] In addition to antiangiogenic agents and cytotoxic peptides or proteins, any other antitumor agents suitable for recombinant expression in the myeloid like progenitor cells can also be used in the invention. For example, the myeloid like cells can be transfected with a gene encoding tumor suppressor gene p53, p21 and BRCA2. It was shown that transfecting p53 into human breast cancer cell lines has led to restored growth suppression in the cells (Casey et al., Oncogene 6: 1791-7, 1991). The myeloid like cells can also be transfected with an exogenous gene encoding an enzyme for anti-tumor activity. For example, the gene can encode a cyclin-dependent kinase (CDK) or a cytosine deaminase. It was shown that restoration of the function of a wild-type cyclin-dependent kinase, pl6INK4, by transfection with a pl6I K4-expressing vector reduced colony formation by some human cancer cell lines (Okamoto, Proc. Natl. Acad. Sci. U.S.A. 91 : 1 1045-9, 1994).

[0066] The exogenous gene encoding the therapeutic agent can be transfected into the myeloid-like progenitor cells (e.g., human CD14⁺ cells or rodent CD44^HI cells) in an appropriate vector. The employed vector should allow introduction of the exogenous gene into the cells and also subsequent expression (transient or stable) of the gene once transfected into the cells. Various vectors are suitable for this aspect of the invention.

Preferably, retroviral vectors and corresponding packaging cell lines well known in the art can be employed. Particularly suitable for the present invention are lentiviral vectors.

Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers.

[0067] Retroviral vectors are comprised of cw-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis -acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HTV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739, 1992; Johann et al., J. Virol. 66: 1635-1640, 1992; Sommerfelt et al. , Virol. 176:58-59, 1990; Wilson et ah, J. Virol. 63:2374-2378, 1989; Miller et al., J. Virol. 65:2220-2224, 1991 ; and PCT US94/05700). Any of these vectors may be employed in the present invention. In particular, a number of retroviral vector have been used for gene transfer in clinical applications in the art. These include pLASN and MFG-S, retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med. 1 : 1017-102 (1995); Malech et al., Proc. Natl. Acad. Sci. U.S.A. 94:22 12133-12138 (1997)). Another vector is PA317/pLASN, which was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science 270:475-480, 1995). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors (Ellem et al., Immunol

Immunother. 44: 10-20, 1997; Dranoff et al., Hum. Gene Ther. 1 : 1 1 1 -2, 1997).

[0068] Vectors suitable for the present invention can also be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques can be found in common molecular biology references such as Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), D. Goeddel, ed., Gene Expression Technology, Methods in Enzymology series, Vol. 185, Academic Press, San Diego, Calif. (1991), and Innis, et al. PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, Calif. (1990). In order to obtain transcription of the gene encoding an anti-tumor agent within a target cell (e.g., a CD44 monocyte), a transcriptional regulatory region capable of driving gene expression in the target cell is utilized. The transcriptional regulatory region can comprise a promoter, enhancer, silencer or repressor element and is functionally associated with a nucleic acid of the present invention. Preferably, the transcriptional regulatory region drives high level gene expression in the target cell. Transcriptional regulatory regions suitable for use in the present invention include but are not limited to the human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polyomavirus promoter, the albumin promoter, PG and the a-actin promoter coupled to the CMV enhancer.

[0069] Many producer cell line or packaging cell line for transfecting retroviral vectors and producing viral particles are also known in the art. The producer cell to be used in the invention needs not to be derived from the same species as that of the target cell (e.g., human target cell). For example, producer or packaging cell lines suitable for the present invention include cell lines derived from human (e.g., HEK 292 cell), monkey (e.g., COS-1 cell), mouse (e.g., NIH 3T3 cell) or other species (e.g., canine). Additional examples of retroviral vectors and compatible packaging cell lines for producing recombinant retroviruses in gene transfers are reported in, e.g., Markowitz et al., Virol. 167:400-6, 1988; Meyers et al., Arch. Virol. 1 19:257-64, 1991 (for spleen necrosis virus (SNV)-based vectors such as vSN021); Davis et al., Hum. Gene. Ther. 8: 1459-67, 1997 (the "293-SPA" cell line); Povey et al., Blood 92:4080-9, 1998 (the "1MI-SCF" cell line); Bauer et al., Biol. Blood Marrow

Transplant. 4: 119-27, 1998 (canine packaging cell line "DA"); Gerin et al., Hum. Gene Ther. 10: 1965-74, 1999; Sehgal et al., Gene Ther. 6:1084-91, 1999; Gerin et al., Biotechnol. Prog. 15:941-8, 1999; McTaggart et al., Biotechnol. Prog. 16:859-65, 2000; Reeves et al., Hum. Gene. Ther. 11 :2093-103, 2000; Chan et al., Gene Ther. 8:697-703, 2001; Thaler et al., Mol. Ther. 4:273-9, 2001; Martinet et al., Eur. J. Surg. Oncol. 29:351-7, 2003; and Lemoine et al., I .Gene Med. 6:374-86, 2004. Any of these and other retroviral vectors and packaing producer cell lines can be used in the practice of the present invention.

