WO2006062946A2

WO2006062946A2 - Incorporation of bone marrow derived stem cells in tumors

Info

Publication number: WO2006062946A2
Application number: PCT/US2005/044066
Authority: WO
Inventors: Christopher R. Cogle; Edward W. Scott
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2004-12-06
Filing date: 2005-12-06
Publication date: 2006-06-15
Also published as: WO2006062946A3

Abstract

Circulating bone marrow cells have been found to contribute to growth of colonic adenomas. Recipients of gender-mismatched transplants showed evidence of Y-chromosome positive epithelial cells, accounting for 1 to 4% of the cells in histologic section. Furthermore, there was no evidence of fusion events by confocal microscopic genotypic analysis. Compositions and methods of treatment of cancer patients, are described.

Description

INCORPORATION OF BONE MARROW DERIVED STEM CELLS IN TUMORS

FIELD OF THE INVENTION

[0001] Human bone" marrow cells may have the potential to serve as a therapeutic source of readily harvestable cells for targeting malignant environments. These cells are useful as vectors for gene therapy of tumors.

BACKGROUND

[0002] Multicellular animals are derived from a clone of cells descended from a single original cell, the fertilized egg. Embryogenesis involves the division and differentiation of multipotential cells, each cell having the ability to develop into multiple cellular lineages. Phenotypically, the cells of such lineages can vary substantially, such as blood cells, muscle cells and neural cells, being specialized.

[0003] A wide spectrum of diseases may be treated based upon both the possession of a population of cells having multi- lineage potential and an understanding of the mechanisms that regulate embryonic cell development. For example, the capacity to generate a new population of hematopoietic cells is the basis of bone marrow transplantation, which is currently used as a treatment for a growing number of diseases including anemia, leukemia and breast cancer. In addition, transplantation of genetically altered multipotential cells has been considered as potential therapy for a variety of different diseases including AIDS. [0004] Mammalian hematopoietic (blood) cells provide a diverse range of physiologic activities. Hematopoietic cells are divided into lymphoid, myeloid and erythroid lineages. The lymphoid lineage, comprising B, T and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes, as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like. The erythroid lineage provides the red blood cells, which act as oxygen carriers.

[0005] Despite the diversity of the nature, morphology, characteristics and function of hematopoietic cells, it is presently believed that these cells are derived from a single cell population, termed hematopoietic "stem cells." Unlike more "mature" blood cells, stem cells are capable of self-regeneration but may also divide into progenitor cells that are no longer pluripotent and have a limited self-regeneration. These progenitor cells divide repeatedly to form more mature cells which eventually become terminally differentiated to form the various mature hematopoietic cells. Thus the large number of mature hematopoietic cells is derived from a small reservoir of stem cells by a process of proliferation and differentiation. [0006] Progenitor cells mature into bipotential cells and then become lineage committed, that is, are incapable of maturing into more than one lineage. The use of the words progenitor or progenitor cells indicates cell populations which are no longer stem cells but which have not yet become terminally differentiated. The use of the word lymphoid, myeloid or erythroid in conjunction with progenitor indicates the potential cell populations into which the progenitor is capable of maturing.

[0007] Highly purified populations of stem cells currently find use in repopulation of the entire hematopoietic system. Purified progenitor cells of individual lineages would find use in repopulating or augmenting the various lineages. Although use of autologous cells would be highly beneficial in therapeutic applications, there is a need in the art to target cells to a desired location in the patient in need of such therapy and to be able to track the location and identify the cells used in such applications.

SUMMARY

[0008] Bone marrow contains stem cells that engraft in various distant organs. These observations prompted us to investigate the cellular origin of post-transplant solid tumor neoplasias found in women having received a bone marrow transplant at our facility. In women having received bone marrow transplantation from male siblings we found colonic adenomas post-transplant. Presence of donor derived cells were examined using a combination of immunohistochemistry for cells expressing the pan-leukocyte antigen, CD45, and the colonocyte antigen, cytokeratin 20, in conjunction with fluorescent in situ hybridization for X and Y chromosomes. Confocal microscopy was used to evaluate for evidence of cell fusion. In these cases, recipients of gender-mismatched transplants showed evidence of Y-chromosome positive cytokeratin positive cells within the adenomas. The male cells accounted for 1 to 4% of the adenoma cells in histologic section. Confocal microscopy permitted evaluation of nuclei (Z-stack steps of 0.25 microns) and demonstrated no male cell with more than one X chromosome. A total of 40 male cells expressing cytokeratin were found throughout the adenomas. When considering how bone marrow derived cells may migrate to neoplasias due to upregulation of tumor growth factors, which also act as inflammatory cytokines and chemokines, such as stromal derived factor- 1 (SDF- 1). Up- or down-regulating SDF-I has profound effects on recruiting circulating bone marrow stem/progenitor cells for engraftment as end-organ epithelia. SDF-I staining of the colonic adenomas found in our patients demonstrated intense, patchy SDF-I expression compared to normal colonic epithelia.

[0009] In a preferred embodiment, bone marrow derived stem cells are transformed with vectors expressing factors which inhibit cell migration. For example, inhibitors of SDF- 1 induced stem cell migration.

[00010] hi another preferred embodiment, stem cells are transformed with vectors expressing factors which induce cell migration. For example, SDF-I induced cell migration. [00011] In another preferred embodiment, the invention provides a composition comprising: isolated cells, a vector encoding for a desired molecule and a fluorescent protein, wherein the isolated cells are transformed with the vector. Preferably the isolated cell is a stem cell and the vector is further comprised of oligonucleotides encoding complementary mRNA to specific target mRNA which codes for cell surface antigens. These cell surface antigens, are for example, MHC molecules, or any other molecule that has been identified as being involved in the disease process. The cell whereby the antigen is not expressed, is referred to as an "antigen depleted cell" or in the case of MHC molecules as an "MHC depleted cell". Essentially, this method is used for depleting any antigen that is involved in a disease process.

[00012] hi another preferred embodiment, a stem cell is armed with an immunoglobulin used for targeting the stem cell to a desired in vivo location, such as a tumor. The antibody can be monoclonal, polyclonal and/or bispecifϊc. hi accordance with the invention, the bispecific antibody, targets the cell to a specific location in vivo. For example, the location can be to myocardial tissues, hepatocyte, kidneys and the like. The bispecific antibody determines the specific antigen to which the isolated cell is targeted. Preferably the cell is a stem cell so once the cell homes to the organ or tissue, the location of which is monitored using the methods discussed in detail in the examples which follow, the microenvironment of the target area induces the differentiation of the stem cell thereby the organ is repopulated with new cells. If the cell is antigen depleted and the antigen involved in the disease process is not present then the disease state is ameliorated.

[00013] In another preferred embodiment, bone marrow stem cells are transfected with vectors that produce cytotoxic molecules. Such cytotoxic molecules can be secreted in the microenvironment, thereby leading to the cytolysis of a tumor cell. [00014] hi another preferred embodiment, the invention provides a method for tracking and targeting cells to organs, and tissues in vivo. The cells are tracked starting from the point of introduction into the animal until the stem cell reaches the target of interest, hi particular, the invention provides for the phenotypic identification of the infused cells at any time point or location post-infusion. A particular advantage is using stem cells, which are assimilated by the target tissue or organ of interest and, due to the in vivo target microenvironment, develop into the cell type of the desired target. The therapeutic advantages are many and include the repair of tissues caused by a variety of factors, organs, cell linings, replacement of necrotic tissues and the like. The method is important especially in the areas of chemotherapy, cancers, autoimmune diseases, rejuvenation of necrotic tissues and organs. Especially of interest is the use of the method to repair organs such that organ transplantation may be overcome.

[00015] hi a preferred embodiment, the method for tracking stem cells comprises the steps of: (a) isolating and purifying stem cells from a subject; (b) providing a chemically heteroconjugated bispecific antibody with a binding site specific for a stem cell antigen and a binding site specific for a target antigen in a patient; and,(c) arming the stem cells with the bispecific antibody under conditions wherein; (i) the bispecific antibody binds to the stem cells via the c-kit ligand; and, (ii) the second antigenic binding site of the bispecific antibody is free to bind to the target antigen; and, (iii) binding of a labeled antibody to the Fc region of the bispecific antibody; or, (iv) fluorescently labeling the bispecific antibody thereby a secondary labeled antibody is not required; and, (d) reinfusing the armed and labeled stem cells into a patient; and, (e) tracking the armed and labeled stem cells by extracting samples from the patient at different time intervals; and, (f) identifying the armed and labeled cells by plienotyping the cells using flow cytometry cell sorting; (g) identifying the armed and labeled cells by immunohistochemical staining or other methods to detect the primary antibody on the cells in various target tissues such as bone marrow, spleen, liver, pancreas, lungs, neural tissue, gastrointestinal track, heart, vascular endothelium, etc.

[00016] hi particular, the use of characteristic phenotypic markers which identify the cell population can be used to track the armed and labeled cells. Examples include, but not limited to: CD2, CD3, CD8, CDlO, CD19, CD20, CD14, CD15, CD16, CD33, CD34, CD38, HLA-DR, C-Kit, Thy, Rho, CD45RA and the like, in animals, such as human, mice, primates and the like. The bispecific antibodies of the invention can recognize any antigenic determinant, desired by the user.

[00017] hi one aspect of the invention, the bispecific antibody is specific for c-kit ligand of stem cells and myocardial antigens. The specificity of the bispecific antibody for myocardial antigens, such as, for example, VCAM-I, NCAM-I, PECAM, etc., targets the stem cell to the heart.

[00018] In another preferred embodiment, a secondary antibody which is fluorescently labeled and specific for a region of the bispecific antibody, such as the Fc portion. In another preferred embodiment, the bispecific antibody is labeled directly thereby, by-passing the need for a second antibody.

[00019] In one aspect of the invention, the armed and labeled stem cells home to, and bind to the target tissue antigens. The stem cells accumulate at the target antigen site and differentiate into cells typical of the targeted tissue or organ. The location of the cells is verified by taking a patient sample, such as a blood sample or a biopsy of the targeted tissue or organ. The sample is subjected to cell sorting assays such as flow cytometry. Preferably, the samples are taken at different time intervals after reinfusion of the stem cells to track the location of the armed and labeled cells and to evaluate their functional capacity of the stem cells in stem cell assays or retransplantation assays. The numbers of armed and labeled cells at a particular time interval and/or in vivo location are quantitatively assessed by comparing the number of armed and labeled cells that were reinfused with the number of armed and labeled cells present in a sample at the particular time interval and/or in vivo location by flow cytometry. In accordance with the invention, the blood samples and target tissue samples taken from a patient at a particular time interval and quantitatively assessed using flow cytometry, is indicative of in vivo homing progress of armed and labeled stem cells to target tissues. In accordance with the invention, the blood samples and target tissue samples taken from a patient at a particular time interval will be sorted and tested for putative specific functions. The sorted cells can be used in any functional assay, depending on the cellular population. For example, cytokine assays, ELISA' s, B cell assays, T-cell assays such as cytotoxic assays, proliferation assays, and the like.

[00020] In another preferred embodiment, the invention provides a method for treating a patient suffering from cancer, comprising the steps of: (a) isolating stem cells from a patient suffering from cancer and/or HLA-matched or partially HLA-matched subject; (b) modifying a stem cell such as by arming the cell with an antibody or introducing a vector encoding a desired molecule; and, (c) reinfusing the stem cells into a patient. [00021] hi one aspect of the invention, the antibody is a bispecific antibody. The bispecific antibody comprises two monoclonal antibodies. However, the bispecific antibody can be comprised of two polyclonal antibodies or an engineered bispecific antibody. Preferably, each of the specificities of the bispecific antibody are directed to a tumor antigen and the T cell receptor complex. Antibodies can be raised against any tumor antigen from a patient. Thus the targeting of the cell can be individually tailored as the tumor displays different antigens. For tracking purposes, the bispecific antibody can be directly labeled or a second antibody specific for a region of the bispecific antibody is labeled. Detection of the armed cell is preferably through cell sorting techniques such as flow cytometry. For example, wherein samples are taken at different time intervals after reinfusion of the cells to track the location of the armed and labeled cells. The numbers of armed and labeled cells at a particular time interval and/or in vivo location are quantitatively assessed by comparing the number of armed and labeled cells that were reinfused with the number of armed and labeled cells present in a sample at the particular time interval and/or in vivo location by flow cytometry.

[00022] hi another preferred embodiment the invention provides for a method for imaging and tracking cells in vivo to a desired location, the method comprising: isolating and purifying cells from a subject; and, providing a chemically heteroconjugated bispecific antibody with a binding site specific for cellular antigen and a binding site specific for a target antigen in any location in a patient; and, arming the isolated cells with the bispecific antibody under conditions wherein; (i) the bispecific antibody binds to a specific antigen on the isolated cell; and, (ii) the second antigenic binding site of the bispecific antibody is free to bind to a target antigen; and, (iii) binding of a labeled antibody to the Fc region of the bispecific antibody; or, (iv) fluorescently labeling the bispecific antibody thereby a secondary labeled antibody is not required; and, (d) reinfusing the armed and labeled cells into a patient; and, (e) tracking the armed and labeled cells by extracting samples from the patient at different time intervals; and, (f) identifying the armed and labeled cells using flow cytometry cell sorting.

[00023] In another preferred embodiment, the cells used are from any source, such as bone marrow cells, stem cells, hematopoietic stem cells, erythroid stem cells and cells of the immune system. Examples include, lymphocytes, NK cells, and the like. [00024] hi another aspect of the invention, the cells are transformed with a vector that encodes for a fluorescent protein. Examples include, green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein.

[00025] In accordance with the invention, the bispecific antibody, targets the cell to a specific location in vivo. For example, the location can be to myocardial tissues, hepatocyte, kidneys and the like. The bispecific antibody determines the specific antigen to which the isolated cell is targeted. Preferably the cell is a stem cell so once the cell homes to the organ or tissue, the location of which is monitored using the methods discussed in detail in the examples which follow, the microenvironment of the target area induces the differentiation of the stem cell thereby the organ is repopulated with new cells. If the cell is antigen depleted and the antigen involved in the disease process is not present then the disease state is ameliorated. For example, MHC antigens.

[00026] In another preferred embodiment, a method of treating a patient suffering from cancer, comprises the steps of: (a) isolating cells from a patient suffering from cancer and/or from HLA-matched or partially HLA-matched subject; (b) modifying a stem cell by arming the cell with an antibody or introducing a vector encoding a desired molecule;(c) reinfusing the stem cells into a patient and, (d) administering an anti-inflammatory compound, thereby, treating a patient suffering from cancer. Preferably, the vector expresses ant-inflammatory agents that inhibit cell migration to the tumor.

[00027] In another preferred embodiment, the anti-inflammatory agents are directed to stromal derived factor 1 (SDF-I), MCP-I, MJP-lα, MJP-lft RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF₃ EPO, tenascin, MAdCAM-I, oA integrins, cό integrins, beta defensins 3 and 4. These can be antibodies which bind to these targets and inhibit the interaction of a migrating cell expressing the ligands which bind to each of these markers, thereby inhibiting cell migration to inflamed and/or tumor sites. [00028] In another preferred embodiment, anti-inflammatory compounds are administered, prior, in conjunction with, and/or after the stem cells. The anti-inflammatory compounds can be antibodies specific for stromal derived factor 1 (SDF-I), MCP-I, MIP- Ice, MIP-IjS, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, α4 integrins, cό integrins, beta defensins 3 and 4. In another aspect the anti-inflammatory compounds are: 5-aminosalicylates, glucocorticoids, thioguanine derivatives, methotrexate (MTX), cyclosporine, antibiotics, and infliximab. The chronic inflammation can be caused by bacterial, viral or parasitic infection or exposure to a carcinogen.

[00029] In another preferred embodiment, a method of treating a chronic inflammatory condition comprises administering to a patient having a chronic inflammatory condition stem cells expressing at least one protein which is an anti-inflammatory agent. The method comprises the steps of (a) obtaining stem cells from the patient; (b) transfecting, infecting or transducing the stem cells with an anti-inflammatory agent; and (c) administering the transfected, infected or transduced stem cells expressing the anti-inflammatory nucleic acid to the patient in an amount sufficient to treat the chronic inflammatory condition. Preferably, the anti-inflammatory agent inhibits cell migration to the inflammatory in vivo site. [00030] In one preferred embodiment, the nucleic acid expressing an anti-inflammatory agent inhibits cellular interactions between a cell expressing markers comprising stromal derived factor 1 (SDF-I), MCP-I, MIP-I α, MJP-IjS, RANTES, exotaxin EL-8, C3a, P- selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, α4 integrins, eδ integrins, beta defensins 3 and 4 and ligands thereof. The anti-inflammatory agent can be expressed by the vector as an antibody which binds to said markers. [00031] In one preferred embodiment, a method of treating epithelial cell inflammation and cancer comprises an anti-inflammatory agent, such as for example, anti-SDF agent. However any anti-inflammatory agent can be used, without limitation such as antibodies specific for stromal derived factor 1 (SDF-I), MCP-I, MIP-I α, MIP-IjS, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, α4 integrins, cδ integrins, beta defensins 3 and 4. hi another aspect the anti-inflammatory compounds are: 5-aminosalicylates, glucocorticoids, thioguanine derivatives, methotrexate (MTX), cyclosporine, antibiotics, and infliximab. For example, an antibody, competitive receptor blocker, anti-inflammatory compound and the like. Delivery of the anti-SDF agent is in sunscreen lotion, lotion, cream and the like.

[00032] In another preferred embodiment, a method of treating colon cancer comprises an anti-inflammatory agent (supra) such as for example, anti-SDF agent that is orally ingested in the form of a tablet, capsule, pill and the like or a suppository. [00033] In another preferred embodiment, a method of treating lung cancer comprises an anti-inflammatory agent (supra) such as, for example, anti-SDF agent that is delivered as an inhalant or aerosol. [00034] Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[00035] The invention is pointed out with particularity in the appended claims. The above and further advantages of this invention may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which: [00036] Figures IA through IF are histochemical stains showing BMDC give rise to epithelial neoplasias in humans. Basal cell skin cancer found in a woman before hematopoietic cell transplantation (Figure IA) showing nucleated (blue, DAPI) cells containing X chromosome (red). Six years after hematopoietic cell transplantation from her brother, she developed another basal cell skin cancer (Figure IB). The skin cancer after transplant is of male marrow origin as indicated by the nucleated cells (blue, DAPI) containing Y chromosomes (green). A squamous cell lung cancer was also found in this patient and demonstrates male cells (green, Y chromosome) in cytokeratin positive (brown) cells (Figure 1C, arrows). Colonic adenomas were also found in additional patients and stained for cells expressing cytokeratin (brown), mucin (magenta), nuclei (blue), and Y chromosome (green) (Figures ID and IE). Adenomas were also stained for the pan- leukocyte antigen, CD45 (brown), nuclei (blue), and Y chromosome (green) (Figure IF). Male colonocytes were recognized by their epithelial orientation, lack of CD45 expression and presence of Y chromosome DNA.

[00037] Figure 2 is a fluorescent micrograph showing epithelial neoplasias of bone marrow origin do not contain fusion karyotype. Typical fluorescent micrographs of adenoma sections found in women having received a gender-mismatched bone marrow transplantation. Left image (M) depicting a merged image of an adenoma with staining for Y chromosome (green), X chromosome (red), and nuclei (DAPI, blue). Arrows indicate adenomatous male cells lining the crypt. Right sided images are Z stack images, in order, from 1 to 16. Z stack steps are 0.5 microns. Indicated cells have only one Y chromosome and one X chromosome throughout their Z stack images.

[00038] , Figure 3A-3F are micrographs showing mouse models demonstrate that bone marrow can be a source of epithelial neoplasia in small bowel, colon, and lung. Micrographs of small bowel adenomas found in APC mutant female mice previously transplanted with male bone marrow (Figure 3A-3D). Male cells are distinguished by their Y chromosome (green). Male adenoma cells are CD45 negative (Figure 3B, arrows) and cytokeratin positive (Figure 3D, arrows). In a mouse model of lung cancer, a single (GFP, green) hematopoietic stem cell gives rise to cytokeratin positive (red) lung cancer cells (Figure 3E and 3F). Colocalization of red and green indicate lung cancer cells of hematopoietic stem cell origin (Figure 3F, arrows).

[00039] Figure 4 is an immunhistochemical stain showing high SDF-I expression in epithelial neoplasias of bone marrow origin. Immunohistochemical staining of adenoma sections, which contain male epithelial cells. Intense, brown (DAB) staining represents SDF- 1 protein expression predominantly located in the epithelial layers of the adenoma.

DETAILED DESCRIPTION

[00040] Compositions and methods for the treatment of tumors, such as solid tumors are disclosed. It was determined that circulating bone marrow cells contribute to growth of colonic adenomas. In particular, colonic adenomas found in women who received a hematopoietic cell transplantation from male siblings were examined for the presence of donor-derived epithelial cells with the combined use of fluorescence in situ hybridization of interphase nuclei, immunohistochemical staining for cytokeratin (CK20) and CD45 (leukocyte common antigen), periodic acid Schiff staining for mucin, and stromal derived factor 1 (SDF-I). Confocal microscopy was used to evaluate for evidence of cell fusion. In these adenoma cases, recipients of gender-mismatched transplants showed evidence of Y- chromosome positive epithelial cells, accounting for 1 to 4% of the cells in histologic section. Furthermore, there was no evidence of fusion events by confocal microscopic genotypic analysis. Bone marrow derived cells can incorporate as epithelia into adenomas of the gastrointestinal tract, without evidence of cell fusion.

Definitions

[00041] Prior to setting forth the invention, the following definitions are provided: [00042] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[00043] As used herein, the term "infectious agent" refers to an organism wherein growth/multiplication leads to pathogenic events in humans or animals. Examples of such agents are: bacteria, fungi, protozoa and viruses.

[00044] As used herein, a "pharmaceutically acceptable" component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. [00045] As used herein, the term "safe and effective amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a compound of the present invention effective to yield the desired therapeutic response. For example, an amount effective to delay the growth of or to cause a cancer, either a sarcoma or lymphoma, or to shrink the cancer or prevent metastasis. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

[00046] As used herein, a "pharmaceutical salt" include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids. Preferably the salts are made using an organic or inorganic acid. These preferred acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. The most preferred salt is the hydrochloride salt.

[00047] As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. Examples of cancers are cancer of the brain, breast, pancreas, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma. As used herein, the terms "cancer," "neoplasm," and "tumor," are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well- established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient.

[00048] The term "leukemia" refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease- acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic). Accordingly, the present invention includes a method of treating leukemia, and, preferably, a method of treating acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross¹ leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

[00049] The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Examples of sarcomas which can be treated with the present compositions and optionally a potentiator and/or chemotherapeutic agent include, but not limited to a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyte sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectatic sarcoma. [00050] The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas which can be treated with the compositions of the invention and optionally a potentiator and/or another chemotherapeutic agent include but not limited to, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

[00051] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Carcinomas which can be treated with the compositions of the invention and optionally a potentiator and/or a chemotherapeutic agent include but not limited to, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum. [00052] Additional cancers which can be treated with the compositions of the invention include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer. [00053] "Diagnostic" or "diagnosed" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. [00054] The terms "patient" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.