[0070] Many of the retroviral vectors and packing cell lines used for gene transfer in the art can be obtained commercially. For example, a number of retroviral vectors and compatible packing cell lines are available from Clontech (Mountain View, CA). Examples of lentiviral based vectors include, e.g., pLVX-Puro, pLVX-IRES-Neo, pLVX-IRES-Hyg, and pLVX-IRES-Puro. Corresponding packaging cell lines are also available, e.g., Lenti-X 293T cell line. In addition to lentiviral based vectors and packaging system, other retroviral based vectors and packaging systems are also commercially available. These include MMLV based vectors pQCXIN, pQCXIQ and pQCXIH, and compatible producer cell lines such as HEK 293 based packaging cell lines GP2-293, EcoPack 2-293 and AmphoPack 293, as well as NIH/3T3-based packaging cell line RetroPack PT67. Any of these and other retroviral vectors and producer cell lines can be employed in the practice of the present invention.

[0071] Once recombinant retroviruses expressing a gene encoding a therapeutic agent are produced, they can be readily used to infect cells in a Lin^" CD44⁺ progenitor cell population. Methods for infecting primary cells with a recombinant retrovirus are well known in the art. For example, the recombinant viruses can be transfected into the cells in accordance with methods well known in the art of gene therapy (see, e.g., Mulligan et al., Hum. Gene Ther. 5:543-563, 1993). Following the transfection, the engineered cells can be administered or implanted into a subject afflicted with a tumor. In some preferred embodiments, the Lin^" CD44⁺ cells to be transfected with the recombinant virus are isolated from a subject to be treated. In some other embodiments, the transfected cells are not autologous to the subject to whom the cells are ultimately administered.

V. Treating rumors with isolated or engineered myeloid-like progenitor cell populations

[0072] The present invention provides methods of inhibiting tumor growth and promoting tumor regression (e.g., killing tumor cells) in a subject. In these methods, an isolated myeloid-like progenitor cell population or its engineered variant that harbors an expression vector operably encoding an anti-tumor agent is administered to the subject. In some embodiments, the isolated or engineered myeloid-like progenitor cell population is directly administered to a subject in treatment. In some other embodiments, the cell population is stimulated or activated prior to being administered to the subject. For example, as disclosed in the Examples, the cell population (e.g., a human CD14⁺ cell population) can be activated with lipopolysaccharide (LPS), a ligand of toll-like receptor 4 (TLR4). Thus, prior to being administrated to a subject, the cell populations to be used in the therapeutic methods of the invention can be treated with a TLR4 ligand, e.g., LPS, monophosphoryl lipid A (MPLA), myeloid related protein 8 (MRP8) or myeloid related protein 14 (MRP 14).

Activation of the myeloid-like progenitor cell populations with a TLR4 ligand can be readily performed in accordance with methods well known in the art or specifically exemplified herein. See, e.g., Umland et al., J. Leu. Biol. 75:671-679, 2004; Chang et al., J. Dent. Res. 84:994, 2005; and Crisostomo et al., Am. J. Physiol. Cell. Physiol. 294:C675-C682, 2008.

[0073] Preferably, the myeloid-like progenitor cells are administered to the subject by injection at a site that is within or close to a solid tumor. In some related embodiments, the invention provides methods for delivering an anti-tumor agent to a solid tumor in a subject. In these methods, a vector expressing an anti-tumor agent as disclosed herein is first constructed. The vector (e.g., a lentivirus based vector) is then transfected into a population of myeloid-like progenitor cells. The cells can then be administered to a subject afflicted with a tumor, e.g., via local or central injection at a site close to or distant from the tumor.

[0074] Methods of administering therapeutic cell populations to a subject can be accomplished based on procedures routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000; Ritter et al., J. Clin. Invest. 1 16:3266-76, 2006; Iwasaki et al., Jpn. J. Cancer Res. 88:861-6, 1997; Jespersen et al., Eur. Heart J. 1 1 :269-74, 1990; and Martens, Resuscitation 27: 177, 1994. Preferably, the myeloid-like progenitor cell population is preferably isolated from the subject in need of treatment of a tumor. In addition, the cells from the myeloid-like cell population are typically administered (e.g., via injection) in a physiologically tolerable medium, such as phosphate buffered saline (PBS). The isolated cells, or their engineered form as disclosed herein, should be administered to the subject in a number sufficient to inhibit the growth and promote the regression of a tumor in the subject. The subject to be treated can be one already suffering from the development of a tumor. The subject can be one that is known to be predisposed to develop a tumor (i.e., through genetic predisposition). In the latter case, the isolated myeloid-like cell population can be stored after isolation, and can then be injected prophylactically during early stages of a later developed tumor. Preferably, administration of therapeutic cell populations of the invention for treating tumors is carried out by local or central injection of the cells into the subject (i.e., as opposed to systemic administration such as peripheral administration). In these embodiments, the site of injection can be within the tumor, close to the tumor or distant from the tumor. For example, to treat a brain tumor located in one hemisphere of the brain, the cells can be injected at a site that is in the opposite hemisphere from the tumor or that is in the same hemisphere but posterior to the tumor. [0075] Various types of tumors are suitable for treatment with methods and