[00055] "Sample" is used herein in its broadest sense. A sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.

[00056] "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy. As used herein, "ameliorated" or "treatment" refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests. For example, amelioration or treatment of a patient suffering from an infectious disease organism, such as for example, Hepatitis B Virus, may be determined by a decrease of viral particles in a sample taken from a patient, as measured by, for example, a decrease in plaque forming units (p.f.u.).

[00057] The "treatment of neoplastic disease or neoplastic cells", refers to one or more of the following effects: (1) inhibition, to some extent, of tumor growth, including, (i) slowing down and (ii) complete growth arrest; (2) reduction in the number of tumor cells; (3) maintaining tumor size; (4) reduction in tumor size; (5) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of tumor cell infiltration into peripheral organs; (6) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of metastasis; (7) enhancement of anti-tumor immune response, which may result in (i) maintaining tumor size, (ii) reducing tumor size, (iii) slowing the growth of a tumor, (iv) reducing, slowing or preventing invasion and/or (8) relief, to some extent, of the severity or number of one or more symptoms associated with the disorder. [00058] "Treatment of an individual suffering from an infectious disease organism" refers to a decrease or elimination of the disease organism from an individual. For example, a decrease of viral particles as measured by plaque forming units or other automated diagnostic methods such as ELISA, etc., may be used to monitor efficacy of treatment. [00059] "Treatment of an individual suffering from graft- versus-host-disease or GVHD" refers to a decrease or cessation of symptoms associated with GVHD. For example, an amelioration of lacy, livid maculopapular rash, jaundice, diarrhoea, abdominal pain, hepatosplenomegaly, alopecia, bullae, desquamation of skin. Treatment or amelioration of GVHD results in clinical downgrading of the disease. For example, acute GVHD, which typically occurs in the first 100 days post-transplant, may be classified according to degree or "stage" of damage in the main target organs of GVHD, the skin, intestine, and liver. For example, liver GVHD is staged from none (stage 0; bilirubin < 2 mg/dl) to severe (stage 4; bilirubin > 15 mg/dl) based on serum bilirubin level. Skin GVHD is staged based upon the percent body surface area that the rash involves, with stage 0 having no rash and stage 4 having rash of up to 100 % body surface area with bullae or desquamation. Intestinal GVHD is staged based upon the volume of daily liquid stool output, with stage 0 being no diarrhea and stage 4 being > 1500 ml liquid stool per day with abdominal pain or ileus. Chronic GVHD, which typically occurs after day 100 post-transplant and can last several years post- transplant, is typically scored based upon number of organ sites that the chronic GVHD involves (limited chronic GVHD, one site; extensive chronic GVHD, two or more sites). Chronic GVHD involves the same organs as acute GVHD, but in addition, chronic GVHD-' may also affect the mucous glands in the eyes, salivary glands in the mouth, and glands that lubricate the stomach lining and intestines.

[00060] As used herein, "an ameliorated symptom" or "treated symptom" refers to a symptom which is approaches a normalized value, e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.

[00061] "Cells of the immune system" or "immune cells" as used herein, is meant to include any cells of the immune system that may be assayed, including, but not limited to, B lymphocytes, also called B cells, T lymphocytes, also called T cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor-infiltrating (TIL) cells, gene modified immune cells including hybridomas, drug modified immune cells, and derivatives, precursors or progenitors of the above cell types.

[00062] "Immune effector cells" refers to cells capable of binding an antigen and which mediate an immune response selective for the antigen. These cells include, but are not limited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes, macrophages, natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.

[00063] "Immune related molecules" refers to any molecule identified in any immune cell, whether in a resting ("non-stimulated") or activated state, and includes any receptor, ligand, cell surface molecules, nucleic acid molecules, polypeptides, variants and fragments thereof.

[00064] "T cells" or "T lymphocytes" are a subset of lymphocytes originating in the thymus and having heterodimeric receptors associated with proteins of the CD3 complex (e.g., a rearranged T cell receptor, the heterodimeric protein on the T cell surfaces responsible for antigen/MHC specificity of the cells). T cell responses may be detected by assays for their effects on other cells (e.g., target cell killing, activation of other immune cells, such as B-cells) or for the cytokines they produce.

[00065] As used herein, "allogeneic" is used to refer to immune cells derived from non- self major histocompatibility complex donors. HLA haplotypes/allotypes vary from individual to individual and it is often helpful to determine the individual's HLA type. The HLA type may be determined via standard typing procedures. [00066] As will be recognized by those in the art, the term "host compatible" or "autologous" cells means cells that are of the same or similar haplotype as that of the subject or "host" to which the cells are administered, such that no significant immune response against these cells occurs when they are transplanted into a host. [00067] As used herein, "partially-mismatched HLA", refers to HLA types that are between about 20% to about 90% compatible to the host's HLA type.

[00068] "CD4" is a cell surface protein important for recognition by the T cell receptor of antigenic peptides bound to MHC class II molecules on the surface of an APC. Upon activation, naϊve CD4 T cells differentiate into one of at least two cell types, ThI cells and Th2 cells, each type being characterized by the cytokines it produces. "ThI cells" are primarily involved in activating macrophages with respect to cellular immunity and the inflammatory response, whereas "Th2 cells" or "helper T cells" are primarily involved in stimulating B cells to produce antibodies (humoral immunity). CD4 is the receptor for the human immunodeficiency virus (HIV). Effector molecules for ThI cells include, but are not limited to, IFN-γ, GM-CSF, TNF-α, CD40 ligand, Fas ligand, IL-3, TNF-β, and IL-2. Effector molecules for Th2 cells include, but are not limited to, IL-4, IL-5, CD40 ligand, IL- 3, GS-CSF, IL-10, TGF-β, and eotaxin. Activation of the ThI type cytokine response can suppress the Th2 type cytokine response, and reciprocally, activation of the Th2 type cytokine response can suppress the ThI type response.

[00069] A "chemokine" is a small cytokine involved in the migration and activation of cells, including phagocytes and lymphocytes, and plays a role in inflammatory responses. [00070] A "cytokine" is a protein made by a cell that affect the behavior of other cells through a "cytokine receptor" on the surface of the cells the cytokine effects. Cytokines manufactured by lymphocytes are sometimes termed "lymphokines." Cytokines are also characterized as Type I (e.g. IL-2 and IFN-γ) and Type II (e.g. IL-4 and IL-10). [00071] By the term "modulate," it is meant that any of the mentioned activities, are, e.g., increased, enhanced, increased, agonized (acts as an agonist), promoted, decreased, reduced, suppressed blocked, or antagonized (acts as an agonist). Modulation can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over baseline values. Modulation can also decrease its activity below baseline values.

[00072] An "epitope", as used herein, is a portion of a polypeptide that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Epitopes may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native polypeptide for the ability to react with antigen-specific antisera and/or T-cell lines or clones. An epitope of a polypeptide is a portion that reacts with such antisera and/or T-cells at a level that is similar to the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may also be predicted via computer analysis.

[00073] "Substrate" refers to any rigid or semi-rigid support to which nucleic acid molecules or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.

[00074] "Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. [00075] As used herein, the term "transplant" includes any cell, organ, organ system or tissue which can elicit an immune response in a recipient subject mammal, hi general, therefore, a transplant includes an allograft or a xenograft cell, organ, organ system or tissue. An allograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of the same species as the recipient. A xenograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of a different species as the recipient. [00076] The term "immune rejection," as used herein, is intended to refer to immune responses involved in transplant rejection, as well as to the concomitant physiological result of such immune responses, such as for example, interstitial fibrosis, chronic graft artheriosclerosis, or vasculitis. The term "immune rejection," as used herein, is also intended to refer to immune responses involved in autoimmune disorders, and the concomitant physiological result of such immune responses, including T cell-dependent infiltration and direct tissue injury; T cell-dependent recruitment and activation of macrophages and other effector cells; and T cell-dependent B cell responses leading to autoantibody production. [00077] The term "transplant rejection," as used herein, refers to T cell-mediated rejection of transplant cells, organs, organ systems or tissue, hi general, such transplant rejection generally includes accelerated, acute and chronic rejection. It is intended that the term, as used herein, also refer to GVHD, and the physiological results of such a disorder. [00078] The term "reducing immune rejection," is meant to encompass prevention or inhibition of immune rejection, as well as delaying the onset or the progression of immune rejection. The term is also meant to encompass prolonging survival of a transplant in a subject mammal, or reversing failure of a transplant in a subject. Further, the term is meant to encompass ameliorating a symptom of an immune rejection, including, for example, ameliorating an immunological complication associated with immune rejection, such as for example, interstitial fibrosis, chronic graft atherosclerosis, or vasculitis. The term is also meant to encompass induction of tolerance in a subject mammal that has undergone a transplant.

[00079] The term "tolerance," as used herein, refers to a state wherein the immune system of a transplant recipient subject mammal is non-responsive to the transplant. This state is considered donor transplant-specific, and, as such, is distinguished from nonspecific immunosuppression. Operatively, the term as used herein, refers to permanent acceptance of a graft without ongoing immunosuppression, wherein, for example, challenge with a second graft of donor origin (especially when the second graft is of the same tissue as the first graft) should be accepted, and challenge with a third party graft should be rejected. [00080] The term "autoimmune rejection," as used herein, refers to immune responses involved in autoimmune disorders, and the concomitant physiological result of such immune responses.

[00081] The term "activated T cell," as used herein, refers to a T cell that expresses antigens indicative of T-cell activation (that is, T cell activation markers). Examples of T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40 and 4-1BB. The expression of activation markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.

[00082] The term "resting T cell," as used herein, refers to a T cell that does not express T-cell activation markers. Resting T cells include, but are not limited to, T cells which are CD25^', CD69^", ICOS^", SLAM^', and 4- IBB^". The expression of these markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.

[00083] The term "T cell activator," as used herein, refers to any compound or factor that is a T cell receptor stimulatory factor, that is, induces T cell receptor signaling. Preferably, the compound or factor also induces co-stimulatory pathways. Non-limiting examples of T cell activators include, but are not limited to, anti-CD3, antibodies (preferably monoclonal antibodies) either alone or in conjunction with anti-CD28 antibodies (preferably monoclonal antibodies), or mitogens such as, for example, phorbol 12-myristate 13 -acetate (PMA), phytohemagglutinin (PHA) or concanavalin-A (Con- A).

[00084] "Acute inflammation" is associated with disorders in which tissue inflammation is generally of relatively short duration, and lasts from about a few minutes to about one to two days, although it may last several weeks. The main characteristics of acute inflammatory disorders include increased blood flow, exudation of fluid and plasma proteins (edema) and emigration of leukocytes, such as neutrophils.

[00085] "Anaplasia" refers to the histological features of cancer. These features include derangement of the normal tissue architecture, the crowding of cells, lack of cellular orientation termed dyspolarity, cellular heterogeneity in size and shape termed "pleomorphism." The cytologic features of anaplasia include an increased nuclear- cytoplasmic ratio (the nuclear-cytoplasmic ratio can be over 50% for malignant cells), nuclear pleomorphism, clumping of the nuclear chromatin along the nuclear membrane, increased staining of the nuclear chromatin, simplified endoplasmic reticulum, increased free ribosomes, pleomorphism of mitochondria, decrease in size and number of organelles, enlarged and increased numbers of nucleoli, and sometimes the presence of intermediate filaments.

[00086] "Biological samples" include solid and body fluid samples. The biological samples used in the present invention can include cells, protein or membrane extracts of cells, blood or biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid). Examples of solid biological samples include, but are not limited to, samples taken from tissues of the central nervous system, bone, breast, kidney, cervix, endometrium, head/neck, gallbladder, parotid gland, prostate, pituitary gland, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid, heart, lung, bladder, adipose, lymph node, uterus, ovary, adrenal gland, testes, tonsils and thymus. Examples of "body fluid samples" include, but are not limited to blood, serum, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, and tears.

[00087] "Bone marrow derived progenitor cell" (BMDC) or "bone marrow derived stem cell" refers to a primitive stem cell with the machinery for self-renewal constitutively active. Included in this definition are stem cells that are totipotent, pluripotent and precursors. A "precursor cell" can be any cell in a cell differentiation pathway that is capable of differentiating into a more mature cell. As such, the term "precursor cell population" refers to a group of cells capable of developing into a more mature cell. A precursor cell population can comprise cells that are totipotent, cells that are pluripotent and cells that are stem cell lineage restricted (i.e. cells capable of developing into less than all hematopoietic lineages, or into, for example, only cells of erythroid lineage). As used herein, the term "totipotent cell" refers to a cell capable of developing into all lineages of cells. Similarly, the term "totipotent population of cells" refers to a composition of cells capable of developing into all lineages of cells. Also as used herein, the term "pluripotent cell" refers to a cell capable of developing into a variety {albeit not all) lineages and are at least able to develop into all hematopoietic lineages (e.g., lymphoid, erythroid, and thrombocytic lineages). Bone marrow derived stem cells contain two well-characterized types of stem cells. Mesenchymal stem cells (MSC) normally form chondrocytes and osteoblasts. Hematopoietic stem cells (HSC) are of mesodermal origin that normally give rise to cells of the blood and immune system (e.g., erythroid, granulocyte/macrophage, magakaryocite and lymphoid lineages). In addition, hematopoietic stem cells also have been shown to have the potential to differentiate into the cells of the liver (including hepatocytes, bile duct cells), lung, kidney (e.g., renal tubular epithelial cells and renal parenchyma), gastrointestinal tract, skeletal muscle fibers, astrocytes of the CNS, Purkinje neurons, cardiac muscle (e.g., cardiomyocytes), endothelium and skin. [00088] "Chronic inflammatory disorders," generally, are of longer duration, e.g., weeks to months to years or even longer, and are associated histologically with the presence of lymphocytes and macrophages and with proliferation of blood vessels and connective tissue. Inflammatory disorders are generally characterized by heat, redness, swelling, pain and loss of function. Examples of causes of inflammatory disorders include, but are not limited to, microbial infections (e.g., bacterial, viral and fungal infections), physical agents (e.g., burns, radiation, and trauma), chemical agents (e.g., toxins and caustic substances), tissue necrosis and various types of immunologic reactions. Examples of chronic inflammatory disorders, include, but are not limited to osteoarthritis, rheumatoid arthritis, asthma, cystic fibrosis, chronic infections (e.g., to Schistosomiasis, Papilloma, Helicobacter, Hepatitis B and C, EBV, HPV); inflammatory bowel disease (FBD), Crohn's disease, psoriasis, atopic eczema, acne, systemic lupus erythematosis, multiple sclerosis, atherosclerosis, restenosis, chronic bronchitis, sinusitis, chronic gastroenteritis and colitis, chronic cystitis and urethritis, hepatitis, chronic dermatitis, chronic conjunctivitis, chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendinitis), uremic pericarditis, chronic cholecystis, chronic vaginitis, and chronic uveitis.

[00089] As used herein "differentiation" refers to the synthesis of proteins that are produced selectively in a single cell type (for example, albumin in hepatocytes). Differentiation is generally reflected in specialized structure and function of cells. [00090] "Dysplasia" refers to a pre-malignant state in which a tissue demonstrates histologic and cytologic features intermediate between normal and anaplastic. Dysplastic cells demonstrate cellular growth abnormality in which the cellular appearance is altered and tissue architecture might be disturbed. Dysplasia is often reversible. [00091] As used herein "ectoderm" refers to the outermost germ layer of the developing embryo which gives rise to the epidermis and nerves; "endoderm" refers to the innermost germ layer of the developing embryo which gives rise to the epithelia of the lung, digestive tract, bladder and urethra; and "mesoderm" refers to the middle germ layer of the developing embryo which gives rise to the musculoskeletal, vascular and urinogenital systems, and connective tissue (e.g., dermis). "Invasive" or "aggressive" as used herein with respect to cancer refers to the proclivity of a tumor to expand beyond its boundaries into adjacent tissue, or to the characteristic of the tumor with respect to metastasis. The invasive property of a tumor is often accompanied by the elaboration of proteolytic enzymes, such as collagenases, that degrade matrix material and basement membrane material to enable the tumor to expand beyond the confines of the particular tissue in which that tumor is located. [00092] "BMDC-dependent metaplasia" refers to metaplastic tissue containing cells of bone marrow derived stem cell origin that demonstrate at least one biological or histological characteristic associated the tissue in which the metaplasia occurs. "BMDC-associated cancer" refers to a neoplastic tissue containing benign or malignant cells of bone marrow derived stem cell origin that demonstrate one or more biological or histological characteristics associated with the tissue in which the neoplasia occurs. [00093] "BMDC-dependent metaplasia" refers to metaplastic tissue containing cells of bone marrow derived stem cell origin that demonstrate at least one biological or histological characteristic associated the tissue in which the metaplasia occurs. "BMDC-associated cancer" refers to a neoplastic tissue containing benign or malignant cells of bone marrow derived stem cell origin that demonstrate one or more biological or histological characteristics associated with the tissue in which the neoplasia occurs. [00094] "BMDC-derived cell" refers to a cell of bone marrow derived stem cell origin that demonstrates at least one biological or histological characteristic associated only with a BMDC cell, and at least one biological or histological characteristic associated only with a non-BMDC cell (e.g., of a tissue other than bone marrow). Similarly, a "Mesenchymal- derived cell" refers to a BMDC-derived cell of mesodermal origin that demonstrates at least one biological or histological characteristic associated only with a mesenchymal stem cell (MSC), e.g., expresses KRT1-19 and TFF2 in the presence of gastric tissue, and at least one biological or histological characteristic associated only with a non-MSC cell (e.g., of a tissue other than bone marrow). Additionally, an "Hematopoietic-derived cell" refers to a BMDC- derived cell of mesodermal origin that demonstrates at least one biological or histological characteristic associated only with a Hematopoietic stem cell (HSC), e.g., does not express KRT 1-19 and TFF2 in the presence of gastric tissue, and at least one biological or histological characteristic associated only with a non-HSC cell (e.g., of a tissue other than bone marrow).

[00095] A "marker nucleic acid" is a gene whose expression (e.g., mRNA, cDNA) is found in BMDC or BMDC-derived cells used in the methods of the invention, and not found in non-BMDC cells. Such marker nucleic acids include DNA comprising the entire or partial sequence of a gene specifically expressed in a BMDC. For example, a "BMDC-specific oligonucleotide" or "BMDC-specific primer" means a DNA sequence that has at least 15 nucleotides from the sequence of a gene specifically expressed in BMDCs. A "marker protein" is a protein encoded by or corresponding to a marker nucleic acid of the invention. A marker protein comprises the entire or a partial sequence of a protein specifically expressed in BMDC. The terms "protein" and "polypeptide" are used interchangeably herein. [00096] As used herein, "metaplasia" refers to the conversion of one cell or tissue type into another, including transdifferentiation and conversion between undifferentiated stem cells of different tissues. "Transdifferentiation" refers to the conversion of one differentiated cell type to another, with or without an intervening cell division. Naturally occurring metaplasias are associated with excessive growth that arises through either wound healing or abnormal response to hormonal stimulation. For example, ectopic bone formation is quite common in surgical scars, muscle that is subjected to repeated trauma, or the walls of sclerotic arteries. The epithelia of respiratory tract or urinary bladder can undergo squamous metaplasia, a precursor to squamous cell carcinoma. Intestinal metaplasia of the stomach can generate patches of intestinal crypts or villi within the stomach. Barrett's metaplasia of the esophagus can develop as a result of duodenal-esophageal reflux and is considered the precursor lesion for the development of esophageal adenocarcinoma. Patches of ectopic epithelium, for example patches of tubal or endocervical epithelium can also develop in the endometrial lining of the uterus.

[00097] The term "metastasis" refers to the condition of spread of cancer from the organ or tissue of origin to additional distal sites in the patient. The process of tumor metastasis is a multistage event involving local invasion and destruction of intracellular matrix, intravasation into blood vessels, lymphatics or other channels of transport, survival in the circulation, extravasation out of the vessels into secondary sites and growth in the new location(s). Increased malignant cell motility has been associated with enhanced metastatic potential in animal as well as human tumors. The term "micrometastatic disease" refers to a locally invasive cancer from the organ or tissue of origin, for example, to proximal tissues or sentinel lymph nodes. "Neoplasia" or "neoplastic transformation" is the pathologic process that results in the formation and growth of a neoplasm, tissue mass, or tumor. Such process includes uncontrolled cell growth, including either benign or malignant tumors. Neoplasms include abnormal masses of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues and persists in the same excessive manner after cessation of the stimuli which evoked the change. Neoplasms may show a partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue.

[00098] Neoplasms tend to morphologically and functionally resemble the tissue from which they originated. For example, neoplasms arising within the islet tissue of the pancreas resemble the islet tissue, contain secretory granules, and secrete insulin. Clinical features of a neoplasm may result from the function of the tissue from which it originated. [00099] By assessing the histologic and other features of a neoplasm, it can be determined whether the neoplasm is benign or malignant. Invasion and metastasis (the spread of the neoplasm to distant sites) are definitive attributes of malignancy. Despite the fact that benign neoplasms may attain enormous size, they remain discrete and distinct from the adjacent non-neoplastic tissue. Benign tumors are generally well circumscribed and round, have a capsule, and have a grey or white color, and a uniform texture. By contrast, malignant tumor generally have fmgerlike projections, irregular margins, are not circumscribed, and have a variable color and texture. Benign tumors grow by pushing on adjacent tissue as they grow. As the benign tumor enlarges it compresses adjacent tissue, sometimes causing atrophy. The junction between a benign tumor and surrounding tissue may be converted to a fibrous connective tissue capsule allowing for easy surgical remove of benign tumors. By contrast, malignant tumors are locally invasive and grow into the adjacent tissues usually giving rise to irregular margins that are not encapsulated making it necessary to remove a wide margin of normal tissue for the surgical removal of malignant tumors. Benign neoplasms tends to grow more slowly than malignant tumors. Benign neoplasms also tend to be less autonomous than malignant tumors. Benign neoplasms tend to closely histologically resemble the tissue from which they originated. More highly differentiated cancers, cancers that resemble the tissue from which they originated, tend to have a better prognosis than poorly differentiated cancers. Malignant tumors are more likely than benign tumors to have an aberrant function (i.e. the secretion of abnormal or excessive quantities of hormones).

Stem Cell Compositions

[000100] hi a preferred embodiment, the stem cells are transformed with nucleic acids which encode for desired chemokines, such as for example, cytokines which attract immune cells to a tumor. As discussed in detail in the Examples which follow, bone marrow stem cells have been shown to migrate to a tumor site in vivo. Population of a tumor with stem cells producing chemoattractants will increase the homing of immune cells to such sites, detecting of the tumor cells and subsequent cytolysis of the tumor cell. [000101] In another preferred embodiment, the invention provides methods for the targeting and tracking of stem cells to specific locations within an animal's body. The methods used herein are also useful in the therapeutic applications of repairing or colonizing specifically targeted areas within an animal, with stem cells, which then differentiate into mature cells of the specific cell type of the targeted area.