compositions of the present invention. These include both benign and malignant tumors. In some preferred embodiments, the methods of the invention are directed to killing tumor cells in a malignant tumor. Examples of solid tumors that are suitable for treatment with methods of the invention include brain tumors, melanoma (e.g., ocular melanoma), gastrointestinal tumors, breast cancer, lung cancer and ovarian cancer. In some preferred embodiments, the methods of the invention are directed to treating and killing cells in primary brain tumors and other vascular malignancies. Specifically, the isolated or further engineered myeloid- like progenitor cells described herein can be employed to treat subjects suffering from brain tumors such as glioma, glioblastoma, medulloblastoma, astrocytoma, other primitive neuroectoderma.

[0076] As noted above, the number of myeloid-like progenitor cells to be administered to a subject afflicted with a tumor should be sufficient for arresting tumor growth and/or for promoting tumor regression in the subject. The sufficient number of cells can be determined based on the type of tumor, its size and stage of development, the route of administration, the age of the specific subject to be treated and other factors that will be readily apparent to one of ordinary skill in the art of treating retinal diseases. As a general guidance for

administration via injection or implantation, from about 1 x 10² to about 1 x 10⁸ cells are administered. In some embodiments, from about 1 x 10³ to about 1 x 10⁷ cells are administered to treat a tumor. In still some other embodiments, about 1 x 10⁴ to 1 x 10⁶ cells are administered. The cells may be administered in a single dose or by multiple dose administration over a period of time, as determined by the clinician in charge of the treatment. Also, the number of cells and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low number of cells may be administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high number of cells at relatively short intervals may be required until progression of the tumor is reduced or terminated, and preferably until the subject shows partial or complete regression of the tumor. Thereafter, the subject can be administered a prophylactic regime.

[0077] The therapeutic regimen of the invention can be used alone in the treatment of a tumor in a subject. Alternatively, the treatment may also employed in combination with other known antitumor drugs or treatment methods. For example, the cell therapy disclosed herein can be used together with surgery, chemotherapies or radiation therapies that have been routinely practiced in the art for the treatment of tumors. Thus, in some embodiments, subjects who have been undergoing surgical procedures to remove a tumor can be administered a population of myeloid-like progenitor cells to kill residual tumor cells and to prevent recurrence or metastasis. In some embodiments, subjects suffering from cancers can be simultaneously treated with a known chemotherapy regimen and the cell therapy of the invention. There are many antineoplastic drugs and cytotoxic agents which can be readily utilized in combination with myeloid-like progenitor cells for treating tumors.

Antineoplastic drugs include classes of agents such as alkylating agents, antimetabolites, antimitotics and topoisomerase II inhibitors. Specific examples include actinomycin, anthracyclines (e.g., doxorubicin, daunorubicin, Valrubicine, Idarubicine and epirubicin) and other cytotoxic antibiotics (e.g., bleomycin, plicamycin and mitomycin). In addition to these individual antitumor drugs, various chemotherapy regiment using two or more drugs can also be employed in combination with the cell therapy disclosed herein. Detailed information about such chemotherapy regimens is readily available from, e.g., National Comprehensive Cancer Network (Jen Kintown, Pennysylvania).

[0078] In some preferred embodiments of the invention, subjects in need of treatment for a tumor can be subject to the combination of a radiation therapy and the cell therapy disclosed herein. For example, the subject to be treated can be first administered with a radiotherapy regimen. Radiotherapy is often used as the primary therapy for the treatment of malignant tumors. Most common cancer types can be treated with radiotherapy in some way. The amount of radiation used in radiation therapy is typically measured in gray (Gy), and varies depending on the type and stage of cancer being treated. For curative cases, the typical dose for a solid epithelial tumor ranges from 60 to 80 Gy, while lymphoma tumors are treated with 20 to 40 Gy. For preventative (adjuvant) purposes, the doses are typically around 45 - 60 Gy in 1.8 - 2 Gy fractions. Many other factors are considered by radiation oncologists when selecting a dose, including whether the patient is receiving chemotherapy, whether radiation therapy is being administered before or after surgery, and the degree of success of surgery. Typically, the total radiotherapy dose is fractionated (spread out over time) for several important reasons. Fractionation allows normal cells time to recover, while tumor cells are generally less efficient in repair between fractions. Fractionation also allows tumor cells that were in a relatively radio-resistant phase of the cell cycle during one treatment to cycle into a sensitive phase of the cycle before the next fraction is given. In some embodiments, a curative dose is used in a radiotherapy as the primary therapy for treating a tumor patient. The patient who has gone through the radiotherapy can be treated with the cell therapy disclosed herein. In some other embodiments, a subject may be administered a preventative or sublethal dose of radiation prior to or simultaneously with treatment with the myeloid-like progenitor cells disclosed herein.

[0079] Additional guidance for preparation and administration of the therapeutic cell populations of the invention are described in the art. See, e.g., Goodman & Gilman's The Pharmacological Bases of Therapeutics , Hardman et al., eds., McGraw-Hill Professional (10^th ed., 2001); Remington: The Science and Practice of Pharmacy, Gennaro, ed.,

Lippincott Williams & Wilkins (20^th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7^th ed., 1999).

EXAMPLES

[0080] The following examples are offered to illustrate, but not to limit the present invention.

Example 1. Tumor-homing of mouse myeloid like progenitor cells

[0081] We first observed that bone marrow derived Lin^" and Lin⁺ cells localize and proliferate within the 9L tumors. A chimeric mouse brain tumor model was established by stereotactically implanting rat 9L gliosarcoma cells into the right frontal lobe of SCID mice brain as described in Barnett, et al. Gene Therapy, 1 1 : 1283-9, 2004. Six days later, 10⁶ Lin^" or Lin⁺ mouse bone marrow cells were injected into the established tumor. At early time points (i.e., within several days of implantation of the cells), both populations were observed to localize near the injection site. At later time points, both populations of cells proliferated within the tumor as determined by Ki67 staining. Both populations show a subset of cells that proliferate within the tumor as demonstrated by KI67 staining. However, the Lin^" cells were able to target the satellite tumor foci, while the Lin⁺ cells were not as readily found within the tumor satellite foci.

[0082] To show that homing of the bone marrow progenitor cells was not due to the potential immunogenic nature of the 9L cell line, we examined the cells for their tumor- targeting activities using brain tumors established with different tumor cell lines.

Specifically, we tested the homing of mouse bone marrow cells to intracerebrally established rat-RG2 brain tumors in SCID mice and also human U87 glioma in SCID mice. Results from the study confirmed that the cells home to the tumors. To show that the observed activity was not an artifact of the chimeric mouse brain tumor model, we observed a similar response in the mouse GL261 gliosarcoma model. In this model, mouse-GL261 gliosarcoma cells are implanted into the cerebrum of c57bl/6 mice. We found that mtratumorally-injected bone marrow cells from actin-GFP labeled mice (c57bl/6 background) home to the tumor cells.

[0083] To determine the migratory abilities of these two populations of cells, we established two different paradigms: coronal and sagittal models. The Lin^" cells and Lin⁺ cells were both identified at the site of injection at 3-4 days following implantation. We found that the Lin^" cells avidly targeted the 9L tumor from either the opposite hemisphere or from implantation in the same hemisphere but posterior to the tumor implantation site. We also observed implanted bone marrow cells between the injection site and the tumor. This suggests that the Lin^" cells migrate through the intervening brain rather than marginating into the blood stream and then exiting at the tumor site (Figure 1). On the other hand, the Lin⁺ cells were not seen within the tumor when injected in either the contralateral hemisphere or posterior to the tumor, indicating that the Lin⁺ cells did not effectively migrate to the tumor when introduced at a distance. These data suggest that the mouse bone marrow progenitor cells (especially Lin^" cells) migrate through normal brain and infiltrate tumor.

Example 2. Tumor homing and non-tumor homing sub-populations of Lin^" cells

[0084] Lin^" myeloid-like progenitor cells can be divided into two subpopulations: CD44^HI and CD44^LO cells (Ritter et al., J. Clin. Invest. 116:3266-76, 2006). We therefore tested these two fractions to determine if they specifically localize within the tumor. We found that CD44^HI cells localize within the tumor. In addition, they replicate within the bed of the tumor as determined by Ki67 staining. Conversely, the CD44^LO cells generally do not localize to the tumor. As observed with Lin^" cells, the CD44^HI subpopulation of cells also migrate from one hemisphere to the other to localize and proliferate within the tumor.

Conversely, the CD44^LO cells do not home to the tumor.

[0085] We further characterized activities of the CD44^HI and CD44^LO cell populations in vivo and in vitro. It was found that the CD44^HI cells divided to a limited extent in vivo and in vitro as determined by Ki67 staining. CD44 cells are known to be mainly of myeloid lineage (Ritter et al., J. Clin. Invest. 116:3266-76, 2006). We observed that these cells express F4/80, a macrophage and dendritic cell marker, in the presence of established 9L tumors in vivo or when co-cultured with 9Ltumor cells in vitro. An extensive evaluation of cell markers for other cell lineages did not show any evidence of alternative differentiation of the CD44^HI cells. In addition, it was found that these cells were positive for CD1 lc, also a dendritic and monocyte/macrophage marker. We additionally observed some early staining of the cells in vivo with CD1 lb and a very scant occasional cell staining with CD45. As described below, it was also found that CD44^HI cells are avid phagocytes of tumor cells damaged by uv or gamma irradiated, but not undamaged tumor cells.