[000102] hi a preferred embodiment, the method of the invention comprises arming stem cells with antibodies specific to antigens in a desired target area and arming the stem cells with a second antibody for the in vivo tracking of the stem cell from any area in the animal's body to the desired target area, such as for example, a tumor. [000103] In accordance with the invention, stem cells from a patient are harvested, sorted, purified and identified. The stem cells are then armed with an antibody which will target the stem cell to the targeted location, hi accordance with the invention, this procedure can be used for different antigen specificities of armed stem cells, e.g. Her2/neu, prostate tumor antigens, pancreatic tumor antigens, etc. Different isotypes of the arming antibody (e.g. IgGl, etc.) can be detected by utilizing secondary antibodies specific for the isotype. The secondary antibody can come from different sources, e.g. rat, sheep, goat etc; the important property being that it is targeted against the species of origin of the primary antibody. Also, secondary antibodies conjugated with different fluorochromes can be used, e.g. PE, FITC, APC, etc.

[000104] Isolation of cells useful in the present invention are well known in the art. For example, peripheral blood mononuclear cells (PBMCs) can be obtained from a subject and isolated by density gradient centrifugation, e.g., with Ficoll/Hypaque. Specific cell populations can be depleted or enriched using standard methods. For example, monocytes/macrophages can be isolated by adherence on plastic. T cells or B cells can be enriched or depleted, for example, by positive and/or negative selection using antibodies to T cell or B cell surface markers, for example by incubating cells with a specific primary monoclonal antibody (mAb), followed by isolation of cells that bind the mAb using magnetic beads coated with a secondary antibody that binds the primary mAb. Peripheral blood or bone marrow derived hematopoietic stem cells can be isolated by similar techniques using stem cell-specific mAbs (e.g., anti-CD34 mAbs). Specific cell populations can also be isolated by fluorescence activated cell sorting according to standard methods. Monoclonal antibodies to cell-specific surface markers known in the art and many are commercially available.

[000105] hi another preferred embodiment, the methods of the invention are used for the repopulation of destroyed cells in an organ in need of repair. For example, kidneys, liver, heart, lungs, intestines and the like. The stem cells are armed with an antibody which is specific to the organ of interest. This is highly advantageous in patients suffering from organ damage due to trauma, car accidents, diseases, burn victims, patients with slow wound healing as in diabetes or hemophiliacs, hi an illustrative example, stem cells are armed with antibodies that target, for example, regions of a damaged liver due to cirrhosis. The stem cells are also armed with a secondary antibody so that the trafficking of the cell is monitored. When the stem cells reach the liver or region of the liver in need of repair, the stem cells repopulate the region of the liver by developing into hepatic cells due to influences of the environment. More importantly, stem cells from closely match HLA donors can tolerize the immune system so the administered stem cells are not recognized as being "foreign." [000106] Any cell can be used in the methods of the invention, including but not limited to, stem cells, thymocytes, precursor cells and the like. A precursor cell population includes cells of a mesodermal derived cellular lineage, more particularly of hematopoietic lineage, endothelial lineage, muscle cell lineage, epithelial cell lineage and neural cell lineage. [000107] A "precursor cell" can be any cell in a cell differentiation pathway that is capable of differentiating into a more mature cell. As such, the term "precursor cell population" refers to a group of cells capable of developing into a more mature cell. A precursor cell population can comprise cells that are totipotent, cells that are pluripotent and cells that are stem cell lineage restricted (i.e. cells capable of developing into less than all hematopoietic lineages, or into, for example, only cells of erythroid lineage). As used herein, the term "totipotent cell" refers to a cell capable of developing into all lineages of cells. Similarly, the term "totipotent population of cells" refers to a composition of cells capable of developing into all lineages of cells. Also as used herein, the term "pluripotent cell" refers to a cell capable of developing into a variety {albeit not all) lineages and are at least able to develop into all hematopoietic lineages (e.g., lymphoid, erythroid, and thrombocytic lineages). For example, a pluripotent cell can differ from a totipotent cell by having the ability to develop into all cell lineages except endothelial cells. A "pluripotent population of cells" refers to a composition of cells capable of developing into less than all lineages of cells but at least into all hematopoietic lineages. As such, a totipotent cell or composition of cells is less developed than a pluripotent cell or compositions of cells. As used herein, the terms "develop", "differentiate" and "mature" all refer to the progression of a cell from the stage of having the potential to differentiate into at least two different cellular lineages to becoming a specialized cell. Such terms can be used interchangeably for the purposes of the present application. [000108] As used herein, the term "population" refers to cells having the same or different identifying characteristics. The term "lineage" refers to all of the stages of the development of a cell type, from the earliest precursor cell to a completely mature cell (i.e. a specialized cell).

[000109] A stem cell population of the present invention is capable of developing into cells of mesodermal cell lineage, of ectodermal cell lineage or of endodermal cell lineage. As used herein, mesodermal cells include cells of connective tissue, bone, cartilage, muscle, blood and blood vessel, lymphatic and lymphoid organ, notochord, pleura, pericardium, peritoneum, kidney and gonad. Ectodermal cells include epidermal tissue cells, such as those of nail, hair, glands of the skin, the nervous system, the external sense organs (e.g., eyes and ears) and mucous membranes (such as those of the mouth and anus). Endodermal cells include cells of the epithelium such as those of the pharynx, respiratory tract (except the nose), digestive tract, bladder and urethra cells. Preferred cells within a stem cell population of the present invention include cells of at least one of the following cellular lineages: hematopoietic cell lineage, endothelial cell lineage, epithelial cell lineage, muscle cell lineage and neural cell lineage. Other preferred cells within a stem cell population of the present invention include cells of erythroid lineage, endothelial lineage, leukocyte lineage, thrombocyte lineage, erythroid lineage (including primitive and definitive erythroid lineages), macrophage lineage, neutrophil lineage, mast cell lineage, megakaryocyte lineage, natural killer cell lineage, eosinophil lineage, T cell lineage, endothelial cell lineage and B cell lineage.

[000110] Various techniques may be employed to separate the cells by initially removing cells of dedicated lineage. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation. [000111] If desired, a large proportion of terminally differentiated cells may be removed by initially using a "relatively crude" separation. For example, magnetic bead separations may be used initially to remove large numbers of lineage committed cells. Desirably, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed. [000112] Procedures for separation may include but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including but not limited to, complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique. [000113] Techniques providing accurate separation include but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. [000114] In another preferred embodiment, the stem cells may be transformed with DNA which codes for different growth factors and/or cytokines which will aid in the differentiation of the stem cells if the organ of interest is damaged to the extent that the microenvironment is not supportive of cell differentiation.

[000115] In another preferred embodiment, the stem cells are transformed with nucleic acids which are complementary to genes which code for antigens which are recognized by the immune system, such that these genes are rendered incapable of producing these antibodies. This is especially advantages in autoimmune diseases whereby the immune system recognizes self-antigens and mounts an immune reaction. Such autoimmune diseases include arthritis, myocarditis, myasthenia gravis and the like. These cells are then armed with antibodies which allows their targeting and detection at the site they home to. In accordance with the invention, the stem cells differentiate into mature cells representative of the target area but do not express the antigen which is inducing an autoimmune reaction thereby alleviating or down-regulating the autoimmune response. In other cases it is desirable to target the stem cells to areas whereby diseases such as cancer have destroyed certain target areas such as for example, colon cancer. Stem cells can be targeted to areas which have been removed by surgery or have been affected by chemotherapy and allowed to repopulate the area. Li other cases, such as in hemophiliacs, it is desirable to target stem cells to the blood vessel lining thereby repairing the blood vessels and prevent further bleeding. [000116] The methods of the invention have many advantages over gene therapy or organ transplantation, skin grafts and the like. The stem cells are immature and are able to repopulate without an immune response being mounted.

[000117] The terms, "patient", "subject" or "animal" are used interchangeably and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.

[000118] In another preferred embodiment, stem cells are harvested, transformed with complementary strand oligonucleotides which deletes certain gene segments (antisense therapy) good for diseases such as autoimmune diseases and the like. By decreasing the expression of the antigen that is recognized by cells involved in the autoimmune process. [000119] The term "DNA construct" and "vector" are used herein to mean a purified or isolated polynucleotide that has been artificially designed and which comprises at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their natural environment.

[000120] As used herein, the term "administering a molecule to a cell" (e.g., an expression vector, nucleic acid, a angiogenic factor, a delivery vehicle, agent, and the like) refers to transducing, transfecting, microinjecting, electroporating, or shooting, the cell with the molecule. In some aspects, molecules are introduced into a target cell by contacting the target cell with a delivery cell (e.g., by cell fusion or by lysing the delivery cell when it is in proximity to the target cell). [000121] A cell has been "transformed", "transduced", or "transfected" by exogenous or heterologous nucleic acids when such nucleic acids have been introduced inside the cell. Transforming DNA may or may not be integrated (covalently linked) with chromosomal DNA making up the genome of the cell, hi prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element, such as a plasmid. hi a eukaryotic cell, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations (e.g., at least about 10).

[000122] The DNA segments typically further include an expression control DNA sequence operably linked to the humanized immunoglobulin coding sequences, including naturally-associated or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences. [000123] As stated previously, the DNA sequences can be expressed in hosts after the sequences have been operably linked to (i.e., positioned to ensure the functioning of) an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., tetracycline or neomycin resistance, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein by reference). [000124] E. coli is one prokaryotic host useful particularly for cloning the DNA sequences of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well- known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta- lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. [000125] Other microbes, such as yeast, may also be used for expression. Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.

[000126] In addition to microorganisms, mammalian tissue cell culture may also be used to express and produce polypeptides (see, Winnacker, "From Genes to Clones," VCH Publishers, New York, N. Y. (1987), which is incorporated herein by reference). Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, preferably myeloma cell lines, etc, and transformed B- cells or hybridomas. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev., 89, 49-68 (1986), which is incorporated herein by reference), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, cytomegalovirus, Bovine Papilloma Virus, and the like.

[000127] The vectors containing the DNA segments of interest (e.g., lymphokines, encoding sequences and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See, generally, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, (1982), which is incorporated herein by reference.)

[000128] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin Remington's Pharm. Sd., 15th Ed. (Mack Publ. Co., Easton (1975)).

[000129] The vectors may also comprise oligonucleotides which express mRNA which is complementary to mRNA coding for surface antigens which are involved, in for example, autoimmune diseases. Transforming cells, which are armed and labeled, with these vectors results in a decrease of such surface antigens, or "antigen depleted" or "MHC depleted" cells and these cells are targeted to specific tissues or organs wherein an autoimmune response is leading to the destruction of the cells comprising the tissue or organ. The antigen depleted cells, once they home to and differentiate into the mature cell type of that tissue or organ, will have decreased surface expressions of the antigens thereby decreasing the autoimmune response. This is advantageous over gene therapy methods, as the cells are autologous, the cells used can be stem cells which will differentiate into the mature cell of the targeted tissue type once they have trafficked or homed to the desired location and they can be tracked using the methods of the invention described infra.

[000130] Examples of antigens involved in autoimmunity are the MHC or HLA antigens. As used herein, the term "transplantation antigen" is used to refer to antigenic molecules that are expressed on the cell surface of transplanted cells, either at the time of transplantation, or at some point following transplantation. Generally these antigenic molecules are proteins and glycoproteins. The primary transplantation antigens are products of the major histocompatibility complex (MHC), located on chromosome 6 in humans. The human MHC complex is also called the human leukocyte antigen (HLA) complex. MHC antigens are divided into MHC class I antigens (in humans, this class includes HLA-A, -B, and -C antigens) and MHC class II antigens (in humans, this class includes HLA-DP, -DQ, and -DR antigens). Thus, the terms "MHC-II antigens", "MHC class II antigens", and "MHC class II transplantation antigens" are used interchangeably herein to refer to the class of proteins, which in humans, includes HLA-DP, -DQ and -DR antigens. While the terms "MHC class II genes" and "MHC-II genes" are used interchangeably herein to refer to the genes which encode the MHC class II transplantation antigens. The term "MHC-II" is used herein to refer to the gene locus which encodes the MHC class II transplantation antigens, as well as the group of proteins encoded by that locus. Transplantation antigens also include cell surface molecules other than MHC class I and II antigens. These antigens include the following: (1) the ABO antigens involved in blood cell recognition; (2) cell adhesion molecules such as ICAM, which is involved in leukocyte cell-cell recognition; and (3) /32 -microglobulin, a polypeptide associated with the 44 kd heavy chain polypeptide that comprises the HLA-I antigens but is not encoded by the MHC complex.

[000131] As used herein, the term "transplantation antigen nucleotide sequence" refers to nucleotide sequences associated with genes encoding transplantation antigens. Nucleotide sequences associated with genes include the region of the gene encoding the structural product, including intron and exon regions, and regions upstream of the structural gene associated with transcription, transcription initiation (including transcription factor binding sites), translation initiation, operator and promoter regions, ribosome binding regions, as well as regions downstream of the structural gene, including termination sites. Nucleotide sequences associated with genes also include sequences found on any form of messenger RNA (mRNA) derived from the gene, including the pre-mRNA, spliced mRNA, and polyadenylated mRNA.

[000132] As used interchangeably herein, the terms "oligo-nucleotides", "polynucleotides", and "nucleic acids" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term "nucleotide" as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term "nucleotide" is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term "nucleotide" is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, all as described herein.

[000133] The phrase "having a length of N bases" or "having a length of N nucleotides" is used herein to describe lengths along a single nucleotide strand, of a nucleic acid molecule, consisting of N individual nucleotides.

[000134] As used herein, the term "bind", refers to an interaction between the bases of an oligonucleotide which is mediated through base-base hydrogen bonding. One type of binding is "Watson-Crick-type" binding interactions in which adenine-thymine (or adenine-uracil) and guanine-cytosine base-pairs are formed through hydrogen bonding between the bases. An example of this type of binding is the binding traditionally associated with the DNA double helix.

[000135] As used herein, the term "oligonucleotide" refers to a polynucleotide formed from naturally occurring bases and pentofuranosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally occurring species or synthetic species formed from naturally occurring subunits or their close homologs. The term "oligonucleotide" may also refer to moieties which function similarly to naturally occurring oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar moieties or intersugar linkages. Exemplary among these are the phosphorothioate and other sulfur-containing species which are known for use in the art. In accordance with some preferred embodiments, at least some of the phosphodiester bonds of the oligonucleotide have been substituted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA or DNA whose activity to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with other structures which are, at once, substantially nonrionic and non-chiral, or with structures which are chiral and enantionierically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.

[000136] Oligonucleotides may also include species which include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-O-alkyl- and 2'-halogen-substituted nucleotides. Some specific examples of modifications at the 2' position of sugar moieties which are useful in the present invention are OH, SH, SCH₃, F, OCH₃, OCN, 0(CH₂) _nNH₂ or 0(CH₂) _nCH₃ where n is from 1 to about 10, and other substituents having similar properties. [000137] In another preferred embodiment, stem cells comprise vectors expressing desired chemokines. Chemokines and cytokines play a powerful role in the development of an immune response. The role of chemokines in leukocyte trafficking is reviewed by Baggiolini (1998) Nature 392:565-8, in which it is suggested that migration responses in the complicated trafficking of lymphocytes of different types and degrees of activation will be mediated by chemokines. The use of small molecules to block chemokines is reviewed by Baggiolini and Moser (1997) J. Exp. Med. 186:1189-1191.

[000138] The role of various specific chemokines in lymphocyte homing has been previously described. For example, Campbell et al. (1998) Science, showed that SDF-I (also called PBSF), 6-C-kine (also called Exodus-2), and MIP-3j8 (also called ELC or Exodus-3) induced adhesion of most circulating lymphocytes, including most CD4⁺ T cells; and MIP -3a; (also called LARC or Exodus-1) triggered adhesion of memory, but not naive, CD4⁺ T cells. Tangemann et al. (1998) J. Immunol. 161:6330-7 disclose the role of secondary lymphoid- tissue chemokine (SLC), a high endothelial venule (HEV)-associated chemokine, with the homing of lymphocytes to secondary lymphoid organs. Campbell et al. (1998) J. Cell Biol 141(4): 1053-9 describe the receptor for SLC as CCR7, and that its ligand, SLC, can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear.

Methods of Isolation of Cells

[000139] Sources of Stem Cells: Except where otherwise required, the invention can be practiced using stem cells of any vertebrate species. Included are stem cells from humans; as well as non-human primates, domestic animals, livestock, and other non-human mammals. [000140] Embryonic Stem Cells: Embryonic stem cells can be isolated from blastocysts of members of the primate species (Thomson et al, Proc. Natl. Acad. Sd. USA 92:7844, 1995). Human embryonic stem (hES) cells can be prepared from human blastocyst cells using the techniques described by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) andReubinoff et al, Nature Biotech. 18:399 (2000)). [000141] Briefly, human blastocysts are obtained from human in vivo preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or one-cell human embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to the blastocyst stage in Gl .2 and G2.2 medium (Gardner et al, Fertil Steril 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma). The inner cell masses are isolated by immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 min, then washed for 5 min three times in DMEM, and exposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3 min (Solter et al, Proc. Natl. Acad. ScL USA 72:5099, 1975). After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers. [000142] After 9 to 15 days, inner cell mass-derived outgrowths are dissociated into clumps, either by exposure to calcium and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then replated on mEF in fresh medium. Growing colonies having undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells are then routinely split every 1-2 weeks by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (.about.200 U/mL; Gibco) or by selection of individual colonies by micropipette. Clump sizes of about 50 to 100 cells are optimal.

[000143] Antibodies are particularity useful for the preparation of substantially pure stem cells. By "substantially pure" herein is meant that at least about 50% of the cells present after sorting are stem cells, with at least about 70% preferred and at least about 90% preferred. [000144] Appropriate markers or antigens for detecting bone marrow derived cells (BMDC ) are polypeptides or nucleic acids not normally found in tissues outside of the bone marrow. Examples of such markers include, but are not limited to, FIk-I (Swissprot: locus VGR2_HUMAN, accession P35968), Sca-1 (Swissprot: locus ICE3_HUMAN, accession P42574), Thy-1 (Swissprot: locus THY1_HUMAN, accession P04216), Patched (Accession NP-000255.1 GL4506247). CXCR (NP--003458.1 GL4503175), survivin (Swissprot: locus BIR5_HUMAN, accession 015392), and the human homolog of mouse nucleostatin (NP-- 705775.1 GL23956324) polypeptides and nucleic acids encoding all or a portion of these proteins. These polypeptides and nucleic acids can be readily obtained using methods well- known to those skilled in the art. Other BMDC markers can also be identified, for example, using transcriptional profiling techniques well-known to those skilled in the art, which can be used to determine the expression of specific subsets of genes in BMDCs and not in non- BMDC tissues. The further elucidation of BMDC-specific markers (e.g., associated with the bone-marrow stem-cell compartment and not historically associated with cancer) using the methods described herein, will allow the detection of BMDC associated metaplasias and cancers at the single level (e.g., by immunohistochemistry or nucleic acid amplification) prior to detection by conventional methods. Immunological based diagnostic and prognostic assays such as those described herein, utilize an antibody that is specific for a BMDC polypeptide (i.e., an antigen normally found only in BMDCs) which can be a polyclonal antibody or a monoclonal antibody and in a preferred embodiment is a labeled antibody. [000145] Polyclonal antibodies are produced by immunizing animals, usually a mammal, by multiple subcutaneous or intraperitoneal injections of an immunogen (antigen) and an adjuvant as appropriate. As an illustrative embodiment, animals are typically immunized against a protein, peptide or derivative by combining about 1 μg to 1 mg of protein capable of eliciting an immune response, along with an enhancing carrier preparation, such as Freund's complete adjuvant, or an aggregating agent such as alum, and injecting the composition intradermally at multiple sites. Animals are later boosted with at least one subsequent administration of a lower amount, as 1/5 to {fraction (1/10)} the original amount of immunogen in Freund's complete adjuvant (or other suitable adjuvant) by subcutaneous injection at multiple sites. Animals are subsequently bled, serum assayed to determine the specific antibody titer, and the animals are again boosted and assayed until the titer of antibody no longer increases (i.e., plateaus).

[000146] Such populations of antibody molecules are referred to as "polyclonal" because the population comprises a large set of antibodies each of which is specific for one of the many differing epitopes found in the immunogen, and each of which is characterized by a specific affinity for that epitope. An epitope is the smallest determinant of antigenicity, which for a protein, comprises a peptide of six to eight residues in length (Berzofsky, J. and I. Berkower, (1993) in Paul, W., Ed., Fundamental Immunology, Raven Press, N. Y., p.246). Affinities range from low, e.g., 10^"6 M, to high, e.g., 10^'11 M. The polyclonal antibody fraction collected from mammalian serum is isolated by well known techniques, e.g., by chromatography with an affinity matrix that selectively binds immunoglobulin molecules such as protein A, to obtain the IgG fraction. To enhance the purity and specificity of the antibody, the specific antibodies may be further purified by immunoaffinity chromatography using solid phase-affixed immunogen. The antibody is contacted with the solid phase-affixed immunogen for a period of time sufficient for the immunogen to immunoreact with the antibody molecules to form a solid phase-affixed immunocomplex. Bound antibodies are eluted from the solid phase by standard techniques, such as by use of buffers of decreasing pH or increasing ionic strength, the eluted fractions are assayed, and those containing the specific antibodies are combined.

[000147] In one preferred embodiment, the population of stem cells is purified. A purified population of stem cells contains a significantly higher proportion of stem cells than the crude population of cells from which the stem cells are isolated. For example, the purification procedure should lead at least to a five fold increase, preferably at least a ten fold increase, more preferably at least a fifteen fold increase, most preferably at least a twenty fold increase, and optimally at least a twenty-five fold increase in stem cells with respect to the total population. The purified population of stem cells should include at least 15%, preferably at least 20%, more preferably at least 25%, most preferably at least 35%, and optimally at least 50% of stem cells.

[000148] The purified population of stem cells may be isolated by contacting a crude mixture of cells containing a population of stem cells that express an antigen characteristic of stem cells with a molecule that binds specifically to the extracellular portion of the antigen. Such a technique is known as positive selection. [000149] Procedures used to isolate stem cells are described in detail in the Examples which follow. However, isolation of cells useful in the present invention can be obtained by any method that is well known in the art. For example, bone marrow derived hematopoietic stem cells can be isolated by density gradient centrifugation, e.g., with Ficoll/Hypaque. Specific cell populations can be depleted or enriched using standard methods using stem cell- specific mAbs (e.g., anti-CD34 mAbs). Specific cell populations can also be isolated by fluorescence activated cell sorting according to standard methods. Monoclonal antibodies to cell-specific surface markers known in the art and many are commercially available. The binding of the stem cells to the molecule permit the stem cells to be sufficiently distinguished from contaminating cells that do not express the antigen to permit isolating the stem cells from the contaminating cells. For example, Lin^", Sca⁺, c-kit⁺, CD34⁺. [000150] The molecule used to separate stem cells from the contaminating cells can be any molecule that binds specifically to the antigen that characterizes the stem cell. The molecule can be, for example, a monoclonal antibody, a fragment of a monoclonal antibody, or, in the case of an antigen that is a receptor, the ligand of that receptor. For example, VEGF. The number of antigens, such as VEGF receptors, characteristic of stem cells found on the surface of such cells, must be sufficient to isolate purified populations of such cells. For example, the number of antigens found on the surface of stem cells should be at least approximately 1,000, preferably at least approximately 5,000, more preferably at least approximately 10,000, most preferably at least approximately 25,000, and optimally at least approximately 100,000. There is no limit as to the number of antigens contained on the surface of the cells. For example, the cells may contain approximately 150,000, 250, 000, 500,000, 1,000,000, or even more antigens on the surface.