[0086] It was further observed that the CD44^HI cells (or a subpopulation of these cells) proliferate when cocultured with various tumor cells. Based on their surface markers, cell morphology and their phagocytic function, these cells are likely to be macrophage precursors. Within the context of the brain, these cells can be called microglial cells. By contrast, the CD44^LO population in mice is mainly erythroid and lymphoid lineage (Ritter et al., J. Clin. Invest. 1 16:3266-76, 2006). The CD44^LO population of cells when implanted into brain tumors, generally do not remain, and typically die off when co-cultured with 9L cells in vitro. Therefore, they cannot be characterized by immunohistochemistry.

Example 3. Killing of sublethally irradiated tumors by CD44⁺ cells

[0087] We examined phagocytotic activity of CD44^HI cells on various tumor cells that have been sublethally irradicated. These tumor cells include rat gliosarcoma cell line 9L, mouse glioma cell line GL261, rat glioma cell line CNSl, rat glioma cell line RG2, and mouse B16 melanoma line. It was found that CD44^HI cells alone or in combination with CD44^LO cells appear to kill and remove the sublethally irradiated tumor cells. As shown in Figure 2, CD44^HI cells combined with CD44 ^LO cells were able to selectively kill 9L cells that have been irradiated at a dose of 4.5Gy. It was also found that, when irradiated with 4.5 Gy and coincubated with CD44^HI cells alone, GL261 cells are reduced in number by 95% (85% if irradiated at 3.0Gy). Similarly, CNSl and RG2 cells were also killed/phagocytosed with 4.5GY sublethal irradiation. In addition, it was found that mouse B16 melanoma cells are more radioresistant and require 6Gy to induce a tumor killing/phagocytosis response. From these studies, it appears that the tumor killing response of the CD44^H1 cells can be broadly applied to various primary brain tumors. In addition, the tumor killing activity can also be exploited against other systemic tumors as exemplified by melanoma which classically metastasizes to the brain.

Example 4. CD44^H1 cells need to be injected centrally to tumors

[0088] We have shown that specific fractions of bone marrow home to the tumor mass when implanted either in the contralateral hemisphere or posterior to the site of tumor implantation. The cells migrate to the tumor within one week of implantation. We wished to determine whether we could inject these cells peripherally and have them home to the tumor. To allow us to test within the same genetic background, we used the GL261 mouse brain tumor model that also leads to localization of CD44^HI cells. In this experiment, we implanted GL261 cells into the cerebrum of C57B1/6 mice. Six days later, we injected GFP labeled CD44^HI cells either directly into the tumor or peripherally into the tail vein. The intratumoral GFP CD44^HI cells proliferated and targeted to the tumor as described previously. However, appreciable numbers GFP cells were not observed in the tumor of peripherally-injected mice.

[0089] Thus, it appears that CD44^HI cells need to be injected centrally in order to home to the tumors. This result could be due to the number of cells delivered peripherally, the relative immune privilege of the brain, or other factors. Signals expressed by the tumor may set up a local gradient that can direct a migrating cell from one hemisphere to the other, but either the signal is not released systemically or the cells cannot cross the blood brain barrier. Alternatively, it is possible that peripheral injection results in a dilution effect so that the injected cells are not at a sufficient concentration in circulation to establish in the tumor.

[0090] We are interested in further characterizing the factors that confer homing ability in the CD44^HI cells. In tissue culture, we have studied the migration of these cells to different cytokines. The results indicate that several cytokines act as chemoattractants for the CD44^HI cells. Examples include SDF-1 (stromal cell-derived factor- 1 ; aka C-X-C motif ligand 12 or CXCL12) and Stem Cell Factor (SCF). It was observed that SDF1 functions better as a chemoattractant than SCF. In addition, we found that media conditioned by 9L cells is more effective at attracting the bone marrow progenitor cells than unconditioned media.

Example 5. Activities of rodent CD44* cells transduced with a lentiviral vector [0091] To enable delivery of therapeutic agents by the tumor-homing CD44⁺ cells, we examined expression of an exogenous gene transfected into the cells via a lentiviral based vector that expresses GFP. Use of the lenti-GFP vector and its transduction into CD44⁺ cells from C57B1/6 mice were carried out as described in Miyoshi et al., Science 283:682-686, 1999. We found that the GFP-expressing FG12 lentiviral vector was efficiently transduced into CD44^HI and CD44^LO cells. Employing a similar vector, we achieved a transduction efficiency of 60-80% in Lin⁺ and Lin^" cell fractions. In addition, we achieved a transduction efficiency of about 20% for both CD44^H1 and CD44^LO cell populations.