[000151] The source of stem cells may be any natural or non-natural mixture of cells that contains stem cells. The source may be derived from an embryonic mammal, or from the post-natal mammal. One source of cells is the hematopoietic micro-environment, such as the circulating peripheral blood, preferably from the mononuclear fraction of peripheral blood, umbilical cord blood, bone marrow, fetal liver, or yolk sac of a mammal. The stem cells, especially neural stem cells, may also be derived from the central nervous system, including the meninges.

[000152] Either before or after the crude cell populations are purified as described above, the population of stem cells may be further concentrated by methods known in the art. For example, the stem cells can be enriched by positive selection for one or more antigens characteristic of stem cells. Such antigens include, for example, FLK-I, CD34, and AC133. For example, human stem cells may be pre-purified or post-purified by means of an anti- CD34 antibody, such as the anti-My-10 monoclonal antibody described by Civin in U.S. Pat. No. 5,130,144. The hybridoma cell line that expresses the anti-My monoclonal antibody is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA. Some additional sources of antibodies capable of selecting CD34⁺ cells include AMAC, Westbrook, Me.; Coulter, Hialea, FIa.; and Becton Dickinson, Mountain View, Calif. CD34⁺ cells may also be isolated by means of comparable antibodies, which may be produced by methods known in the art, such as those described by Civin in U.S. Pat. No. 5,130,144.

[000153] In addition, or as an alternative to, the enrichment with anti-CD34 antibodies, populations of stem cells may also be further enriched with anti- Sea antibodies; with the AC 133 antibodies described by Yin et al, Blood 90, 5002-5112 (1997) and by Miraglia et al, Blood, 90, 50135021 (1997). The AC133 antibodies may be prepared in accordance with Yin et al; ibid, or purchased from Miltenyi Biotec.

[000154] In accordance with the invention, stem cells can also be detected using for example, antibodies to c-kit. The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor for an unidentified ligand and is a member of the colony stimulating factor- 1 (CSF- l)~platelet-derived growth factor (PDGF)--kit receptor subfamily, c-kit was shown to be allelic with the white-spotting (W) locus of the mouse. Mutations at the W locus affect proliferation and/or migration and differentiation of germ cells, pigment cells and distinct cell populations of the hematopoietic system during development and in adult life. The effects on hematopoiesis are on the erythroid and mast cell lineages as well as on stem cells, resulting in a macrocytic anemia which is lethal for homozygotes of the most severe W alleles, and a complete absence of connective tissue and mucosal mast cells. W mutations exert their effects in a cell autonomous manner, and in agreement with this property, c-kit RNA transcripts were shown to be expressed in targets of W mutations (Nocka, K., Majumder, S., Chabot, B., Ray, P., Cervone, M., Bernstein, A. and Besmer, P. (1989) Genes & Dev. 3, 816- 826.). High levels of c-kit RNA transcripts were found in primary bone marrow derived mast cells and mast cell lines. Somewhat lower levels were found in melanocytes and erythroid cell lines. The identification of the ligand for c-kit is of significance and interest because of the pleiotropic effects it might have on the different cell types which express c-kit and which are affected by W mutations in vivo. The demonstration of identity of c-kit with the W locus implies a function for the c-kit receptor system in various aspects of melanogenesis, gametogenesis and hematopoiesis during embryogenesis and in the adult animal. [000155] The ligand of the c-kit receptor, KL, has been identified and characterized, based on the known function of c-kit/W in mast cells (Zsebo, K. M., et al., (1990a) Cell 63, 195- 201; Zsebo, K. M., et al, Cell 63, 213-214 (1990B). The c-kit receptor in hematopoiesis KL stimulates the proliferation of bone marrow derived and connective tissue mast cells and in erythropoiesis, in combination with erythropoietin, KL promotes the formation of erythroid bursts (day 7-14 BFU-E). Furthermore, recent in vitro experiments with KL have demonstrated enhancement of the proliferation and differentiation of erythroid, myeloid and lymphoid progenitors when used in combination with erythropoietin, GM-CSF, G-CSF and IL-7 respectively suggesting that there is a role for the c-kit receptor system in progenitors of several hematopoietic cell lineages.

[000156] As used herein, c-kit ligand protein and polypeptide encompasses both naturally occurring and recombinant forms, i.e., non-naturally occurring forms of the protein and the polypeptide which are sufficiently identically to naturally occurring c-kit to allow possession of similar biological activity. Examples of such polypeptides includes the polypeptides designated KL-1.4 and S-KL, but are not limited to them. Such protein and polypeptides include derivatives and analogs. In one embodiment of this invention, the purified mammalian protein is a murine protein. In another embodiment of this invention, the purified mammalian protein is a human protein.

[000157] Cells may be further enriched for stem cells by removing cells that are Lin⁺. Such a method is known as negative selection. Negative selection may be used either before or after positive selection. Thus, molecules, such as antibodies or fragments of antibodies, that bind to all or any combination of CDl, CD2, CD3, CD4, CD5, CD8, CDlO, CDlIb, CD13, CD14, CD15, CD16, CD19, CD20, CD24, CD25, CD28, CD29, CD33, CD36, CD38, CD41, CD41a, CD56, CD66b, CD66e, CD69, and glycophorin A maybe used to remove the unwanted Lin⁺ cells by the same methods described above for positive selection.

Stern cell Maturity and Cell Differentiation

[000158] In accordance with the invention, it is desirable to determine cell maturity and differentiation. Several different ways, to assess maturity and cell differentiation, are available. For example, one such method is by measuring cell phenotypes such as, of immune cells. The phenotypic changes can be evaluated by flow cytometry after immunofiuorescent staining using monoclonal antibodies that will bind membrane proteins characteristic of various immune cell types. [000159] A means of assessing cell differentiation is by measuring cell function. This may be done biochemically, by measuring the expression of enzymes, mRNA's, genes, proteins, or other metabolites within the cell, or secreted from the cell. Bioassays may also be used to measure functional cell differentiation or measure specific antibody production directed at a patient's tumor, tumor cell lines or cells from fresh tumors. As used herein, "fresh tumors" refer to tumors removed from a host by surgical or other means. [000160] In accordance with the invention, stem cells can be detected using for example, bispecific antibodies comprised of anti-c-kit x anti-VCAM-1, in addition to the methods detailed in the Examples section which follows. The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor for an unidentified ligand and is a member of the colony stimulating factor- 1 (CSF- l)--platelet-derived growth factor (PDGF)--kit receptor subfamily, c-kit was shown to be allelic with the white-spotting (W) locus of the mouse. Mutations at the W locus affect proliferation and/or migration and differentiation of germ cells, pigment cells and distinct cell populations of the hematopoietic system during development and in adult life. The effects on hematopoiesis are on the erythroid and mast cell lineages as well as on stem cells, resulting in a macrocytic anemia which is lethal for homozygotes of the most severe W alleles, and a complete absence of connective tissue and mucosal mast cells. W mutations exert their effects in a cell autonomous manner, and in agreement with this property, c-kit RNA transcripts were shown to be expressed in targets of W mutations (Nocka, K., Majumder, S., Chabot, B., Ray, P., Cervone, M., Bernstein, A. and Besmer, P. (1989) Genes & Dev. 3, 816-826.). High levels of c-kit RNA transcripts were found in primary bone marrow derived mast cells and mast cell lines. Somewhat lower levels were found in melanocytes and erythroid cell lines.

[000161] The identification of the ligand for c-kit is of importance and interest because of the pleiotropic effects it might have on the different cell types which express c-kit and which are affected by W mutations in vivo. The demonstration of identity of c-kit with the, W locus implies a function for the c-kit receptor system in various aspects of melanogenesis, gametogenesis and hematopoiesis during embryogenesis and in the adult animal. [000162] The ligand of the c-kit receptor, KL, has been identified and characterized, based on the known function of c-kit/W in mast cells (Zsebo, K. M., et al., (1990a) Cell 63, 195- 201; Zsebo, K. M., et al., (1990B) Cell 63, 213-214). The c-kit receptor in hematopoiesis KL stimulates the proliferation of bone marrow derived and connective tissue mast cells and in erythropoiesis, in combination with erythropoietin, KL promotes the formation of erythroid bursts (day 7-14 BFU-E). Furthermore, recent in vitro experiments with KL have demonstrated enhancement of the proliferation and differentiation of erythroid, myeloid and lymphoid progenitors when used in combination with erythropoietin, GM-CSF, G-CSF and IL- 7 respectively suggesting that there is a role for the c-kit receptor system in progenitors of several hematopoietic cell lineages..

[000163] As used herein, c-kit ligand protein and polypeptide encompasses both naturally occurring and recombinant forms, i.e., non-naturally occurring forms of the protein and the polypeptide which are sufficiently identically to naturally occurring c-kit to allow possession of similar biological activity. Examples of such polypeptides includes the polypeptides designated EX-1.4 and S-KL, but are not limited to them. Such protein and polypeptides include derivatives and analogs. In one embodiment of this invention, the purified mammalian protein is a murine protein. In another embodiment of this invention, the purified mammalian protein is a human protein.

[000164] hi another preferred embodiment of the invention, patient cells such as for example, stem cells, lymphocytes and the like can be modified to either express certain molecules or the suppression of expression of certain molecules, such as for example, antigens involved in autoimmune diseases. Stem cells may also be modified to express any receptor desired, such as for example, a modified T cell receptor, which would be advantageous in autoimmune diseases, or targeting of tumors, or antigens of infectious disease organisms such as viruses and the like. Hematopoietic stem cell specific regulatory elements are known in the art. Preferably regulatory elements derived from the CD34 gene are used (see e.g., Satterthwaite, A. B. et al. (1992) Genomics 12:788-794; Burn, T. C. et al. (1992) Blood 80:3051-3059).

[000165] In another preferred embodiment, isolated stem cells can express recombinant T cell receptor expressing constructs and these stem cells will home to the tumor site, as described in detail in the Examples which follow. Described and provided herein are recombinant T cell receptor (TCR) constructs suitable for use in transducing the cells. Li addition, the stem cells can be induced to mature into T-cells specific for tumor antigens Any cells may be used including T cells, B cells and the like. Suitable constructs and uses thereof are described in International application no. US94/10033, which is hereby incorporated herein by reference. The recombinant TCR can be put under the control of a T cell specific promoter so that it is only expressed in T cells. For example, the promoter could be Granzyme A or Granzyme B, which would cause the recombinant TCR to be expressed predominantly in NK cells and cytotoxic T lymphocytes (CTLs). Cytotoxic lymphocytes require Granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Heusel et al. (1994) Cell 76:977-987. The T-cells derived from transduced cells should home and circulate properly since they have matured in vivo and have not been directly manipulated subsequently ex vivo. They can then be expanded in number by administering cytokines in vivo. Since primarily antigen-activated cells proliferate in response to cytokines, modified T cells recognizing the target antigen should be relatively amplified. Also, it may be possible to get a stronger response from the T cells derived from the transduced cells. If more mature T cells are transduced with the recombinant TCR, they may have a dampened response if they are "memory" cells (i.e. previously exposed to antigens) and, therefore, "biased."

[000166] Another advantage to genetically modified progenitor cells over mature T cells would be the ability to express the recombinant TCR in more than one hematopoietic lineage. For example, since macrophages are known to have the ability to engulf tumor cells, it may be useful to express the recombinant TCR in macrophages.

[000167] The constructs can be prepared in a variety of conventional ways. Numerous vectors are now available which provide the desired features, such as long terminal repeats, marker genes, and restriction sites, which may be further modified by techniques known in the art. The constructs can encode a signal peptide sequence in addition to the antigenic specificity region and cytoplasmic signaling sequence, to ensure that the recombinant TCR is properly processed post-translationally and expressed on the cell surface. Preferably, the construct is under the control of a T cell specific promoter. Suitable T cell specific promoters include, but are not limited to, Granzyme A, Granzyme B and CD8. [000168] In one embodiment, the signal transducing region and antigenic specificity region are both obtained from TCRs ("classic TCR"). In another embodiment the constructs encode chimeric polypeptides comprising the signal transducing region obtained from a T cell specific receptor or the Fcγ receptor and an antigen binding portion of an immunoglobulin or of a NK receptor ("chimeric TCR").

[000169] The recombinant classic TCRs are functional, preferably full length, TCRα and γ, or γ and δ, polypeptides which have been derived from a T cell with known antigenic specificity. Suitable sources of antigen-specific receptors include, but are not limited to, cytotoxic T lymphocytes, T helper cells and NK cells. In another embodiment, the polypeptides may be recombined so as to form a single functional polypeptide with the Va and Vβ regions forming the antigen binding site. In another embodiment, the Va and V/3 regions from different TCRs may be recombined to endow the TCR with a different specificity. [000170] The T cell progeny of the cells containing the recombinant classic TCR polypeptides are "MHC restricted", that is, they will only recognize antigen in the presence of MHC. Thus, when using these cells to treat a patient, the TCRs must be able to recognize the same haplotype as that of the host. It is well within the skill of one in the art to determine if the haplotype of the host will be compatible with a particular TCR. The classic TCR approach is advantageous where the antigen is expressed as a short peptide on the cell surface by processing internally and presented in the groove of an MHC molecule. [000171] In the case of the chimeric TCR, the chimeric molecule contains an antigen binding sequence from an antibody or another receptor, a transmembrane sequence and a sequence that can transduce a signal and elicit a function. A variety of these and related molecules have been cloned and expressed in various T cell lines. Kuwana et al. (1987) Biochem. Biophys. Res. Comm. 149:960-968; Gross et al. (1989) Trans. Proc. 21:127-130; Becker et al. (1989) Cell 58:911-921; Gross et al. (1989) Proc. Natl. Acad. ScL USA 86:10024-10028; and Goverman et al. (1990) Cell 60:929-939. Several chimeric TCRs have been created and found to be active in targeting T cells to the antigen recognized by the antibody binding site. Eshhar (1993) Proc. Natl. Acad. ScL USA 90:720-724; and Hwu et al. (1993) J. Exp. Med. 178:361-366.

[000172] Suitable signal transducing regions can be obtained from receptors that have activation capacity through a specific chain including, but not limited to, the γ chain of the Fc receptor, the CD3 ζ chain, EL-2 receptor γ chain, CD8 or CD28. Alternatively, the antigen binding domain may be associated with TCR a or β chain constant regions, which will transduce signal via association with endogenous CD3 f chain. Preferably, the functional portion of the chimeric molecule is the signaling region of a F_cγor f polypeptide and the antigen binding domain is a variable region of an antibody. The variable region may be either the V_H or V_L regions or, preferably, a single chain recombinant thereof. Methods for recombination in mammalian cells may be found in Molecular Cloning, A Laboratory Manual (1989) Sambrook, Fritsch and Maniatis, Cold Spring Harbor, N. Y. [000173] The T cell progeny of the cells containing the chimeric TCR molecules, recognize antigen in the absence of MHC when the antigen binding site is derived from an antibody and thus may not be MHC restricted. These molecules are suitable for use in all hosts regardless of haplotype. Upon reintroduction of the genetically modified cells into the host and subsequent differentiation, T cells are produced that are specifically directed against the specific antigen. Generally, suitable antigens include those found on specific cancer cells. More specifically, suitable antigens include, but are not limited to, viral coat proteins and specific surface proteins of cancer cells.

[000174] In accordance with the invention, the stem cells are removed, modified, armed with desired antibodies for targeting and tracking of the stem cells to the desired location, and the like. A second antibody or label may used for monitoring the location of the stem cells. In many situations, cell immunotherapy involves removal of bone marrow or other source of hematopoietic cells from a human host, isolating the progenitor cells from the source and optionally expanding the isolated cells. Meanwhile, the host can be treated to partially, substantially or completely ablate native hematopoietic capability. The isolated cells can be modified during this period of time, so as to provide for cells having the desired genetic modification. In the case of complete hematopoietic ablation, stem cell augmentation is also be required. After completion of the treatment of the host, the modified cells may then be restored to the host to provide for the new capability. The methods of hematopoietic cell removal, host ablation and stem/progenitor cell repopulation are known in the art. If necessary, the process may be repeated to ensure the substantial repopulation of the modified cells. The present invention is advantageous in that it provides for the specific targeting and monitoring of cells in vivo.

[000175] The modified cells may be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Usually, at least about 1x10⁵ cells are administered, preferably IxIO⁶ or more. The cells may be introduced by injection, catheter, or the like. If desired, although not required, factors may also be included, including, but not limited to, interleukins, e.g. IL-2, IL-3, IL-6, and IL-Il, as well as the other interleukins, the colony stimulating factors, such as GM-CSF, interferons, e.g. γ-interferon, erythropoietin. [000176] Immune cells express a variety of cell surface molecules which can be detected with either monoclonal antibodies or polyclonal antisera. Immune cells that have undergone differentiation or activation can also be enumerated by staining for the presence of characteristic cell surface proteins by direct immunofluorescence in fixed smears of cultured cells.

[000177] Armed stem cells of the invention include antibodies that target the stem cells to a desired location. However, in certain therapeutic applications, an expression vector encoding a desired tissue specific molecule, such as VCAM-I, can be used to transform a stem cell. [000178] Vectors can be constructed which also comprise a detectable/selectable marker gene. In preferred embodiments these marker genes are fluorescent proteins such as green fluorescent protein (GFP), cyan- (CFP)₅ yellow- (YFG), blue- (BFP), red- (RFP) fluorescent proteins; enhanced green fluorescent protein (EGFP), EYFP, EBFP, Nile Red, dsRed, mutated, modified, or enhanced forms thereof, and the like.

[000179] As used herein, the "green-fluorescence protein" is a gene construct which in transfected or infected cells, respectively, shines green under ultraviolet light and thus enables the detection of a cell transfected or infected, respectively, with GFP in a simple manner.

[000180] These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93/04701, which has a targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage etc. The vectors can be chromosomal, non-chromosomal or synthetic-

[000181] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses. DNA viral vectors are preferred. Viral vectors can be chosen to introduce the cytokine or chemokine to cells of choice. Such vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as herpes simplex I virus (HSV) vector (Geller, A.I et al, J. Neurochem., 64:487(1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford, England) (1995); Geller, A.I. et al., Proc. Natl. Acad. Sd. USA 87:1149 (1990)) Adenovirus vectors (LeGaI LaSaIIe et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3:219 (1993); Yang et al., J Virol. 69:2004 (1995)) and Adeno-associated virus vectors (Kaplitt, M.G. et al., Nat. Genet. 8:148 (1994)).

[000182] Pox viral vectors introduce the gene into the cells cytoplasm. Avipox virus vectors result in only short term expression of the nucleic acid. Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus vectors are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The vectors can be introduced by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include for example, naked DNA calcium phosphate precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection and viral vectors. [000183] Uses of green fluorescent protein for the study of gene expression and protein localization are well known. The compact structure makes GFP very stable under diverse and/or harsh conditions such as protease treatment, making GFP an extremely useful reporter in general.

[000184] New versions of green fluorescent protein have been developed, such as a "humanized" GFP DNA, the protein product of which has increased synthesis in mammalian cells. One such humanized protein is "enhanced green fluorescent protein" (EGFP). Other mutations to green fluorescent protein have resulted in blue-, cyan- and yellow-green light emitting versions.

[000185] Endogenously fluorescent proteins have been isolated and cloned from a number of marine species including the seapansies Renilla reniformris, R. kollikeri andi?. mullerei and from the sea pens Ptilosarcus, Stylatula and Acanthoptilum, as well as from the Pacific Northwest jellyfish, Aequorea victoria; Szent-Gyorgyi et al. (SPIE conference 1999), D. C. Prasher et al., Gene, 111 :229-233 (1992) and several species of coral (Matz et al. Nature Biotechnology, 17 969-973 (1999). These proteins are capable of forming a highly fluorescent, intrinsic chromophore through the cyclization and oxidation of internal amino acids within the protein that can be spectrally resolved from weakly fluorescent amino acids such as tryptophan and tyrosine.

[000186] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen. Antibodies include recombinant proteins comprising the binding domains, as wells as fragments, including Fab, Fab¹, F(ab)₂, and F(ab') ₂ fragments.

[000187] Antibodies directed against surface antigens for detection of tumors and the like membrane proteins can also be fluorescently labeled. Specificity for particular cell types is likely to be easier to achieve with antibodies than with other molecules because antibodies can be raised against nearly any surface marker. Also, microinjected antibodies could label sites on the cytoplasmic face of the plasma membrane, blood vessels and the like. Treatment

[000188] The amount of stem cells administered to the patient will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about IxIO⁶ to about IxIO¹², more preferably about IxIO⁸ to about IxIO¹¹, more preferably, about IxIO⁹ to about IxIO¹⁰ stem cells are utilized for adult humans. These amounts will vary depending on the age, weight, size, condition, sex of the patient, the type of tumor to be treated, the route of administration, whether the treatment is regional or systemic, and other factors. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient.

[000189] Methods of re-introducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example, administration of activated CD8⁺ cells via intravenous infusion is appropriate.

Therapeutic Molecules

[000190] In another preferred embodiment, anti-inflammatory molecules which inhibit cell recruitment at a particular in vivo site are preferred. The relationship between chronic inflammation and cancer has long been recognized. In 1863, Virchow hypothesized that the origin of cancer was at sites of chronic inflammation (Balkwill, F. & Montovani, A. (2001) Lancet 357:539-545). Virchow suggested that the "lymphoreticular infiltrate" reflected the origin of cancer at sites of chronic inflammation. The notion was based in part on his idea that some classes of irritants, together with the tissue injury and ensuing inflammation they caused, enhanced cell proliferation. Thus, while acute injury or normal types of inflammation are usually self-limiting, chronic injury or inflammation over decades leads to a sustained expansion of tissue proliferative zones and predisposes to neoplastic progression (Coussens, L. M. & Wer, Z. (2002) Nature 420:860-867). Sustained proliferation, with or without chronic inflammation, has been generally accepted as a risk factor, or at least an early biomarker, for cancer

[000191] There is in fact a growing body of evidence that many, if not most, malignancies are initiated by tissue injury or chronic inflammation. In many cases, this inflammation can be attributed to known bacterial, viral or parasitic infections. Overall, approximately 15% of malignancies worldwide can be attributed specifically to chronic infections, yielding a global total of 1.2 million cases per year. Examples of cancers linked to infection include bladder cancer due to schistosomiasis, liver cancer due to hepatitis B and C infection, cervical cancer due to HPV, and gastric cancer due to Helicobacter pylori. Many other cancers are initiated by chronic inflammation secondary to other etiologies—for example, esophageal adenocarcinoma due to gastroesophageal reflux disease (GERD), colon cancer in the setting of inflammatory bowel disease (IBD), and lung cancer due to chronic smoking. [000192] These inflammation-associated cancers develop in the setting of a damaged organ that has lost a large proportion of normal cell types. For example, in chronic H. pylori infection of the stomach, the normal secretory cell types such as parietal cells are replaced by metaplastic cell lineages, i.e., cells that are not typical of the normal glandular cell types. Thus, prior to the development of gastric cancer, the gastric fundus shows an abundance of cells that resemble intestinal cells (intestinal metaplasia) or pyloric glands (pseudopyloric metaplasia or SPEM). Intestinal metaplasia (Barrett's esophagus) is also a precursor lesion for esophageal adenocarcinoma.

[000193] Certainly chronic inflammation leads to increased oxidative stress, whereby leukocytes and other phagocytic cells induce DNA damage in proliferating cells, through the generation of reactive oxygen and nitrogen species that are produced normally by these cells to fight infection. In addition, chronic inflammation appears to promote apoptosis of normal cells that can then lead to a compensatory proliferative response by the remaining tissue. However, a more important factor may be the association with chemokines and cytokines, which have been shown to induce not only monocyte/leukocyte migration but also influence cancer cells and interestingly, bone marrow stem cells (Balkwill, F. & Montovani, A. (2001) Lancet 357:539-545). Chemokines in particular have been shown to bind to receptors (such as CXCR 2 and 4) on cancer cells and stem cells and influence their homing (or invasion/metastases). Thus, chronic inflammation has many parallels with wound healing, and both host responses involve an expansion of undifferentiated descendants of progenitor cells.

[000194] Thus, in a preferred embodiment of this first aspect of the invention, the inflammatory condition is a chronic inflammatory condition. In further preferred embodiments, the chronic inflammatory disorders that can be treated according to the methods of the invention include those associated with bacterial, viral infection or parasite infection, for example, but not limited to, Schistosomiasis, Papilloma, Helicobacter, Hepatitis B and C, EBV, HPV infection. Other disorders that can be treated according to the methods of the invention chronic inflammation due, for example, but are not limited to osteoarthritis, rheumatoid arthritis, asthma, cystic fibrosis, juvenile chronic arthritis, ankylosing spondylitis, psoriatic arthropathy, Reiter's syndrome, Adult Still's disease, Behcet's syndrome,inflammatory bowel disease (IBD), Crohn's disease, psoriasis, atopic eczema, acne, systemic lupus erythomatosis, multiple sclerosis, atherosclerosis, restenosis; chronic bronchitis, sinusitis, chronic gastroenteritis and colitis, chronic cystitis and urethritis; hepatitis, chronic dermatitis; chronic conjunctivitis, chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendinitis), uremic pericarditis, chronic cholecystis, chronic vaginitis, and chronic uveitis, emphysema, Sjorgen's disease, systemic lupus erythematosus, infection, strain, sprain, cartilage damage, trauma, and recent orthopedic surgery. Anti-inflammatory agents include, but not limited to diclofenac, fenuprofen, flubiprofen, ibufprofen, indomethacin, ketoprofen, meclofenamate, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tolmetin, Cox-2 inhibitors (including but not limited to CELEBREX™, BEXTRA™, gold compounds, hydroxychloroquine, sulfasalazine, penacillamine, corticosteroids, pain medications, and cytotoxic or immunsuppressive drugs (including, but not limited to, methotrexate, azathiprine, and cyclosporine).

[000195] In a preferred embodiment, molecules that are anti-inflammatory agents can be administered to a tumor site in vivo, and/or used to transduce a stem cell to produce these molecules and inhibit further stem cell migration. For example, cyclooxygenase (COX) is a key enzyme that catalyzes the biosynthesis of prostaglandins (PGs) from arachidonic acid. Two isoforms of COX, designated COX-I and COX-2, have been identified. COX-I is constitutively expressed in most tissues and seems to be responsible for housekeeping roles in normal physiological functions (Amiram R., J.Biol.Chem., 263:3022-2024, 1988). In contrast, COX-2 is not detectible in most normal tissues, but is induced by proinflammatory cytokines, growth factors, oncogenes, carcinogens, and tumor promoters, implying a role for COX-2 in both inflammation and control of cell growth (Subbaramaiah K., Cancer Res., 56:4424-4429, 1996). The increased level of PGs in tumors is due, at least in part, to increased expression of COX-2. Overexpression of COX-2 also inhibits apoptosis and increases the invasiveness of malignant cells (Tsujii M., et al., Proc. Natl. Acad. Sd. USA, 94:3336-3340, 1997). Accordingly, compounds that inhibit selectively the activity or expression of COX-2 might be an important focus for cancer chemoprevention or anti- inflammation.

[000196] Nitric oxide synthase (NOS) is another important enzyme involved in regulation of inflammation, vascular tone, neurotransmission, tumor cells and other homeostasis of human body. NOS also exists in the two forms of constitutive form and inducible form. The excessive generation of nitric oxide (NO) is related with pathological vasodilation, cytotoxicity and tissue injury. According to the recent results, NOS increases the permeability of a blood vessel, causes inflammatory reaction such as edema, and promotes the activation of COX to stimulate the biosynthesis of inflammatory mediator such as prostaglandin to induce severe inflammatory reaction. In various cancer tissue, the activation of iNOS is highly increased. Therefore, agents which significantly inhibit the activity of COX-2 and INOS could be utilized not only for prevention of cancer, but also for treatment of inflammation and cancer. Other inflammatory cues include, but are not limited to C3a, P- selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, alpha4 integrins, alpha5 integrins, beta defensins 3 and 4.

[000197] The integrins are a family of cell-surface glycoproteins involved in cell- adhesion, immune cell migration and activation. Alpha-4 integrin is expressed by all circulating leukocytes except neutrophils, and forms heterodimeric receptors in conjunction with either the /31 or /37 integrin subunits; both alpha-4 beta-1 (α4/51) and alpha-4 beta-7 iμAβl) dimers play a role in the migration of leukocytes across the vascular endothelium (Springer et al., 1994 Cell 76: 301-14; and Butcher et al., 1996 Science 272: 60-6) and contribute to cell activation and survival within the parenchyma (Damle et al., 1993 J. Immunol. 151 : 2368-79; Koopman et al, 1994 J. Immunol. 152: 3760-7; and Leussink et al., 2002 Acta Neuropathol. 103:131-136).

[000198] Specifically, alpha-4 beta-1 (also known as very late antigen-4 [VLA-4]), binds to vascular cell adhesion molecule-1 (VCAM-l)(Lobb et al., 1994 J. Clin. Invest. 94:1722-8), which is expressed by the vascular endothelium at many sites of chronic inflammation (Bevilacqua et al., 1993 Ann. Rev. Immunol. 11: 767-804; and Postigo et al., 1993 Res. Immunol. 144:723-35). The alpha-4 beta-7 dimer interacts with mucosal addressin cell adhesion molecule (MAdCAM-I), and mediates homing of lymphocytes to the gut (Farstad et al., \991 Am. J. Pathol. 150: 187-99; and lssekutz et al., 1991 J. Immunol. 147: 4178-84). Expression of MAdCAM-I on the vascular endothelium is also increased at sites of inflammation in the intestinal tract of patients with inflammatory bowel disease (EBD) (Briskin et al., 1997 Am. J. Pathol. 151: 97-110).

[000199] hi other preferred embodiments, anti-SDFl is used as therapy to inhibit homing of stem cells to epithelial tissue cancers, such as melanoma. The therapy includes sunscreen for patients prone to skin cancer.

[000200] In another preferred embodiment, anti-MAdCAMl therapies for patients prone to colon cancer can be used by providing the agent in orally ingestible formulations. [000201] With regards to metastasis, epithelial cancers originating from a BMDC would likely have more potential to spread given the multipotent capabilities of BMDCs to migrate. Preferred embodiments include interfering with processes involved with BMDC migration. These processes include, but are not limited to, SDF-1/CXCR4 axis, MMP activation, c-Kit, G-CSF₅ GM-CSF, SCF, HIF-I, and nitric oxide synthase activity (eNOS, iNOS). [000202] Pharmaceutical composition of the present invention for preventing cancer and treating cancer and inflammation could further comprise a pharmaceutically permissible vector and a diluent. Solvent, dispersion medium, absorption retardant and the like which are commercially used in the field of medicine industry can be used as a vector. [000203] Pharmaceutical compositions of the present invention for preventing cancer and treating cancer and inflammation could be administered through whatever general route to reach the target tissue. Therefore, the composition of the present invention could be administered through an affected part of the body, oral administration, parenteral administration, intra-nasal, intravenous injection, intramuscular injection, subcutaneous injection and intrascleral administration. The composition could be formulated as solution, suspended solution, tablet, pill, capsule and sustained releasing agent. The preferred formulation is an injection, and the dosage content of the composition should be determined in consideration of the skill in the art according to the kinds and degree of disease, age, sex and so forth.

[000204] In another preferred embodiment, the vectors expressing desired therapeutic molecules include but not limited to: endostatin, angiogenin, angiostatin, chemokines, angioarrestin, angiostatin (plasminogen fragment), anti-angiogenic antithrombin EI, cartilage-derived inhibitor (CDI), CD59 complement fragment, fibronectin fragment, gro- beta, heparinases, heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon inducible protein (IP-10), interleukin-12, kringle 5 (plasminogen fragment), metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prolactin 16kD fragment, proliferin-related protein (PRP), various retinoids, tetrahydrocortisol-S, thrombospondin-1 (TSP-I), transforming growth factor-beta (TGF-b), vasculostatin, vasostatin (calreticulin fragment) and the like.

[000205] Cytolytic molecules that can be expressed, include, but are not limited to TNF-α, TNF-jS, suitable effector genes such as those that encode a peptide toxin—such as ricin, abrin, diphtheria, gelonin, Pseudomonas exotoxin A, Crotalus durissus terrϊficus toxin, Crotalus adamenteus toxin, Naja naja toxin, and Naja mocambique toxin. (Hughes et al., Hum. Exp. Toxicol. 15:443, 1996; Rosenblum Qt al, Cancer Immunol. Immunother. 42:115, 1996; . Rodriguez et al, Prostate 34:259, 1998; Mauceri et al, Cancer Res. 56:4311; 1996). [000206] Also suitable are genes that induce or mediate apoptosis-such as the ICE-family of cysteine proteases, the Bcl-2 family of proteins, Bax, bclXs and caspases (Favrot et al., Gene Ther. 5:728, 1998; McGiIl et al, Front. Biosci. 2:D353, 1997; McDonnell et al, Semin. Cancer Biol. 6:53, .1995). Another potential anti-tumor agent is apoptin, a protein that induces apoptosis even where small drug chemotherapeutics fail (Pietersen et al, Adv. Exp. Med. Biol 465:153, 2000). Koga et al. (Hu. Gene Ther. 11:1397, 2000) propose a telomerase-specific gene therapy using the hTERT gene promoter linked to the apoptosis gene Caspase-8 (FLICE).

[000207] Also of interest are enzymes present in the lytic package, that cytotoxic T lymphocytes or LAK cells deliver to their targets. Perforin, a pore-forming protein, and Fas ligand are major cytolytic molecules in these cells (Brandau et al, Clin. Cancer Res. 6:3729, 2000; Cruz et al, Br. J. Cancer 81:881, 1999). CTLs also express a family of at least 11 serine proteases termed granzymes, which have four primary substrate specificities (Kam et al, Biochim. Biophys. Acta 1477:307, 2000). Low concentrations of streptolysin 0 and pneumolysin facilitate granzyme B-dependent apoptosis (Browne et al., MoI Cell Biol. 19:8604, 1999).

[000208] Other suitable effectors encode polypeptides having activity that is not itself toxic to a cell, but renders the cell sensitive to an otherwise nontoxic compound— either by metabolically altering the cell, or by changing a non-toxic prodrug into a lethal drug. Exemplary is thymidine kinase (tk), such as may be derived from a herpes simplex virus, and catalytically equivalent variants. The HSV tk converts the anti-herpetic agent ganciclovir (GCV) to a toxic product that interferes with DNA replication in proliferating cells. [000209] If desired, although not required, factors may also be included, such as, but not limited to, interleukins, e.g. IL-2, IL-3, IL-6, and IL-Il, as well as the other interleukins, the colony stimulating factors, such as GM-CSF, interferons, e.g. γ-interferon, erythropoietin. [000210] In other preferred embodiments, anti-angio genie proteins are expressed by vectors used to transduce stem cells, hi preferred embodiments, the anti-angiogenic protein expressed by the vectors are angiostatin, endostatin or Tubedown-1. [000211] However, a wide variety of molecules may be utilized within the scope of the present invention as anti-angiogenic factors, including for example Anti-Invasive Factor, retinoic acids and their derivatives, paclitaxel including analogues and derivatives thereof, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-I and Plasminogen Activator Inhibitor-2, and lighter "d group" transition metals. Similarly, a wide variety of polymeric carriers may be utilized, representative examples of which include poly (ethylene- vinyl acetate) (40% cross-linked), poly (D,L-lactic acid) oligomers and polymers, poly (L-lactic acid) oligomers and polymers, poly (glycolic acid), copolymers of lactic acid and glycolic acid, poly (caprolactone), poly (valerolactone), poly (anhydrides), copolymers of poly (caprolactone) or poly (lactic acid) with polyethylene glycol, and blends thereof.

[000212] As noted above, the present invention provides compositions comprising an anti- angiogenic factor, and a polymeric carrier. Briefly, a wide variety of anti-angiogenic factors may be readily utilized within the context of the present invention. Representative examples include Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor- 1, Plasminogen Activator Inhibitor-2, and various forms of the lighter "d group" transition metals. These and other anti-angiogenic factors will be discussed in more detail below.

[000213] Briefly, Anti-Invasive Factor, or "AIF" which is prepared from extracts of cartilage, contains constituents which are responsible for inhibiting the growth of new blood vessels. These constituents comprise a family of 7 low molecular weight proteins (<50,000 daltons) (Kuettner and Pauli, "Inhibition of neovascularization by a cartilage factor" in Development of the Vascular System, Pitman Books (CIBA Foundation Symposium 100), pp. 163-173, 1983), including a variety of proteins which have inhibitory effects against a variety of proteases (Eisentein et al, Am. J Pathol. 81:337-346, 1975; Langer et al, Science 193:70-72, 1976; and Horton et al., Science 199:1342-1345, 1978). AIF suitable for use within the present invention may be readily prepared utilizing techniques known in the art (e.g., Eisentein et al, supra; Kuettner and Pauli, supra; and Langer et al, supra). Purified constituents of AEF such as Cartilage-Derived Inhibitor ("CDI") (see Moses et al, Science 248: 1408-1410, 1990) may also be readily prepared and utilized within the context of the present invention.

[000214] Cartilage derived inhibitors include those described by Sorgente N, Dorey C. Inhibition of endothelial cell growth by a factor isolated from cartilage. Exp Cell Res. 1980;128:63-71, which is incorporated herein by reference.

[000215] Combination therapy using the vectors of the invention is in accordance with the invention. The following can be used in conjunction with the vectors. [000216] Retinoic acids alter the metabolism of extracellular matrix components, resulting in the inhibition of angiogenesis. Retinoic acid, as well as derivatives thereof which may also be utilized in the context of the present invention, may be readily obtained from commercial sources, including for example, Sigma Chemical Co. (# R2625).

[000217] Paclitaxel is a highly derivatized diterpenoid (Wani et ah, J. Am. Chem. Soc. 93:2325, 1971) which has been obtained from the harvested and dried bark of Taxis brevifolia (Pacific Yew.) and Taxomyces Andreanae and Endophytic Fungus of the Pacific Yew (Stierle et ah, Science 60:214-216, 1993). Generally, paclitaxel acts to stabilize microtubular structures by binding tubulin to form abnormal mitotic spindles. "Paclitaxel" (which should be understood herein to include analogues and derivatives such as, for example, TAXOL™, TAXOTERE™, 10-desacetyl analogues of paclitaxel and 3¹N- desbenzoyl-3'N-t-butoxy carbonyl analogues of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076, U.S. Pat. Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137; 5,202,448, 5,200,534, 5,229,529, and EP 590267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402-from Υaxus brevifolia).

[000218] Suramin is a polysulfonated naphthylurea compound that is typically used as a trypanocidal agent. Briefly, Suramin blocks the specific cell surface binding of various growth factors such as platelet derived growth factor ("PDGF"), epidermal growth factor ("EGF"), transforming growth factor ("TGF-/3"), insulin-like growth factor ("IGF-I"), and fibroblast growth factor ("/3FGF"). Suramin may be prepared in accordance with known techniques, or readily obtained from a variety of commercial sources, including for example Mobay Chemical Co., New York, (see Gagliardi et al, Cancer Res. 52:5073-5075, 1992; and Coffey, Jr., et al, J. of Cell. Phys. 132:143-148, 1987).

[000219] Tissue inhibitor of metalloproteinases can be expressed in the expression vector and/or administered in conjunction with the compositions of the invention. Tissue Inhibitor of Metalloproteinases- 1 ("TIMP") is secreted by endothelial cells which also secrete MTPases. TIMP is glycosylated and has a molecular weight of 28.5 kDa. TIMP-I regulates angiogenesis by binding to activated metalloproteinases, thereby suppressing the invasion of blood vessels into the extracellular matrix. Tissue Inhibitor of Metalloproteinases-2 ("TIMP- 2") may also be utilized to inhibit angiogenesis. Briefly, TIMP-2 is a 21 kDa nonglycosylated protein which binds to metalloproteinases in both the active and latent, proenzyme forms. Both TIMP-I and TIMP-2 may be obtained from commercial sources such as Synergen, Boulder, Colo.

[000220] Plasminogen Activator Inhibitor-1 (PA) is a 50 kDa glycoprotein which is present in blood platelets, and can also be synthesized by endothelial cells and muscle cells. PAI-I inhibits t-PA and urokinase plasminogen activator at the basolateral site of the endothelium, and additionally regulates the fibrinolysis process. Plasminogen Activator Inhibitor-2 (PAI-2) is generally found only in the blood under certain circumstances such as in pregnancy, and in the presence of tumors. Briefly, PAI-2 is a 56 kDa protein which is secreted by monocytes and macrophages. It is believed to regulate fibrinolytic activity, and in particular inhibits urokinase plasminogen activator and tissue plasminogen activator, thereby preventing fibrinolysis.

[000221] Lighter "d group" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes. Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate (i.e., VO₃ ^"1) and orthovanadate (i.e., VO₄ ^3") complexes such as, for example, ammonium metavanadate (i.e., NH₄ VO₃), sodium metavanadate (i.e., NaVO₃), and sodium orthovanadate (i.e., Na₃ VO₄). Suitable vanadyl (i.e., VO²⁺) complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

[000222] Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate (i.e., WO₄ ^") complexes include ammonium tungstate (i.e., (NH₄)₂ WO₄), calcium tungstate (i.e., CaWO₄), sodium tungstate dihydrate (i.e., Na₂ WO₄.2H₂O), and tungstic acid (i.e., H₂ WO₄). Suitable tungsten oxides include tungsten (IV) oxide (i.e., WO₂) and tungsten (VI) oxide (i.e., WO₃). Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate (i.e., MoO₄ ^2') complexes include ammonium molybdate (i.e., (NH₄) ₂ MoO₄) and its hydrates, sodium molybdate (i.e., Na₂MoO₄) and its hydrates, and potassium molybdate (i.e., K₂MoO₄) and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide (i.e., MoO₂), molybdenum (VI) oxide (i.e., MoO₃), and molybdic acid. Suitable molybdenyl (i.e., MoO₂ ²⁺) complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

[000223] A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include Platelet Factor 4 (Sigma Chemical Co., #F1385); Protamine Sulphate (Clupeine) (Sigma Chemical Co., #P4505); Sulfated Chitin Derivatives (prepared from queen crab shells), (Sigma Chemical Co., #C3641; Murata et ah, Cancer Res. 51:22-26, 1991); Sulfated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine (Sigma Chemical Co., #S4400); Modulators of Matrix Metabolism, including for example, proline analogs {[(L- azetidine-2-carboxylic acid (LACA) (Sigma Chemical Co., #A0760)), cishydroxyproline, d,L-3,4-dehydroproline (Sigma Chemical Co., #D0265), Thiaproline (Sigma Chemical Co., #T0631)], α,α-dipyridyl (Sigma Chemical Co., #D7505), jS-aminopropionitrile fumarate (Sigma Chemical Co., #A3134)]}; MDL 27032 (4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Merion Merrel Dow Research Institute); Methotrexate (Sigma Chemical Co., #A6770; Hirata et ah, Arthritis and Rheumatism 32:1065-1073, 1989); Mitoxantrone (Polverini and Novak, Biochem. Biophys. Res. Comm. 140:901-907); Heparin (Folkman, £/o. Phar. 34:905-909, 1985; Sigma Chemical Co., #P8754); Interferons (e.g., Sigma Chemical Co., #13265); 2 Macroglobulin-serum (Sigma Chemical Co., #M7151); ChIMP-3 (Pavloff et ah, J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Sigma Chemical Co., #C7268; Tomkinson et ah, Biochem J. 286:475-480, 1992); jS-Cyclodextrin Tetradecasulfate (Sigma Chemical Co., #C4767); Eponemycin; Camptothecin; Fumagillin (Sigma Chemical Co., #F6771; Canadian Patent No. 2,024,306; Ingber et ah, Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST"; Sigma:G4022; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); (D- Penicillamine ("CDPT"; Sigma Chemical Co., #P4875 or P5OOO(HC1)); jS-l-anticollagenase- serum; cβ-antiplasmin (Sigma Chem. Co.:A0914; Holmes et ah, J. Biol. Chem. 262(4):1659- 1664, 1987); Bisantrene (National Cancer histitute); Lobenzarit disodium (N-(2)- carboxyphenyl-4-chloroanthronilic acid disodium or "CCA"; Takeuchi et ah, Agents Actions 36:312-316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminohnidazole; metalloproteinase inhibitors such as BB94.

[000224] Although the above anti-angiogenic factors have been provided for the purposes of illustration, it should be understood that the present invention is not so limited. In particular, although certain anti-angiogenic factors are specifically referred to above, the present invention should be understood to include analogues, derivatives and conjugates of such anti-angiogenic factors. For example, paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogues (e.g., taxotere, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylos).