[0092] We then examined presence of the lenti-GFP vector transduced Lin^" cells and GFP expression in tumors in vivo. The cells were implanted into previously established 9L gliosarcoma brain tumors in SCID mice. The animals were sacrificed 15 days later (21 days after tumor implantation). The brains were then removed from the mice, tissue fixed, sectioned and assessed by fluoresence microscopy for GFP expression. As shown in Figure 3, the transduced Lin- cells were found within the established tumor and also targeted tumor satellites in vivo. Although the cells weren't implanted in the opposite hemisphere of the brain, their presence within the tumor and ability to target tumor satellites indicate that transduction with the exogenous gene-expressing lentivurus vector does not change the tumor homing ability of the Lin- cells. In addition, the results also showed that morphology of the transduced cells in the tumors appear to be similar to that of non-transduced cells as described in Example 1 (Fig. 1).

Example 6 Anti-tumor activities of human myeloid-like progenitor cell population

[0093] In addition to tumor homing and tumor killing activities of rodent myeloid-like progenitor cells described above, we also examined anti-tumor activities of a human myeloid-like progenitor cell population. The human cell population was isolated from human cord blood. It was found that, when activated with LPS, the cells appropriately homes to the tumor in an analogous fashion to the rodent CD44^HI cells. Human cord blood was obtained from NDRI. Mononuclear cells were separated from by density gradient using Ficoll Paque (Amersham). To isolate the CD14⁺ fraction human Cord Blood Mono Nuclear Cells (CBMNCs) were incubated with beads anti human CD 14 (Miltenyi) and purified following the MACS separation system. To characterize the CD14⁺ cells, different subpopulation were analyzed using a 2 color flow cytometry antibodies against CD33, CD1 lb, CD44, CD31, CD34 and CD80 (all from BD Biosciences-Pharmingen) and

VEGFR-2 and CD 144 (R&D system).

[0094] Upon isolating the myeloid-like population from human cord blood, the cells were transduced with a GFP-expressing adenoviral vector. Specifically, the freshly isolated human CD14⁺ cells from cord blood were incubated for 4-6 hr in Ml 99 (Invitrogen) and 20% FBS with AD5 F16 (Adenovirus expressing the fiber 16 and encoding eGFP (Ad5 F16 eGFP) at 5000 MOI. After the infection the cells were washed out from the virus and plated overnight. The percentage of GFP-expressing cells was analyzed by flow cytometry. We found that 70% of the CD14⁺ cells were efficiently transduced and expressed high level of the eGFP.

[0095] The human myeloid-like cells expressing the exogenous gene were then examined for their ability to home to tumor cells and control the growth of tumor cells in vitro. The results indicate that the Ad5F16 eGFP CD14⁺ cells were able to target to 9L tumor cells in culture (Fig. 4A). In addition, the transduced CD14⁺ cells reduced the growth of sub-lethally irradiated tumor cells in vitro (Fig. 4B). The cells also inhibited the proliferation of 7.5gy and lOgy irradiated 9L tumors cells, as evidenced by 60% decrease in the expression level of proliferating cell nuclear antigen (PCNA), a marker of DNA synthesis in the nucleus (Fig. 4C). Further, when stimulated with LPS, the cells killed tumor cells in vitro (Fig. 5). The cells were found to secret high level of important proinflammatory cytokines such as IL-Ι β and IL-6 (Fig. 5 and Table 1). When in contact with 7.5gy and lOgy irradiated 9L tumor cells, the human CD44⁺ cells also up-regulated the expression of human IL-8 when in contact with 7.5gy and lOgy irradiated 9L tumor cells and activated pro inflammatory pathways (Table 2). Additional to the in vitro studies, we also examined the in vivo appearance and behavior of these transduced human cells. It was found that, when co injected into SCID mice with irradiated 9L tumor cells, the CD14⁺ cells appear to localize to the tumor cells and also display a morphology that is similar to the rodent myeloid-like cells (CD44⁺ cells) described above (Fig. 7).

Table 1

7.5Gy CB Cntrl 1 1 1 1

7.5Gy CB LPS 294.56 89.01 77.54 1

lOGy CB Cntrl 1 1 1 1

lOGy CB LPS 334.80 94.97 95.84 1

Table 2

Example 7. Anti-tumor activities of subpopulations of bone marrow cells

[0096] After earlier work showed activity in different bone marrow subpopulations, we decided to test the parent whole bone marrow population for anti-tumor activity. It was found that whole bone marrow had strong anti-tumor activity. We then broke cells from the whole bone marrow down into its 3 subpopulations: lymphocytes, monocytes and granulocytes. As detailed below, it was determined that the monocytes are the anti-tumor effector cells, and that lymphocytes (and less cleanly granulocytes) from both bone marrow and spleen did not have anti-tumor activity and could serve as negative control cells.