[000225] Anti-angiogenic compositions of the present invention may additionally comprise a wide variety of compounds in addition to the anti-angiogenic factor and polymeric carrier. For example, anti-angiogenic compositions of the present invention may also, within certain embodiments of the invention, also comprise one or more antibiotics, antiinflammatories, anti-viral agents, anti-fungal agents and/or anti-protozoal agents. Representative examples of antibiotics included within the compositions described herein include: penicillins; cephalosporins such as cefadroxil, cefazolin, cefaclor; aminoglycosides such as gentamycin and tobramycin; sulfonamides such as sulfamethoxazole; and metronidazole. Representative examples of antiinflammatories include: steroids such as prednisone, prednisolone, hydrocortisone, adrenocorticotropic hormone, and sulfasalazine; and non-steroidal anti-inflammatory drugs ("NSAIDS") such as aspirin, ibuprofen, naproxen, fenoprofen, indomethacin, and phenylbutazone. Representative examples of antiviral agents include acyclovir, ganciclovir, zidovudine. Representative examples of antifungal agents include: nystatin, ketoconazole, griseofulvin, flucytosine, miconazole, clotrimazole. Representative examples of antiprotozoal agents include: pentamidine isethionate, quinine, chloroquine, and mefloquine.

[000226] Within certain preferred embodiments of the invention, anti-angiogenic compositions are provided which contain one or more compounds which disrupt microtubule function. Representative examples of such compounds include paclitaxel (discussed above), estramustine (available from Sigma; Wang and Stearns Cancer Res. 48:6262-6271, 1988), epothilone, curacin-A, colchicine, methotrexate, vinblastine and 4-tert-butyl-[3-(2- chloroethyl) ureido] benzene ("tBCEU").

[000227] Anti-angiogenic compositions of the present invention may also contain a wide variety of other compounds, including for example: α-adrenergic blocking agents, angiotensin II receptor antagonists and receptor antagonists for histamine, serotonin, endothelin; inhibitors of the sodium/hydrogen antiporter (e.g., amiloride and its derivatives); agents that modulate intracellular Ca²⁺ transport such as L-type (e.g., diltiazem, nifedipine, verapamil) or T-type Ca²⁺ channel blockers (e.g., amiloride), calmodulin antagonists (e.g., H₇) and inhibitors of the sodium/calcium antiporter (e.g., amiloride); ap-1 inhibitors (for tyrosine kinases, protein kinase C, myosin light chain kinase, Ca²⁺ /calmodulin kinase π, casein kinase II); anti-depressants (e.g. arnytriptyline, fluoxetine, LUVOX™ and PAXIL™); cytokine and/or growth factors, as well as their respective receptors, (e.g., the interleukins, a-, β- or γ-IFN, GM-CSF, G-CSF, epidermal growth factor, transforming growth factors alpha and beta, TNF, and antagonists of vascular epithelial growth factor, endothelial growth factor, acidic or basic fibroblast growth factors, and platelet dervived growth factor); inhibitors of the IP3 receptor (e.g., heparin); protease and collagenase inhibitors (e.g., TIMPs, discussed above); nitrovasodilators (e.g., isosorbide dinitrate); anti-mitotic agents (e.g., colchicine, anthracyclines and other antibiotics, folate antagonists and other anti-metabolites, vinca alkaloids, nitrosoureas, DNA alkylating agents, topoisomerase inhibitors, purine antagonists and analogs, pyrimidine antagonists and analogs, alkyl sulfonates); immunosuppressive agents (e.g., adrenocorticosteroids, cyclosporine); sense or antisense oligonucleotides (e.g., DNA, RNA, nucleic acid analogues (e.g., peptide nucleic acids) or any combinations of these); and inhibitors of transcription factor activity (e.g., lighter d group transition metals).

Combination Therapies

[000228] The compositions of the invention can be used with effective amounts of other therapeutic agents against acute and chronic inflammation. Such agents include other antagonists of adhesion molecules (e.g., other integrins, selectins, and immunoglobulin (Ig) super family members (see Springer, 1990 Nature 346: 425-433; Osborn, 1990 Cell 62: 3; Hynes, 1992 Cell 9: 11). Integrins are heterodimeric transmembrane glycoproteins consisting of an αchain (120-180 kDa) and a /3 chain (90-110 kDa), generally having short cytoplasmic domains. For example, three important integrins (i.e., LFA-I, Mac-1 and P150,95) have different alpha subunits, designated CDl Ia, CDl Ib and CDl Ic, and a common beta subunit designated CD 18. LFA-I {.diβi) is expressed on lymphocytes, granulocytes and monocytes, and binds predominantly to an Ig-family member counter-receptor termed ICAM-I and related ligands. ICAM-I is expressed on many cells, including leukocytes and endothelial cells, and is up-regulated on vascular endothelium by cytokines such as TNF and IL-I . Mac- 1 is distributed on neutrophils and monocytes, and also binds to ICAM-I. The third βι integrin, P 150,95 (αχ/3₂), is also found on neutrophils and monocytes. The selectins consist of L-selectin, E-selectin and P-selectin.

[000229] Other anti-inflammatory agents that can be used include antibodies and other antagonists of cytokines or stem cells transduced with vectors expressing such molecules, such as interleukins IL-I through IL-13, tumor necrosis factors a and β, interferons .a, β and γ, tumor growth factor beta (TGF-/3), colony stimulating factor (CSF) and granulocyte monocyte colony stimulating factor (GM-CSF). Other anti-inflammatory agents include antibodies and other antagonists of chemokines such as MCP-I, MEP- lot, MB?- 1/3, RANTES, exotaxin and EL-8. Other anti-inflammatory agents include NSAEDS, steroids and other small molecule inhibitors of inflammation.

[000230] Drugs can also be used in conjunction with the therapies described herein, include but are not limited to 5 -aminosalicylates, glucocorticoids, thioguanine derivatives, methotrexate (MTX), cyclosporine, antibiotics, and infliximab.

[000231] 5 -Aminosalicylates include sulfasalazine (also known as Azulfidine) which is a conjugate of mesalamine linked to sulfapyridine by a diazo bond and is usually administered in an amount of 500 mg/day to about 6 g/day. 5-Arninosalicylates can also be coadministered with a glucocorticoid. Preferably, a 5-aminosalicylate is used in combination therapy with one of the other agents discussed herein to treat ulcerative colitis, however it can also be used to treat Crohn's disease. Non-sulfonamide containing formulations of mesalamine include but are not limited to AS ACOL™, CLAVERSA, SALOFALK, PENTAS A™, DEPENTUM™, COLAZEDE and ROWASA™. [000232] Glucocorticoids have been a mainstay of treatment for acute severe exacerbations of EBD since 1955, when they first where shown to be efficacious in UC. Oral prednisone can be administered in conjunction with any of the agents disclosed herein. Typically, 20 to 40 mg of oral prednisone is administered once a day. Glucocorticoids can also be administered intravenously and via enemas in combination with or concurrently with or within a short time before/after an anti-alpha-4 integrin agent is administered. For example, hydrocortisone is available as a retention enema (100 mg/60 mL) and the usual dose is one 60-mL enema per night for 2 to 3 weeks. This can be altered when used in combination with the therapies and agents discussed herein as would be understood by the artisan of ordinary skill. Other steroids that can be used include, but are not limited to, prednisolone methasulfobenzoate, tixocortol pivalate, fluticasone propionate, beclomethasone dipropionate, and budesonide.

[000233] Thioguanine derivatives are also useful in the treatment of EBD, CD and UC. These include but are not limited to 6-mercaptopurine (6-MP) and azathioprine (IMURAN). The two drugs can be used interchangeably in combination with any of the alpha-4 integrin modulating agents discussed herein.

[000234] Methotrexate (MTX) is also contemplated for use in combination with the alpha- 4 integrin regulatory agents discussed herein. Preferably, MTX is administered via intramuscular injection (i.m.) to the subject in combination with an anti-alpha-4 integrin agent. MTX is effective in steroid-dependent CD, but not as useful in UC. MTX can be administered in amounts of about 15 to about 25 mg per week per subject or as necessary as determined by the artisan of ordinary skill.

[000235] Cyclosporines (e.g., SANDIMMUNE™, NEORAL™) can also be used in combination to treat pathological inflammation of the bowel. This can be used to treat acute, severe UC, which does not respond to glucocorticoids.

[000236] infliximab (i.e., REMICADE™) can also be used to treat CD. Infliximab is an immunoglobulin that binds to TNF and thereby neutralizes its activity. Another anti-TNF antibody is CDP571.

[000237] Antibiotics are also contemplated , such as for example, metronidazole or ciprofloxacin (or pharmacological equivalents thereof).

[000238] For treatment of MS, agents utilized to treat, ameliorate or palliate symptoms associated with MS, include but are not limited to: muscle relaxants (e.g., Diazepam, cyclobenzaprine, Clonazepam, clonidine, primidone, and the like), anticholinergics (e.g., propantheline, dicyclomine, and the like), central nervous system stimulants (e.g., Pemoline), non-steroidal anti-inflammatory agents (NS such as ibuprofen, naproxen and ketoprofen), interferons, immune globulin, glatiramer (Copaxone™), mitoxantrone (Novantrone™), misoprostol, tumor necrosis factor-alpha inhibitors (e.g., Pirfenidone, infliximab and the like) and corticosteroids (e.g., glucocorticoids and mineralocorticoids).

[000239] Common agents for treating multiple sclerosis include interferon beta- Ib

(Betaserong), interferon beta- Ia (Avonex™), high-dose interferon beta- Ia (Rebif),

Glatiramer (Copaxone™), immune globulin, mitoxantrone (Novantrone™), corticosteroids

(e.g., prednisone, methylprednisolone, dexamethasone and the like). Other corticosteroids may also be used and include but are not limited to Cortisol, cortisone, fludrocortisone, prednisolone, 6α-methylprednisolone, triamcinolone, and betamethasone.

[000240] NS or NSAIDs contemplated for use with this invention include but are not limited to non-selective COX inhibitors and selective COX-2 inhibitors. Non-selective COX inhibitors include but are not limited to salicylic acid derivatives (e.g., aspirin, sodium salicylates, choline magnesium trisalicylate, salsalate, diflunisal, sulfasalazine, and olsalazine), para-aminophenol derivatives (e.g., acetaminophen), indole and indene acetic acids (e.g., tolmetin, diclofenac, and ketorolac), heteroaryl acetic acids (e.g., abuprofen, naproxen, flurbiprofen, ketoprofen, fenprofen, and oxaprozin), anthranilic acids or fenamates

(e.g., mefenamic acid and meclofenamic acid), enolic acids (e.g., oxicams such as piroxicam and meloxicam), and alkanones (e.g., nabumetone). Selective COX-2 inhibitors include diaryl-substituted furanones (e.g., rofecoxib), diaryl-substituted pyrazoles (e.g., celecoxib), indole acetic acids (e.g., etodolac), and sulfonanilides (e.g., nimesulide). NS are oftentimes administered in combination with interferon to lessen the flu-like symptoms experienced by patients receiving, for example, Avonex™. Common NS agents include naproxen, ibuprofen and ketoprofen. Paracetamol is also frequently administered to patients. See, Rees et al., 2002 Mult. Scler. S-Λ5-S.

[000241] Selection of the genes to be introduced into stem cells will depend on the application of the gene therapy. For example, gene therapy with endothelial stem cells may be used to promote angiogenesis, inhibit angiogenesis, or to inhibit the growth of tumors. [000242] Some examples of genes useful for promoting angiogenesis include the genes that encode HIF-I, SDF-I, the VEGFs, the cadherins, the integrins, FGFα, FGF/3, FGF4, HGF, TGFa, EGF, angiopoietin-1, B61, IL-8, and angiogenin.

[000243] Some examples of genes useful for inhibiting angiogenesis for treatment of tumors include the genes that encode soluble KDR, soluble flt-1, KDR antibodies, TGF-/3, lymphotoxin, interferon-γ, platelet factor 4, angiopoietin-2, angiostatin, endostatin, thrombospondin, inducible protein-10, and IL-12.

[000244] Some examples of genes useful for treating genetic diseases, for example hemophilia or diabetes, include the genes that encode factor VIII/von Willebrand, factor IX, and insulin.

[000245] The gene can be delivered at a desired site of vascularization. The site of vascularization may be a natural site or an artificially created site. Natural sites of neovascularization include cardiac and peripheral ischemia, tumors, vascular ulcers and other vascular wounds as described above.

[000246] The stem cells transfected with a gene therapy vector may be naturally or artificially recruited to the site where the protein expressed by the gene is desired. Recruiting the vector to the site can be induced artificially by administering a suitable chemokine systemically or at the desired site. A suitable molecule is hypoxia inducible factor- 1, a chemokine such as stromal derived factor- 1 (SDF-I). The endothelial stem cells may also be recruited to the desired site by means of an interleukin, such as IL-I or IL-8. Other methods include the administration of compounds that regulate the nitric oxide pathway or induce hypoxia. Regulation of the nitric oxide pathway by compounds include, but not limited to: sildenafil (Viagra®), vardenafil (Levitra®), tadalafil (Cialis®), apolipoprotein-E, nitroglycerine, L-argenine, nitrate esters, isoamylynitrite, SIN-I, cysteine, dithiothreitol, N- acetylcysteine, mercaptosuccinic acid, thiosalicylic acid, and methylthiosalicylic acid. The administration of such compounds may be administered to a patient prior to the administration of stem cells, in conjunction with the stem cells after the administration of stem cells or combinations thereof.

[000247] The transfected stem cells that are administered to a mammal for gene therapy may be autologous or heterologous. Preferably, the transfected stem cells are autologous. [000248] Other methods for carrying out gene therapy in mammals have been described in the prior art, for example, in Mulligan, et al., U.S. Pat. No. 5,674,722. The methods described in Mulligan, et al., U.S. Pat. No. 5,674,722 for carrying out gene therapy are incorporated herein by reference.

Antibodies specific for Tumor Antigens

[000249] In another preferred embodiment, the stem cells can express antibodies specific for desired tumor antigens. Many tumor antigens are well known in the art. See for example, Van den Eynde BJ, van der Bruggen P. Curr Opin Immunol 1997; 9: 684-93; Houghton AN, Gold JS, Blachere NE. Curr Opin Immunol 2001; 13: 134-140; van der Bruggen P, Zhang Y, Chaux P, Stroobant V, Panichelli C, Schultz ES, Chapiro J, Van den Eynde BJ, Brasseur F, Boon T. Immunol Rev 2002; 188: 51-64, which are herein incorporated by reference. Alternatively, many antibodies directed towards tumor antigens are commercially available. [000250] Non-limiting examples of tumor antigens, include, tumor antigens resulting from mutations, such as: alpha-actinin-4 (lung carcinoma); BCR-ABL fusion protein (b3a2) (chronic myeloid leukemia); CASP-8 (head and neck squamous cell carcinoma); beta-catenin (melanoma); Cdc27 (melanoma); CDK4 (melanoma); dek-can fusion protein (myeloid leukemia); Elongation factor 2 (lung squamous carcinoa); ETV6-AML1 fusion protein (acute lymphoblastic leukemia); LDLR-fucosyltransferaseAS fusion protein (melanoma); overexpression of HLA- A2^d (renal cell carcinoma); hsp70-2 (renal cell carcinoma); KIAAO205 (bladder tumor); MART2 (melanoma); MUM-If (melanoma); MUM-2 (melanoma); MUM-3 (melanoma); neo-PAP (melanoma); Myosin class I (melanoma); 0S-9g (melanoma); pml-RARalpha fusion protein (promyelocytic leukemia); PTPRK (melanoma); K-ras (pancreatic adenocarcinoma); N-ras (melanoma). Examples of differentiation tumor antigens include, but not limited to: CEA (gut carcinoma); gplOO / Pmell7 (melanoma); Kallikrein 4 (prostate); mammaglobin-A (breast cancer); Melan-A / MART-I (melanoma); PSA (prostate carcinoma); TRP-I / gp75 (melanoma); TRP-2 (melanoma); tyrosinase (melanoma). Over or under-expressed tumor antigens include but are not limited to: CPSF (ubiquitous); EphA3 ; G250 / MN / CATX (stomach, liver, pancreas); HER-2/neu; Intestinal carboxyl esterase (liver, intestine, kidney); alpha-foetoprotein (liver ); M-CSF (liver, kidney); MUCl (glandular epithelia); p53 (ubiquitous); PRAME (testis, ovary, endometrium, adrenals); PSMA (prostate, CNS, liver); RAGE-I (retina); RU2AS (testis, kidney, bladder); survivin (ubiquitous); Telomerase (testis, thymus, bone marrow, lymph nodes); WTl (testis, ovary, bone marrow, spleen); CA125 (ovarian).

Bispecific Molecules

[000251] In another preferred embodiment, stem cells express bispecific molecules. For example, a molecule comprising a modulatory or cytolytic domain is fused to a bispecific antibody domain or fragments thereof. In one aspect of the invention, the bispecific antibody comprises two monoclonal antibodies. However, the bispecific antibody can comprise two polyclonal antibodies or an engineered bispecific antibody. Preferably, each of the specificities of the bispecific antibody are directed to one or more tumor antigens and/or specific cell or tissue. Antibodies can be raised against any tumor antigen from a patient. Thus the targeting of the chimeric molecule can be individually tailored as the tumor displays different antigens.

[000252] For tracking purposes, the bispecific antibody can be directly labeled or a second antibody specific for a region of the bispecific antibody is labeled. Detection of the localization of the chimeric molecule is preferably through cell sorting techniques such as flow cytometry. For example, wherein samples are taken at different time intervals after administration of the chimeric molecule for imaging and diagnostic purposes. [000253] In accordance with the invention, the bispecific antibody, targets molecules to a specific location in vivo. For example, the location can be to tumors in myocardial tissues, breast, ovaries, testis, hepatocyte, kidneys and the like. The bispecific antibody determines the specific antigen to which the molecule is targeted.

[000254] As described above, the specificity of the antibody domain can be directed to a specific tissue antigen wherein the tumor has been detected coupled with specificity for that particular tumor antigen. Alternatively, the bispecific antibody domain is directed to two tumor antigens that are expressed by the tumor. The bispecific domain can be fused to any modulatory or cytolytic domain discussed above.

[000255] hi another embodiment of the invention, the bispecific antibody (BiAb) construct is a bispecific antibody that binds to one or more tumor antigens as a first or second antigen and a cell or tissue specific antigen a second antigen. The antibody may be covalently bound to the a modulatory or cytolytic molecule and the chimeric molecule may be constructed by chemical coupling, producing a fusion protein or a mosaic protein from the antibody and from a modified or unmodified prokaryotic or eukaryotic modulatory or cytotoxic molecule. Furthermore, the antibody may be joined to modulatory or cytotoxic molecule via multimerization domains.

[000256] In another embodiment of the invention, the polypeptide of the invention, e.g., a endostatin construct, is a fusion construct of a modified or an unmodified endostatin with a modified or an unmodified modulatory or cytotoxic molecule. The construct may be bound in vitro and/or in vivo, e.g., by a multimerization domain, to bispecific antibody domain. The chimeric molecule constructs may, inter alia, result from chemical coupling, may be recombinantly produced (as shown in the appended examples), or may be produced as a fusion protein as described above. In one aspect, the moiety specifically binds to at least one tumor antigen.

[000257] The compositions of the invention can comprise stem cells expressing any cytotoxic agent as described infra. For example, in one aspect, the toxin may be a polypeptide toxin, e.g., a Pseudomonas exotoxin, like PE38, PE40 or PE37, or a truncated version thereof, or a ribosome inactivating protein gelonin (e.g., Boyle (1996) J. Immunol. 18:221-230), and the like. The compositions of the invention can be conjugated to any cytotoxic pharmaceuticals, e.g., radiolabeled with a cytotoxic agents, such as, e.g., .¹³¹I (e.g., Shen (1997) Cancer 80(12 Suρρl):2553-2557), copper-67 (e.g., Deshpande (1988) J. Nucl. Med. 29:211 -225).

[000258] In one embodiment, the construct is a fusion (polypeptide or a mosaic (polypeptide. The fusion (polypeptide may comprise merely the domains of the constructs as described herein, as well as (a) functional fragment(s) thereof. However, it is also envisaged that the fusion (polypeptide comprises further domains and/or functional stretches. Therefore, the fusion (polypeptide can comprise at least one further domain, this domain being linked by covalent or non-covalent bonds. The linkage as well as the construction of such constructs, can be based on genetic fusion according to the methods described herein or known in the art (e.g., Sambrook et al., loc. cit, Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N. Y. (1989)) or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686. The additional domain present in the construct may be linked by a flexible linker, such as a (polypeptide linker, wherein the (polypeptide linker can comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of the further domain and the N-terminal end of the peptide, (polypeptide or antibody or vice versa. The linker may, inter alia, be a Glycine, a Serine and/or.a Glycine/Serine linker. Additional linkers comprise oligomerization domains. Oligomerization domains can facilitate the combination of two or several antigens or fragments thereof in one functional molecule. Non-limiting examples of oligomerization domains comprise leucine zippers (like jun-fos, GCN4, E/EBP; Kostelny, J. Immunol. 148 (1992), 1547-1553; Zeng, Proc. Natl. Acad. Sd. USA 94 (1997), 3673-3678, Williams, Genes Dev. 5 (1991), 1553-1563; Suter, "Phage Display of Peptides and Proteins", Chapter 11, (1996), Academic Press), antibody-derived oligomerization domains, like constant domains C_H1 and C_L (Mueller, FEBS Letters 422 (1998), 259-264) and/or tetramerization domains like GCN₄-LI (Zerangue, Proc. Natl. Acad. ScL USA 97 (2000), 3591-3595). [000259] Furthermore, the chimeric fusion constructs to be used in the present invention, as described herein, may comprise at least one further domain, inter alia, domains which provide for purification means, like, e.g. histidine stretches. The further domain(s) may be linked by covalent or non-covalent bonds.

[000260] The linkage can be based on genetic fusion according to the methods known in the art and described herein or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686. The additional domain present in the construct may be linked by a flexible linker, such as a polypeptide linker to one of the binding site domains; the polypeptide linker can comprise plural, hydrophilic or peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of one of the domains and the N-terminal end of the other of the domains when the polypeptide assumes a conformation suitable for binding when disposed in aqueous solution.

Immune Activating Chimeric Fusion Molecules

[000261] It is also envisaged that the constructs disclosed for uses, compositions and methods of the present invention comprises (a) further domain(s) which may function as immunomodulators. The immunomodulators comprise, but are not limited to cytokines, lymphokines, T cell co-stimulatory ligands, etc. Preferably, the desired molecule targets and delivers a modulatory or cytolytic molecule to the tumor cell and also recruits immune cells and/or activated immune cells to the tumor. For example, adequate activation resulting in priming of naϊve T-cells is critical to primary immunoresponses and depends on two signals derived from professional APCs (antigen-presenting cells) like dendritic cells. The first signal is antigen-specific and normally mediated by stimulation of the clonotypic T-cell antigen receptor (TCR) that is induced by processed antigen presented in the context of MHC class-I or MHC class-II molecules. However, this primary stimulus is insufficient to induce priming responses of naϊve T-cells, and the second signal is required which is provided by an interaction of specific T-cell surface molecules binding to co-stimulatory ligand molecules on antigen presenting cells (APCs), further supporting the proliferation of primed T-cells. The term "T-cell co-stimulatory ligand" therefore denotes in the light of the present invention molecules, which are able to support priming of naϊve T-cells in combination with the primary stimulus and include, but are not limited to, members of the B7 family of proteins, including B7-1 (CD80) and 137-2 (CD86).

[000262] The desired chimeric molecule construct described herein may comprise further receptor or ligand function(s), and may comprise immuno-modulating effector molecule or a fragment thereof. An immuno-modulating effector molecule positively and/or negatively influences the humoral and/or cellular immune system, particularly its cellular and/or non- cellular components, its functions, and/or its interactions with other physiological systems. The immuno-modulating effector molecule may be selected from the group comprising cytokines, chemokines, macrophage migration inhibitory factor (MIF; as described, inter alia, in Bernhagen (1998), MoI Med 76(3-4); 151-61 or Metz (1997), Adv Immunol 66, 197- 223), T-cell receptors and soluble MHC molecules. Such immuno-modulating effector molecules are well known in the art and are described, inter alia, in Paul, "Fundamental immunology", Raven Press, New York (1989). hi particular, known cytokines and chemokines are described in Meager, "The Molecular Biology of Cytokines" (1998), John Wiley & Sons, Ltd., Chichester, West Sussex, England; (Bacon (1998). Cytokine Growth Factor Rev 9(2):167-73; Oppenheim (1997). Clin Cancer Res 12, 2682-6; Taub, (1994) Ther. Immunol. 1(4), 229-46 or Michiel, (1992). Semin Cancer Biol 3(1), 3-15). [000263] Immune cell activity that may be measured include, but is not limited to, (1) cell proliferation by measuring the DNA replication; (2) enhanced cytokine production, including specific measurements for cytokines, such as IFN-γ, GM-CSF, or TNF-ας (3) cell mediated target killing or lysis; (4) cell differentiation; (5) immunoglobulin production; (6) phenotypic changes; (7) production of chemotactic factors or chemotaxis, meaning the ability to respond to a chemotactin with chemotaxis; (8) immunosuppression, by inhibition of the activity of some other immune cell type; and, (9) apoptosis, which refers to fragmentation of activated immune cells under certain circumstances, as an indication of abnormal activation. Modified Chimeric Molecules

[000264] The constructs of the present invention may comprise domains originating from one species, e.g., from mammals, such as human. However, chimeric and/or humanized constructs are also envisaged and within the scope of the present invention. [000265] Furthermore, the polynucleotide/nucleic acid molecules of the invention may comprise, for example, thioester bonds and/or nucleotide analogues. The modifications may be useful for the stabilization of the nucleic acid molecule, e.g., against endo- and/or exonucleases in the cell. These nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of the nucleic acid molecule in the cell. The polynucleotide/nucleic acid molecules of the invention may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination. The polynucleotide may be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. The polynucleotide can be part of a vector, e.g., an expression vector, including, e.g., recombinant viruses. The vectors may comprise further genes, such as marker genes, that allow for the selection of the vector in a suitable host cell and under suitable conditions.

[000266] In one aspect, the polynucleotides of the invention are operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide comprises transcription of the polynucleotide into a translatable niRNA. Regulatory elements ensuring expression in cells, including eukaryotic cells, such as mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription, and, optionally, poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Exemplary regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOXl or GALl promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. The nucleic acids of the invention can also comprise, in addition to elements responsible for the initiation of transcription, other elements, such regulatory elements and transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site (termination sequences are typically downstream of the polynucleotide coding sequence). Furthermore, depending on the expression system used, nucleic acid sequences encoding leader sequences capable of directing the polypeptide to a cellular compartment, or secreting it into the medium, may be added to the coding sequence of the polynucleotide of the invention; such leader sequences are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences. In one aspect, the leader sequence is capable of directing secretion of translated chimeric protein, or a portion thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product; see supra. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDM8, pRc/CMV, pcDNAl, pcDNA3 (hi-vitrogene), or pSPORTl (GIBCO BRL). Expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells; control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host, the host can be maintained under conditions suitable for high level expression of the nucleotide sequences; and, as desired, the collection and purification of the polypeptide of the invention may follow.

[000267] As described above, the polynucleotide of the invention can be used alone or as part of a vector (e.g., an expression vector or a recombinant virus), or in cells, to express the chimeric fusion molecules of the invention. The polynucleotides or vectors containing the DNA sequence(s) encoding any one of the chimeric fusion molecules of the invention can be introduced into the cells, which in turn produce the polypeptide of interest. [000268] The present invention can use any type of suitable vectors, e.g., plasmids, cosmids, viruses and bacteriophages, or any expression system used conventionally in genetic engineering, that comprise a polynucleotide encoding a chimeric fusion molecule of the invention. The vector can be an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno- associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vectors of the invention into targeted cell populations. Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N. Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells. The vectors containing the polynucleotides of the invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, supra.

[000269] Once expressed, the chimeric fusion molecules of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "Protein Purification", Springer-Verlag, N.Y. (1982). rn alternative aspects, the invention is directed to substantially pure chimeric polypeptides of at least about 90% to about 95% homogeneity; between about 95% to 98% homogeneity; and about 98% to about 99% or more homogeneity; these "substantially pure" polypeptides can be used in the preparation of pharmaceuticals. Once purified, partially or to a homogeneity as desired, the polypeptides may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures.

[000270] In a still further embodiment, the present invention relates to a stem cell containing the polynucleotide or vector of the invention, hi alternative aspects, the host/cell is a eukaryotic cell, such as a mammalian cell, particularly if therapeutic uses of the polypeptide are envisaged. Of course, yeast and prokaryotic, e.g., bacterial cells, may serve as well, in particular, if the produced polypeptide is used for non-pharmaceutical purposes, e.g., as in diagnostic tests or kits or in screening methods.

[000271] The polynucleotide or vector in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally, e.g., as an episome. [000272] In one aspect, the nucleic acids encoding the chimeric polypeptide of the invention (including those sequences in vectors, e.g., plasmid or virus) further comprise, genetically fused thereto, sequences encoding an epitope tag, e.g., an N-terminal FLAG-tag and/or a C-terminal His-tag. In one aspect, the length of the FLAG-tag is about 4 to 8 amino acids; or, is about 8 amino acids in length. Methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1989). The genetic constructs and methods described therein can be utilized for expression of the polypeptide of the invention in eukaryotic or prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. Furthermore, transgenic non-human animals, such as mammals (e.g., mice, goats), comprising nucleic acids or cells of the invention may be used for the large scale production of the chimeric polypeptides of the invention.

Administration of Compositions to Animals

[000273] For targeting a tumor cell in situ, the compositions described above may be administered to animals including human beings in any suitable formulation. For example, compositions for targeting a tumor cell may be formulated in pharmaceutically acceptable carriers or diluents such as physiological saline or a buffered salt solution. Suitable carriers and diluents can be selected on the basis of mode and route of administration and standard pharmaceutical practice. A description of exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington's Pharmaceutical Sciences, a standard text in this field, and in USP/NF. Other substances may be added to the compositions to stabilize and/or preserve the compositions. [000274] The compositions of the invention may be administered to animals by any conventional technique. The compositions may be administered directly to a target site by, for example, surgical delivery to an internal or external target site, or by catheter to a site accessible by a blood vessel. Other methods of delivery, e.g., liposomal delivery or diffusion from a device impregnated with the composition, are known in the art. The compositions may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously). For parenteral administration, the compositions are preferably formulated in a sterilized pyrogen-free form.

Nucleic Acid-Based Diagnostic and Prognostic Methods

[000275] Also encompassed by this invention is a method of diagnosing BMDC- dependent metaplasia or BMDC-associated cancer in a subject, comprising: detecting a level of an BMDC nucleic acid in a biological sample; and comparing the level of BMDC in the biological sample with a level of BMDC marker in a control sample, wherein an elevation in the level of BMDC marker in the biological sample compared to the control sample is indicative of diagnosing BMDC-dependent metaplasia or BMDC-associated cancer. [000276] Preferred methods are discussed in the examples which follow. However, other embodiments are also preferred.

[000277] In an embodiment, the BMDC nucleic acid in a biological sample includes amplifying an BMDC RNA. In another embodiment of the above methods, the detecting a level of BMDC nucleic acid in a biological sample includes hybridizing the BMDC RNA with a probe.

[000278] As an alternative to making determinations based on the absolute expression level of the BMDC marker, determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-BMDC-dependent metaplasia or BMDC-associated cancer sample, or between samples from different sources.

[000279] Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a marker, the level of expression of the marker is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the biological sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level.

[000280] One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. Probes based on the sequence of a nucleic acid molecule can be used to detect transcripts corresponding to an BMDC marker. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a marker of the present invention. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed. In an embodiment, the probe includes a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.

[000281] In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose, hi an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.

[000282] "Amplifying" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal. As used herein, the term template-dependent process is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well- known rules of complementary base pairing (see., for example, Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Maniatis, T. et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982.

[000283] A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR) which is described in detail in Mullis, ^{et al}, U.S. Pat. No. 4,683,195, Mullis, ^{et a1}., U.S. Pat. No. 4,683,202, and Mullis, et al, U.S. Pat. No. 4,800,159, and in hmis et al, PCR Protocols, Academic Press, Inc., San Diego Calif, 1990. Briefly, in PCR, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase (e.g.,¹ Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated. Preferably a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of rnRNA amplified. Polymerase chain reaction methodologies are well known in the art. [000284] Another method for amplification is the ligase chain reaction (LCR), disclosed in European Patent No. 320,308Bl . In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. Whiteley, et al, U.S. Pat. No. 4,883,750 describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence.

[000285] Qβ Replicase, described in PCT Application No. PCT/US87/00880 may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which can then be detected.

[000286] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e. nick translation. A similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. BMDC specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having a 3' and 5' sequences of non-prostate specific DNA and middle sequence of prostate specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNaseH, and the products of the probe identified as distinctive products generating a signal which are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Thus, CPR involves amplifying a signal generated by hybridization of a probe to a BMDC- dependent metaplasia or BMDC-associated cancer specific expressed nucleic acid. [000287] Still other amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025 may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR like, template and enzyme dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

[000288] Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh D., et al, Proc. Natl. Acad. ScL, U.S.A. 1989, 86:1173, Gingeras T. R., et al., PCT Application WO 88/1D315), including nucleic acid sequence based amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has prostate specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second prostate specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate BMDC-dependent metaplasia or BMDC-associated cancer specific sequences.

[000289] Davey, C, et al, European Patent No. 329,822Bl disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

[000290] Miller, H. L, et al. , PCT Application WO 89/06700 discloses a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts. Other amplification methods include "race" disclosed by Frohman, M. A., In: PCR Protocols: A Guide to Methods and Applications 1990, Academic Press, New York) and "one-sided PCR" (Ohara, O., et al, Proc. Natl. Acad. ScI, U.S.A. 1989, 86:5673-5677). [000291] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu, D. Y. et al, Genomics 1989, 4:560), may also be used in the amplification step of the present invention.

[000292] Following amplification, the presence or absence of the amplification product may be detected. The amplified product may be sequenced by any method known in the art, including and not limited to the Maxam and Gilbert method. The sequenced amplified product is then compared to a sequence known to be in a BMDC-dependent metaplasia or BMDC-associated cancer specific sequence. Alternatively, the nucleic acids may be fragmented into varying sizes of discrete fragments. For example, DNA fragments may be separated according to molecular weight by methods such as and not limited to electrophoresis through an agarose gel matrix. The gels are then analyzed by Southern hybridization. Briefly, DNA in the gel is transferred to a hybridization substrate or matrix such as and not limited to a nitrocellulose sheet and a nylon membrane. A labeled probe is applied to the matrix under selected hybridization conditions so as to hybridize with complementary DNA localized on the matrix. The probe may be of a length capable of forming a stable duplex. The probe may have a size range of about 200 to about 10,000 nucleotides in length, preferably about 200 nucleotides in length. Various labels for visualization or detection are known to those of skill in the art, such as and not limited to fluorescent staining, ethidium bromide staining for example, avidin/biotin, radioactive labeling such as ³²P labeling, and the like. Preferably, the product, such as the PCR product, may be run on an agarose gel and visualized using a stain such as ethidium bromide. The matrix may then be analyzed by autoradiography to locate particular fragments which hybridize to the probe.

[000293] hi addition to being useful for the detection of primary tumors, amplification based detection methods are especially useful for the detection of micrometastatic disease. The presence of micrometastatic disease can be detected in tissues proximal to the primary site of the original tumor, or by the presence of tumor cells in circulating body fluids such as blood or lymph. Detection of such micrometastatic cells can be accomplished for example, using RT-PCR alone or in combination with inimunomagnetically enriched cells as described for example by Klein et al. {Proc. Natl. Acad. ScL, USA 96:4494-4499 (1999); Raynor et al. BMC Cancer 2:14 (2002); and U.S. Pat. No. 5,674,694).

Monitoring of Effects During Clinical Trials

[000294] Monitoring the influence of agents (e.g., drugs or compounds) on an BMDC- dependent metaplasia or BMDC-associated cancer can be applied not only in basic drug screening, but also in clim^'cal trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase BMDC gene expression or protein levels can be monitored in clinical trials of subjects exhibiting decreased BMDC gene expression or protein levels. Alternatively, the effectiveness of an agent determined by a screening assay to decrease BMDC gene expression protein levels, downregulate BMDC proliferation, or increase BMDC differentiation or apoptosis can be monitored in clim^'cal trials of subjects exhibiting BMDC dependent metaplasia or BMDC-associated cancer, hi such clinical trials, the expression or activity of a BMDC gene, and preferably, other genes that have been implicated in a disorder can be used as a "read out" or markers of the phenotype of a particular cell.

[000295] For example, and not by way of limitation, genes that are modulated in an BMDC by treatment with an agent (e.g., compound, drug or small molecule) which modulates BMDC proliferation, differentiation or apoptosis (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on a BMDC- dependent metaplasia or BMDC-associated cancer, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT- PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of one or more BMDC genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent. [000296] In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising obtaining a pre- administration sample from a subject prior to administration of the agent; detecting the level of expression or activity of a BMDC protein, mRNA, genomic DNA, BMDC proliferation, differentiation or apoptosis in the pre-administration sample; obtaining one or more post- administration samples from the subject; detecting the level of expression or activity of the BMDC protein, mRNA, genomic DNA, BMDC proliferation, differentiation or apoptosis in the post-administration samples; comparing the level of expression or activity of the level of expression or activity of the BMDC protein, mRNA, genomic DNA, BMDC proliferation, differentiation or apoptosis in the pre-administration sample with level of expression or activity of the BMDC protein, mRNA, genomic DNA, BMDC proliferation, differentiation or apoptosis in the post administration sample or samples; and altering the administration of the agent to the subject accordingly.

Methods of Treatment

[000297] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant BMDC expression or activity (e.g., metaplasia, or malignant cell growth, tumors, cancer).

[000298] Also provided by this invention is a method for treating cancer in a subject comprising administering to a subject an effective amount of a combination of a BMDC inhibitor and a hyperplastic inhibitory agent such that the cancer is treated. In an embodiment of the above methods of treating abnormal BMDC growth or cancer, the treating includes inhibiting tumor growth and/or preventing the occurrence of tumor growth in the subject.

[000299] In another embodiment of the above methods of treating abnormal cell growth or cancer, the treating includes a combination treatment in which a BMDC inhibitor is administered to a subject in combination with radiation therapy. [000300] In another embodiment of the above methods of treating abnormal cell growth or cancer, the abnormal cell growth or tumor growth or cancer is caused by repopulation of inflamed tissue with BMDC. In a preferred embodiment, the abnormal cell growth or tumor growth or cancer being treated is epithelial.

[000301] The methods of treatment of the invention comprise the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a BMDC dependent metaplasia or a BMDC-associated cancer, a symptom thereof, or a predisposition toward a the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype".) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the BMDC modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[000302] Prophylactic Methods: In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant BMDC activity, by administering to the subject an agent which modulates BMDC proliferation, differentiation or apoptosis. Subjects at risk for a disease which is caused or contributed to by aberrant BMDC activity can be identified by, for example, any of a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of BMDC aberrancy, for example, an BMDC agonist or BMDC antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[000303] Therapeutic Methods: Another aspect of the invention pertains to methods of modulating BMDC proliferation, differentiation or apoptosis for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting an BMDC or BMDC-derived cell with an agent that modulates one or more of the activities of the BMDC or BMDC-derived cell. An agent that modulates BMDC activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an BMDC, a BMDC antibody, a BMDC agonist or antagonist, a peptidomimetic of a BMDC agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more BMDC activities. Examples of such stimulatory agents include active BMDC protein and a nucleic acid molecule encoding BMDC that has been introduced into the cell, hi another embodiment, the agent inhibits one or more BMDC activities. Examples of such inhibitory agents include antisense BMDC nucleic acid molecules, anti-BMDC antibodies, and BMDC inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant activity of a BMDC. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) BMDC activity. In another embodiment, the method involves administering a BMDC protein or nucleic acid molecule as therapy to compensate^" for reduced or aberrant BMDC activity.

[000304] Stimulation of BMDC activity is desirable in situations in which BMDC are abnormally downregulated and/or in which increased BMDC activity is likely to have a beneficial effect. Likewise, inhibition of BMDC activity is desirable in situations in which BMDC is abnormally upregulated and/or in which decreased BMDC activity is likely to have a beneficial effect. For example, in particular embodiments, treatment of BMDC-dependent metaplasia and/or BMDC-associated cancer would comprise administering an agent that inhibits the proliferation of BMDCs at the site of the metaplasia or cancer. In other embodiments, treatment would involve administering an agent that promotes the differentiation of the BMDCs in the cancer tissue, or alternatively, induce apoptosis of the cancerous cells. [000305] The present invention further includes therapeutic methods which utilize a combination of therapeutic agents of the invention, as described herein, and further therapeutic agents which are known in the art. Specifically, an BMDC modulator of the present invention can be used in combination with a second modulator or with a second "abnormal cell growth inhibitory agent" (ACI agent). The ACI agent can be any therapeutic agent which can be used to treat the selected BMDC-dependent metaplasia or BMDC- associated cancer. One skilled in the art would be able to select appropriate ACI agents for combination therapy with a BMDC modulator. For example, an ACI agent may be a second BMDC modulator, or it may be an art-recognized agent which does not modulate BMDC. [000306] The terms "abnormal cell growth inhibitory agent" and "ACI agent" are used interchangeably herein and are intended to include agents that inhibit the growth of proliferating cells or tissue wherein the growth of such cells or tissues is undesirable. For example, the inhibition can be of the growth of malignant cells such as in neoplasms or benign cells such as in tissues where the growth is inappropriate. Examples of the types of agents which can be used include chemotherapeutic agents, radiation therapy treatments and associated radioactive compounds and methods, and immunotoxins.

[000307] The language "chemotherapeutic agent" is intended to include chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable. Chemotherapeutic agents are well known in the art (see e.g., Gilman A. G., et al. The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treat neoplastic diseases, tumors, and cancers. [000308] The language "radiation therapy" is intended to include the application of a genetically and somatically safe level of X-rays, both localized and non-localized, to a subject to inhibit, reduce, or prevent symptoms or conditions associated with undesirable cell growth. The term X-rays is intended to include clinically acceptable radioactive elements and isotopes thereof, as well as the radioactive emissions therefrom. Examples of the types of emissions include alpha rays, beta rays including hard betas, high energy electrons, and gamma rays. Radiation therapy is well known in the art (see e.g., Fishbach, F., Laboratory Diagnostic Tests, 3rd Ed., Ch. 10: 581-644 (1988)), and is typically used to treat neoplastic diseases, tumors, and cancers.

[000309] The term "immunotoxins" includes immunotherapeutic agents which employ cytotoxic T cells and/or antibodies, e.g., monoclonal, polyclonal, phage antibodies, or fragments thereof, which are utilized in the selective destruction of undesirable rapidly proliferating cells. For example, immunotoxins can include antibody-toxin conjugates (e.g., Ab-ricin and Ab-diphtheria toxin), antibody-radiolabels (e.g., Ab-I¹³⁵) and antibody activation of the complement at the tumor cell. The use of immunotoxins to inhibit, reduce, or prevent symptoms or conditions associated with neoplastic diseases are well known in the art (see e.g., Harlow, E. and Lane, D., Antibodies, (1988)).

[000310] The language "inhibiting undesirable cell growth" is intended to include the inhibition of undesirable or inappropriate cell growth. The inhibition is intended to include inhibition of proliferation including rapid proliferation. For example, the cell growth can result in benign masses or the inhibition of cell growth resulting in malignant tumors. [000311] Pharmacogenomics: Agents, or modulators which have a stimulatory or inhibitory effect on BMDC activity (e.g., proliferation, differentiation, apoptosis) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., proliferative disorders such as cancer) associated with aberrant BMDC activity, hi conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an BMDC modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an BMDC modulator.

[000312] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10- ll):983-985 and Linder, M. W. et al. (1997) Clin. Chetn. 43(2):254-266. In general, two types of pharmaco genetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans. [000313] One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome-wide association", relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi-allelic" gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants). Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[000314] Alternatively, a method termed the "candidate gene approach", can be utilized to identify genes that predict a drug response. According to this method, if a gene that encodes a drug target is known, (e.g., a BMDC gene) all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[000315] Alternatively, a method termed the "gene expression profiling", can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.

[000316] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an BMDC modulator, such as a modulator identified by one of the exemplary screening assays described herein. Pharmaceutical Compositions

[000317] The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a marker nucleic acid or protein. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a marker nucleic acid or protein. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a marker nucleic acid or protein and one or more additional active compounds. [000318] The invention also provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, peptoids, small molecules or other drugs) which (a) bind to the marker, or (b) have a modulatory (e.g., stimulatory or inhibitory) effect on the activity of the marker or, more specifically, (c) have a modulatory effect on the interactions of the marker with one or more of its natural substrates (e.g., peptide, protein, hormone, co-factor, or nucleic acid), or (d) have a modulatory effect on the expression of the marker. Such assays typically comprise a reaction between the marker and one or more assay components. The other components may be either the test compound itself, or a combination of test compound and a natural binding partner of the marker.

[000319] The test compounds of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Test compounds may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one- bead one-compound¹ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145).

[000320] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sd. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. ScL USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sd USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sd. 87:6378-6382; Felici, 1991, J MoI. Biol. 222:301-310; Ladner, supra.).

[000321] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a protein encoded by or corresponding to a marker or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to a protein encoded by or corresponding to a marker or biologically active portion thereof. Determining the ability of the test compound to directly bind to a protein can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to the marker can be determined by detecting the labeled marker compound in a complex. For example, compounds (e.g., marker substrates) can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, assay components can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. [000322] In another embodiment, the invention provides assays for screening candidate or test compounds which modulate the expression of a marker or the activity of a protein encoded by or corresponding to a marker, or a biologically active portion thereof. In all likelihood, the protein encoded by or corresponding to the marker can, in vivo, interact with one or more molecules, such as but not limited to, peptides, proteins, hormones, cofactors and nucleic acids. For the purposes of this discussion, such cellular and extracellular molecules are referred to herein as "binding partners" or marker "substrate".