[0097] Bone Marrow derived cells consist of hematopoietic stem/progenitor cells and their differentiated progenies of blood cells (monocytes, granulocytes, lymphocytes and erythrocytes). We wanted to determine if a single cell population from the bone marrow was active against tumor cells or whether synergism between cell populations was necessary. Therefore we separated the bone marrow into its 3 constituent populations and tested these against whole bone marrow. Specifically, after histopaque purification, rat bone marrow contains 26% granulocytes, 50% lymphocytes and 23% monocytes. Several cell surface antigens were used to identify pure sub populations in the granulocyte, lymphocyte and monocyte gates. Specifically, a fraction of lymphocytes and all monocytes were considered positive for CD54 (also called ICAM-1). All monocytes and granulocytes were positive for CD1 lb. The clone HIS48 stained all granulocytes and monocytes. Next, granulocytes, lymphocytes and monocytes were purified by FACS using antibodies against CD54 and HIS48 antigens. The purified cell populations include double negative cells (CD54^", HIS48^") or single positive for CD54 (CD54⁺ HIS48^") which are lymphocytes and confirmed by expression of CD3. Purified double positive cells (CD54⁺, HIS48⁺) were monocytes, and purified single positive cells for HIS48 (HIS48⁺ CD54^" ) were granulocytes. These sub- populations of bone marrow cells were then subject to further studies to investigate their anti-tumor activities, as described below.

[0098] In addition to identifying effector cells from bone marrow, we also tested splenocytes since the spleen serves in part as a reservoir for immune cells. We

characterized the cells after histopaque purification using the same antigens. The splenic cells were only 7% monocytes (CD54⁺HIS48⁺) and 5.7% granulocytes (CD54^"HIS48^"). The majority of the cells were lymphocytes (CD54⁺HIS48\ CD54^"fflS48^", CD3⁺) and other cells. Using an in vitro (in tissue culture) cell adhesion assay (see below) we found that this population had little if any anti-tumor activity. Therefore, the splenocytes with the splenic lymphocytes in particular, can serve as an additional negative control population.

[0099] We devised a simple cell adhesion fluorometric assay to quantify tumor killing in vitro. Results from the studies indicate that the observed anti-tumor activity resides in the monocyte population. Specifically, as shown in Figure 8, the isolated monocyte population (marked "M" in the figure) displayed strong anti-tumor activities. In addition, the data indicate that lymphocytes ("L") or granulocytes ("G") have little or no activities. As expected, a combination of the monocyte cells with lymphocytes and/or granulocytes) ("L+M", "G+M" or "G+M+L") also showed anti-tumor activity while a combination of lymphocytes and granulocytes did not lead to any activity. Results from the studies also indicate that monocytes act alone without any synergism with lymphocytes or granulocytes.

[00100] In another study, we found that a 1 :30 tumor to effector cell ratio has profound anti-tumor activity (Figures 9 and 10). There is evidence of a graded response with a lower effector to tumor ratio showing less activity against tumor cells in tissue culture. It also shows that CD54⁺ lymphocytes (ICAM-1 involved in cell-cell adhesion) do not have antitumor activity. In this study, the granulocytes showed some anti-tumor activity. However, in several other similar experiments, the granulocyte population did not have significant antitumor activity. For example, the study as shown in Fig. 10 confirms that the monocytes have potent anti-tumor activity, that lymphocytes either positive or negative for CD54 (ICAM-1) are negative effector cells, and that the granulocyte population also has no activity. It is likely that the observed activity for granulocyte in Fig. 9 was due to monocyte contamination, specific conditions of the animal from which the bone marrow was obtained, or other reasons.

Example 8 Treatment of established 9L gliosarcoma and melanoma

[00101] We first undertook studies to examine in vivo therapeutic activities of the myeloid- like cells or monocytes for treating gliosarcoma. In the first study, CD44 hi and CD44 low cells were coinjected with irradiated 9L gliosarcoma into the right frontal lobe of syngeneic CD fisher rats. The rats exhibited no outward signs of neurological injury, cachexia or dehydration. Two weeks later, the rats were euthanized, brains isolated, fixed and serially sectioned. Tumor volumes were determined. The results indicate that the size of tumors in rats treated with either the CD44 hi or the CD44 low populations was reduced. It is known that both the CD44 hi and the CD44 low populations contain monocytes. We are working to determine if the anti-tumor activity identified against 9L glioma cells in vivo is purely related to the monocytic fraction or whether there is in vivo synergism.

[00102] In another study, an established brain tumor was used and treated with

unfractionated whole bone marrow after histopaque purification. On day 0, 100K 9L- luciferase gliosarcoma cells were stereotactically implanted into the right frontal lobe of CD Fisher rats. On day 4, we implanted 6 million bone marrow cells into the same location. On day 7, we used Caliper imaging system to visualize the tumors. The results indicate that there is a reduction in tumor size (Figure 11). Specifically, all four imaged control rats had visualized tumors. In the experimental group, 5/7 showed minimal tumor at this time point.

[00103] We also investigated anti-tumor activity of the isolated myeloid-like progenitor cells against melanoma in tissue culture and in vivo. We examined the effector cell population for activity against melanoma cell lines in vitro (or in culture) and in vivo. Using syngeneic C57/B6 mice, we established intracranial B16/F10 melanoma tumors in the right frontal lobe and treated them six days later with CD44 hi or CD 44 low cells. With both populations we found anti-tumor activity (Figure 12). As both the CD44 hi and CD44 low cell populations contain monocytes, we are working to determine if the anti-tumor activity identified against melanoma in vivo is purely related to the monocytic fraction or whether there is in vivo synergism.

Example 9. Anti-tumor activities of cell populations of human bone marrow [00104] We studied several human bone marrow samples taken from discarded bone marrow from iliac crest (spine fusion operations) and from femurs during total hip replacement operations (IRB approved). We used a similar isolation and FACS analysis as described above for rodent bone marrow.

[00105] Using one cell surface marker, we are able to isolate the three different cell populations. As shown in Figure 13, human bone marrow monocytes are highly positive for the transmembrane receptor CD33, while granulocytes are medium/lower positive and lymphocytes do not express this molecule. The granulocytes in some samples, are represented by two distinct populations. The monocytes represent about 11% of the total bone marrow. Upon isolation of the different cell populations, preliminary work has been performed to confirm a similar profile of anti-tumor activity against human glioma cells as that described above for rodent cells.

[00106] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[00107] All publications, GenBank sequences, ATCC deposits, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted.

Claims

WE CLAIM:

1. A method of treating a solid tumor in a subject, comprising administering to the subject with a solid tumor an effective amount of a population of myeloid-like progenitor cells, thereby treating the solid tumor in the subject.

2. The method of claim 1 , wherein the population of myeloid-like progenitor cells is a population of human CD14⁺ cells or a population of rodent CD44^HI cells.

3. The method of claim 1, wherein the population of myeloid-like progenitor cells is a population of monocytes.

4. The method of claim 1 , wherein the myeloid-like progenitor cells are isolated from the subject.

5. The method of claim 1, wherein the subject is a human.

6. The method of claim 5, wherein the subject is administered with a population of human CD33^HI monocytes isolated from bone marrow.

7. The method of claim 5, wherein the subject is administered with a population of human CD14⁺ cells isolated from blood.

8. The method of claim 7, wherein at least 75% of cells in the population of human CD14⁺ cells express CD14⁺.

9. The method of claim 7, wherein at least 90% of cells in the population of human CD14⁺ cells express CD14⁺.

10. The method of claim 7, wherein the cells are administered via central injection to the subject.

11. The method of claim 7, wherein the cells are treated with a toll-like receptor 4 (TLR4) ligand prior to being administered to the subject. The method of claim 1 1, wherein the TLR4 ligand is lipopolysaccharide

(LPS).

13. The method of claim 1 , wherein the solid tumor is a brain tumor.

14. The method of claim 13, wherein the brain tumor is glioma, glioblastoma, ocular melanoma or a metastatic brain tumor.

15. The method of claim 1, further comprising treating the tumor with sublethal radiation therapy prior to administration of the cells.

16. A method of treating a solid tumor in a subject, comprising administering to the subject afflicted with a solid tumor an effective amount of a population of transfected myeloid-like progenitor cells, the cells being transfected with a gene that operably encodes an anti-tumor agent, thereby treating the solid tumor in the subject.

17. The method of claim 16, wherein the myeloid-like progenitor cells are human CD14⁺ cells or rodent CD44^HI cells.

18. The method of claim 16, wherein the myeloid-like progenitor cells are monocytes.

19. The method of claim 16, wherein anti-tumor agent is an antiangiogenic agent, a cytotoxic agent or a tumor suppressor.

20. The method of claim 16, wherein the gene encoding the anti-tumor agent is transfected into the cells via an expression vector.

21. The method of claim 20, wherein the expression vector is a lentiviral vector.

22. The method of claim 16, wherein the transfected cells are injected centrally to the subject.

23. The method of claim 16, wherein the subject is a human.

24. The method of claim 23, and the myeloid-like progenitor cells are human CD14⁺ cells isolated from blood of the subject or CD33^HI monocytes isolated from bone marrow of the subject.

25. The method of claim 16, wherein the solid tumor is a brain tumor.

26. The method of claim 25, wherein the brain tumor is glioma, glioblastoma, ocular melanoma or a metastatic brain tumor.

27. A method for delivering an anti-tumor agent to a solid tumor in a subject, comprising (1) constructing a vector expressing the anti-tumor agent; (2) transfecting the vector into a population of myeloid-like progenitor cells; and (3) administering the transfected cells to the subject.

28. The method of claim 27, wherein the myeloid-like progenitor cells are human CD14⁺ cells or rodent CD44^H1 cells.

29. The method of claim 27, wherein the myeloid-like progenitor cells are monocytes.

30. The method of claim 27, wherein the anti-tumor agent is an

antiangiogenic agent, a cytotoxic agent or a tumor suppressor.

31. The method of claim 27, wherein the vector is a lentiviral vector.

32. The method of claim 27, wherein the transfected cells are injected centrally to the subject.

33. The method of claim 27, wherein the subject is a human, and the myeloid- like progenitor cells are isolated from the subject.

34. The method of claim 27, wherein the solid tumor is a brain tumor.