[000323] One embodiment of the invention in order to facilitate such screening is the use of a protein encoded by or corresponding to marker to identify the protein's natural in vivo binding partners. There are many ways to accomplish this which are known to one skilled in the art. One example is the use of the marker protein as "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al, 1993, Cell 72:223-232; Madura et al, 1993, J. Biol. Chem. 268:12046-12054; Bartel et al ,1993, Biotechniques 14:920-924; Iwabuchi et al, 1993 Oncogene 8:1693-1696; Brent WO94/10300) in order to identify other proteins which bind to or interact with the marker (binding partners) and, therefore, are possibly involved in the natural function of the marker. Such marker binding partners are also likely to be involved in the propagation of signals by the marker protein or downstream elements of a marker protein-mediated signaling pathway. Alternatively, such marker protein binding partners may also be found to be inhibitors of the marker protein. [000324] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that encodes a marker protein fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a marker-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be readily detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the marker protein. [000325] In a further embodiment, assays may be devised through the use of the invention for the purpose of identifying compounds which modulate (e.g., affect either positively or negatively) interactions between a marker protein and its substrates and/or binding partners. Such compounds can include, but are not limited to, molecules such as antibodies, peptides, hormones, oligonucleotides, nucleic acids, and analogs thereof. Such compounds may also be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. The preferred assay components for use in this embodiment is an cervical cancer marker protein identified herein, the known binding partner and/or substrate of same, and the test compound. Test compounds can be supplied from any source. [000326] The basic principle of the assay systems used to identify compounds that interfere with the interaction between the marker protein and its binding partner involves preparing a reaction mixture containing the marker protein and its binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test an agent for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the marker protein and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the marker protein and its binding partner is then detected. The formation of a complex in the control reaction, but less or no such formation in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the marker protein and its binding partner. Conversely, the formation of more complex in the presence of compound than in the control reaction indicates that the compound may enhance interaction of the marker protein and its binding partner.

[000327] The assay for compounds that interfere with the interaction of the marker protein with its binding partner may be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the marker protein or its binding partner onto a solid phase and detecting complexes anchored to the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the marker proteins and the binding partners (e.g., by competition) can be identified by conducting the reaction in the presence of the test substance, i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the marker and its interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below. [000328] hi a heterogeneous assay system, either the marker protein or its binding partner is anchored onto a solid surface or matrix, while the other corresponding non-anchored component may be labeled, either directly or indirectly. In practice, microtiter plates are often utilized for this approach. The anchored species can be immobilized by a number of methods, either non-covalent or covalent, that are typically well known to one who practices the art. Non-covalent attachment can often be accomplished simply by coating the solid surface with a solution of the marker protein or its binding partner and drying. Alternatively, an immobilized antibody specific for the assay component to be anchored can be used for this purpose. Such surfaces can often be prepared in advance and stored. [000329] hi related embodiments, a fusion protein can be provided which adds a domain that allows one or both of the assay components to be anchored to a matrix. For example, glutathione-S-transferase/marker fusion proteins or glutathione-S-transferase/binding partner can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed marker or its binding partner, and the mixture incubated under conditions conducive to complex formation (e.g., physiological conditions). Following incubation, the beads or microtiter plate wells are washed to remove any unbound assay components, the immobilized complex assessed either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of marker binding or activity determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a marker protein or a marker protein binding partner can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated marker protein or target molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the protein-immobilized surfaces can be prepared in advance and stored.

[000330] hi order to conduct the assay, the corresponding partner of the immobilized assay component is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted assay components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non- immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized component is not pre- labeled, an indirect label can be used to detect complexes anchored on the surface, e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g;, a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which modulate (inhibit or enhance) complex formation or which disrupt preformed complexes can be detected. [000331] In an alternate embodiment of the invention, a homogeneous assay may be used. This is typically a reaction, analogous to those mentioned above, which is conducted in a liquid phase in the presence or absence of the test compound. The formed complexes are then separated from unreacted components, and the amount of complex formed is determined. As mentioned for heterogeneous assay systems, the order of addition of reactants to the liquid phase can yield information about which test compounds modulate (inhibit or enhance) complex formation and which disrupt preformed complexes. [000332] hi such a homogeneous assay, the reaction products may be separated from unreacted assay components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, complexes of molecules maybe separated from uncomplexed molecules through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., Trends Biochem Sd August 1993;18(8):284- 7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the complex as compared to the uncomplexed molecules may be exploited to differentially separate the complex from the remaining individual reactants, for example through the use of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, 1998, JMoI. Recognit. 11:141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. ScL Appl, 699:499-525). Gel electrophoresis may also be employed to separate complexed molecules from unbound species (see, e.g., Ausubel et al (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York. 1999). In this technique, protein or nucleic acid - complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, nondenaturing gels in the absence of reducing agent are typically preferred, but conditions appropriate to the particular interactants will be well known to one skilled in the art. Immunoprecipitation is another common technique utilized for the isolation of a protein-protein complex from solution (see, e.g., Ausubel et al (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York. 1999). In this technique, all proteins binding to an antibody specific to one of the binding molecules are precipitated from solution by conjugating the antibody to a polymer bead that may be readily collected by centrifugation. The bound assay components are released from the beads (through a specific proteolysis event or other technique well known in the art which will not disturb the protein-protein interaction in the complex), and a second immunoprecipitation step is performed, this time utilizing antibodies specific for the correspondingly different interacting assay component. In this manner, only formed complexes should remain attached to the beads. Variations in complex formation in both the presence and the absence of a test compound can be compared, thus offering information about the ability of the compound to modulate interactions between the marker protein and its binding partner.

[000333] Also within the scope of the present invention are methods for direct detection of interactions between the marker protein and its natural binding partner and/or a test compound in a homogeneous or heterogeneous assay system without further sample manipulation. For example, the technique of fluorescence energy transfer may be utilized (see, e.g., Lakowicz et al, U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No. 4,868,103). Generally, this technique involves the addition of a fluorophore label on a first 'donor' molecule (e.g., marker or test compound) such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule (e.g., marker or test compound), which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor¹ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). A test substance which either enhances or hinders participation of one of the species in the preformed complex will result in the generation of a signal variant to that of background. In this way, test substances that modulate interactions between a marker and its binding partner can be identified in controlled assays.

[000334] In another embodiment, modulators of marker expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of marker mRNA or protein in the cell, is determined. The level of expression of marker mRNA or protein in the presence of the candidate compound is compared to the level of expression of marker mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of marker expression based on this comparison. For example, when expression of marker mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of marker mRNA or protein expression. Conversely, when expression of marker mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of marker mRNA or protein expression. The level of marker mRNA or protein expression in the cells can be determined by methods described herein for detecting marker mRNA or protein.

[000335] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell- based or a cell free assay, and the ability of the agent to modulate the activity of a marker protein can be further confirmed in vivo, e.g., in a whole animal model for cellular transformation and/or tumorigenesis.

[000336] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a marker modulating agent, an antisense marker nucleic acid molecule, a marker-specific antibody, or a marker-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[000337] It is understood that appropriate doses of small molecule agents and protein or polypeptide agents depends upon a number of factors within the knowledge of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of these agents will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the agent to have upon the nucleic acid or polypeptide of the invention. Exemplary doses of a small molecule include milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). Exemplary doses of a protein or polypeptide include gram, milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 5 grams per kilogram, about 100 micrograms per kilogram to about 500 milligrams per kilogram, or about 1 milligram per kilogram to about 50 milligrams per kilogram). It is furthermore understood that appropriate doses of one of these agents depend upon the potency of the agent with respect to the expression or activity to be modulated. Such appropriate doses can be determined using the assays described herein. When one or more of these agents is to be administered to an animal (e.g. a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[000338] Toxicity and therapeutic efficacy of the compounds or compositions can be determined by standard pharmaceutical, pharmacological, and toxicological procedures in cell cultures or experimental animals. For example, numerous methods for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population) exist. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio between LD₅₀ and ED₅₀. Compounds and compositions exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays or animal studies can be used in formulating a range of dosages for use in humans. [See, for example, Fingl et al, in The Pharmacological Basis of Therapeutics, Ch. 1 p. 1 (1975)].

[000339] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[000340] In a preferred embodiment, an anti-inflammatory agent inhibits cellular interactions between a cell expressing markers comprising stromal derived factor 1 (SDF-I), MCP-I, MIP-Ia, MlP-lft RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM- CSF, SCF, EPO, tenascin, MAdCAM-I, o4 integrins, cδ integrals, beta defensins 3 and 4 and ligands thereof, is administered to a patient. The anti-inflammatory agent can be expressed by the vector as an antibody which binds to said markers.

[000341] hi one preferred embodiment, a method of treating epithelial cell inflammation and cancer comprises an anti-inflammatory agent, such as, for example, anti-SDF agent. For example, an antibody, competitive receptor blocker, anti-inflammatory compound and the like. Delivery of the anti-SDF agent is in sunscreen lotion, lotion, cream and the like. However any anti-inflammatory agent can be used, without limitation such as antibodies specific for stromal derived factor 1 (SDF-I), MCP-I, MIP-Ia, MIP-1|3, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, oA integrins, cδ integrins, beta defensins 3 and 4. In another aspect the anti-inflammatory compounds are: 5-aminosalicylates, glucocorticoids, thioguanine derivatives, methotrexate (MTX), cyclosporine, antibiotics, and infliximab.

[000342] hi another preferred embodiment, a method of treating colon cancer comprises an anti-inflammatory agent, such as, for example, anti-SDF agent that is orally ingested in the form of a tablet, capsule, pill and the like or a suppository. However any anti-inflammatory agent can be used, without limitation such as antibodies specific for stromal derived factor 1 (SDF-I), MCP-I, MIP-Ia, MIP-IjS, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G- CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, α4 integrins, cδ integrins, beta defensins 3 and 4. hi another aspect the anti-inflammatory compounds are: 5-aminosalicylates, glucocorticoids, thioguanine derivatives, methotrexate (MTX), cyclosporine, antibiotics, and infliximab.

[000343] In another preferred embodiment, a method of treating lung cancer comprises an anti-inflammatory agent, such as, for example, anti-SDF agent that is delivered as an inhalant or aerosol. However any anti-inflammatory agent can be used, without limitation such as antibodies specific for stromal derived factor 1 (SDF-I), MCP-I, MIP-Ia, MIP-1/3, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, α4 integrins, o5 integrins, beta defensins 3 and 4. In another aspect the antiinflammatory compounds are: 5 -aminosalicylates, glucocorticoids, thioguanine derivatives, methotrexate (MTX), cyclosporine, antibiotics, and infliximab. [000344] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[000345] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[000346] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium, and then incorporating the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [000347] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

[000348] Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as niicrocrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[000349] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[000350] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and flisidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. [000351] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[000352] hi one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes having monoclonal antibodies incorporated therein or thereon) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[000353] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[000354] For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the cervical epithelium). A method for lipidation of antibodies is described by Cruikshank et al. (1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193.

[000355] The marker nucleic acid molecules can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al., 1994, Proc. Natl. Acad. Sd. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. [000356] For the compounds, methods, and uses of the present invention, any administration regimen regulating the timing and sequence of drug delivery can be used and repeated as necessary to effect treatment. Such regimen may include pretreatment and/or coadministration with additional therapeutic agents.

[000357] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Li addition, the pharmaceutical compositions of the invention may be administered in combination with standard cancer therapy, such as, but not limited to, chemotherapeutic agents and radiation therapy. The language "chemotherapeutic agent" is intended to include chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable or otherwise treat at least one resulting symptom of such a growth. Chemotherapeutic agents are well known in the art (see e.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treat neoplastic diseases. Examples of chemotherapeutic agents include: bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate, etoposide, finasteride (Proscar), flutamide (Eulexin), gemcitabine (Gemzar), goserelin acetate (Zoladex), granisetron (Kytril), irinotecan (Campto/Camptosar), ondansetron (Zofran), paclitaxel (Taxol), pegaspargase (Oncaspar), pilocarpine hydrochloride (Salagen), porfimer sodium (Photofrin), interleukin-2 (Proleukin), rituximab (Rituxan), topotecan (Hycamtin), trastuzumab (Herceptin), tretinoin (Retin-A), Triapine, vincristine, and vinorelbine tartrate (Navelbine).

[000358] Other examples of chemotherapeutic agents include alkylating drugs such as Nitrogen Mustards (e.g., Mechlorethamine (HN.sub.2), Cyclophosphamide, Ifosfamide, Melphalan (L-sarcolysin), Chlorambucil, etc.); ethylenimines, methylmelamines (e.g., Hexamethylmelamine, Thiotepa, etc.); Alkyl Sulfonates (e.g., Busulfan, etc.), Nitrosoureas (e.g., Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), Streptozocin (streptozotocin), etc.), triazenes (e.g., Decarbazine (DTIC; dimethyltriazenoimi- dazolecarboxamide)), Alkylators (e.g., cis-diamminedichloroplatinum II (CDDP)), etc. [000359] Other examples of chemotherapeutic agents include antimetabolites such as folic acid analogs (e.g., Methotrexate (arnethopterin)); pyrimidine analogs (e.g., fluorouracil ('5- fluorouracil; 5-FU); floxuridine (fluorode-oxyuridine); Fudr; Cytarabine (cyosine arabinoside), etc.); purine analogs (e.g., Mercaptopurine (6-mercaptopurine; 6-MP); Thioguanine (6-thioguanine; TG); and Pentostatin (2'-deoxycoformycin)), etc. [000360] Other examples of chemotherapeutic agents also include vinca alkaloids (e.g., Vinblastin (VLB) and Vincristine); topoisomerase inhibitors (e.g., Etoposide, Teniposide, Camptothecin, Topotecan, 9-ammo-campotothecin CPT-Il, etc.); antibiotics (e.g., Dactinomycin (actinomycin D), adriamycin, daunorubicin, doxorubicin, bleomycin, plicamycin (mithramycin), mitomycin (mitomycin C), Taxol, Taxotere, etc.); enzymes (e.g., L- Asparaginase); and biological response modifiers (e.g., interferon-; interleukin 2, etc.). Other chemotherapeutic agents include cis-diaminedichloroplatinum II (CDDP); Carboplatin; Anthracendione (e.g., Mitoxantrone); Hydroxyurea; Procarbazine (N-methylhydrazine); and adrenocortical suppressants (e.g., Mitotane, aminoglutethimide, etc.). [000361] Other chemotherapeutic agents include adrenocorticosteroids (e.g., Prednisone); progestins (e.g., Hydroxyprogesterone caproate,; Medroxyprogesterone acetate, Megestrol acetate, etc.); estrogens (e.g, diethylstilbestrol; ethenyl estradiol, etc.); antiestrogens (e.g. Tamoxifen, etc.); androgens (e.g., testosterone propionate, Fluoxymesterone, etc.); antiandrogens (e.g., Flutamide); and gonadotropin-releasing hormone analogs (e.g., Leuprolide).

[000362] The language "radiation therapy" includes the application of a genetically and somatically safe level of x-rays, both localized and non-localized, to a subject to inhibit, reduce, or prevent symptoms or conditions associated with cancer or other undesirable cell growth. The term "x-rays" includes clinically acceptable radioactive elements and isotopes thereof, as well as the radioactive emissions therefrom. Examples of the types of emissions include alpha rays, beta rays including hard betas, high energy electrons, and gamma rays. Radiation therapy is well known in the art (see e.g., Fishbach, F.. Laboratory Diagnostic Tests, 3rd Ed., Ch. 10: 581-644 (1988)), and is typically used to treat neoplastic diseases. [000363] The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. [000364] All publications and patent documents cited in this application are incorporated by reference in pertinent part for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention.

EXAMPLES Materials and Methods [000365] Patient Characteristics

[000366] Colon biopsies from two women who received therapeutic hematopoietic cell transplantation from sibling male donors were identified.

[000367] The first patient was a 55 year old Caucasian female with a history of acute myelogenous leukemia (AKL) arising from myelodysplastic syndrome (MDS) who received a human leukocyte antigen (HLA) matched allogeneic bone marrow transplant from her brother. Her parity information is unavailable. Neither the patient nor her brother had a personal history of colorectal malignancies. The patient's post-transplant course was complicated by acute graft- versus-host disease involving her skin and liver. Forty seven days post-transplant, the patient also developed diarrhea and a sigmoidoscopy was performed. Random colon biopsy found tissue with pathologic changes consistent with grade 2 graft versus host disease. In addition a 3 mm polyp was removed from the left colon, demonstrating histologic features of tubular adenocarcinoma. For her graft- versus-host disease, she was treated with high dose corticosteroids, cyclosporine and thymocyte globulin; however, she died 57 days post-transplant due to complications from acute graft-versus-host disease.

[000368] The second patient is a 28 year old Caucasian woman with a history of Hodgkin's disease (HD) who received an HLA-matched allogeneic peripheral blood stem cell transplant from her brother. The patient had a history of delivering a male child prior to transplant. In addition, she had no recognized pre-transplant personal history of colorectal malignancy. Her post-transplant course was complicated by pneumonia and acute graft- versus-host disease involving her skin and colon. Thirty days post-transplant the patient developed diarrhea and underwent sigmoidoscopy for evaluation. Random biopsy of the left colon demonstrated tissue with pathology consistent with grade 3 graft versus host disease. Additionally, a tubular adenoma was found and biopsied. The patient died of pneumonia 153 days post-transplant.

[000369] Normal colon and tubular adenomas from female and male patients who did not receive hematopoietic cell transplantation were identified as controls for immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH).

Immunohistochemistry

[000370] Zinc formalin fixed, paraffin-embedded adenoma sections were cut at 4 - 6 μm and air-dried overnight. After deparaffinization and rehydration, endogenous peroxidase activity was quenched by application of 3% hydrogen peroxide in methanol for 10 minutes at room temperature. Tissues to be stained for CD45 (leukocyte common antigen, LCA DakoCytomation, Carpinteria, CA) were antigen retrieved using Trilogy unmasking solution (Cell Marque, Hot Springs, AK). Sections stained for CK20 (cytokeratin 20, DakoCytomation, Carpinteria, CA) were sequentially retrieved with citrate buffer (DakoCytomation,Carpinteria, CA) and trypsin (Digest-all 2, Zymed laboratories, San Francisco CA). Endogenous biotin was blocked with a kit (Dako, Carpinteria, CA), and primary antibody was then applied for one hour at room temperature (1:50 for CD45 and 1:25 for CK20). Primary antibody was detected using an LSAB2-HRP kit (DakoCytomation, Carpinteria, CA) and Diaminobenzidene (DAB). Isotype- matched negative controls were run with each of the antibodies, finding no non-specific binding. An appropriate positive control slide was also stained with each staining run. [000371] For SDF-I staining, sections were stained as previously described. (See for example, Kollet O, Shivtiel S, Chen YQ, et al. HGF, SDF-I, and MMP-9 are involved in stress induced human CD34+ stem cell recruitment to the liver. J CHn Invest 2003; 112:160-9). Briefly, the adenoma blocks were cut into 5 micron sections, deparaffinized and then incubated with mouse anti-human SDF-I Ab K15C at 1:400. Overnight incubation at 4⁰C was followed by incubation with biotin-labeled rabbit anti-mouse IgG (1:100; Dako Corp.) for 30 minutes at room temperature. T he sections were then incubated with ABC complex (Vector Laboratories Inc.) and developed with DAB as substrate. They were then counterstained in hematoxylin and covered with coverslips.

FISH for X and Y Chromosomes

[000372] Slides were treated to two rounds of a five-minute incubation in Lugol's solution (Sigma, St. Louis, MO) followed by destaining in 2.5 M sodium thiocyanate. Tissue was further prepared by incubation in 0.2 N hydrochloric acid for 30 minutes at room temperature, and incubation in 1 M sodium thiocyanate for 30 minutes at 85 °C. Pretreatment concluded with a digestion in pepsin at 4 mg/mL (Sigma, St. Louis, MO) in 0.9% sodium chloride, pH 2.0 for up to 60 minutes at 37°C. Slides were next rinsed with distilled water and equilibrated in 2x saline sodium citrate (SSC). After serial dehydration in ethanol, slides were placed on the heat plate of a Hybrite oven (Vysis Inc, Downers Grove, IL). CEP probes for X and Y chromosomes (Vysis me, Downers Grove, IL) were added to the sections and coverslips were sealed over the slides with rubber cement. Tissue sections and probes were co-denatured at 75 ⁰C for 6 minutes before being hybridized overnight at 37 °C. Slides were then washed in 50% formamide in 2x SSC at 46 °C thrice for 7 minutes each, followed by 2x SSC at 46⁰C for 5 minutes, and the 4x SSC + 0.1 % Igepal (Sigma, St. Louis, MO) at 46 ⁰C for 5 minutes. Slides were air dried in the dark and then mounted with Vectashield containing 4,6-daminidino-2-phenylidole (DAPI) (Vector Laboratories, Burlingame, CA). '

Tissue Analysis

[000373] Slides were analyzed using a Leica laser scanning spectral confocal microscope (Leica Microsystems, Bannockburn, IL). DAB staining for tissue specific antigens and characteristic cellular morphology were used to specifically classify cells. Paraffin-embedded adenoma blocks were sectioned and immunohistochemically stained with specific antibodies to identify adenomatous colonocytes (cytokeratin) and leukocytes (CD45). Adenomatous colonocytes appeared elongated and large, with an epithelial orientation and staining positive with anti-cytokeratin antisera and periodic acid Schiff (PAS) staining. Leukocytes appeared small and round with positive anti-CD45 antisera staining. Y-chromosome signal was nuclear, punctate and green. X chromosome signal was similarly nuclear and punctate, but red. A total of 1000 nonoverlapping adenomatous colonocytes (usually 25 - 30 per slide) with distinct nuclei and absence of CD45 staining were examined for X- and Y-signals. Total numbers of adenomatous cells scored by sex chromosome content are presented for the controls and the two patients (Table 1). Example 1: Identification of Stem Cell Populations in Tumors [000374] Fluorescence in situ hybridization (FISH) assays for X- and Y-chromosomes identified cells of male and female origin. Y-chromosome positive cells in adenoma epithelia were found in both female patients (Fig. 1). These male epithelial cells co4ocalized with staining for cytokeratin and mucin (PAS). In addition, male epithelial cells did not express the pan- leukocyte antigen, CD45. After cytokeratin, FISH, and PAS staining, cells in the lamina propria demonstrated a ragged appearance after the extensive immunohistochemical process. However, slide preparations of adenomas just stained for CD45 and FISH for X and Y chromosomes demonstrated male leukocytes in the lamina propria of the adenomas. [000375] To enumerate male epithelial cells, a minimum of 200 cells were counted per patient (typically 3 to 5 slides per patient) (Table 1). Male adenoma cells were found in both patients receiving hematopoietic cell transplantation, with rates of incorporation ranging from 1 to 4% of total adenoma epithelial cells. There did not appear to be any clustering of male epithelial cells in any tissue section. Additionally, confocal microscopy Z stack analysis (0.25 micron steps) was performed on thin (4 - 6 microns) and thick sections (15 microns) to follow nuclei over their entire thickness to evaluate for additional X or Y chromosomes. No evidence for cell fusion (XXY or XXXY karyotype) was found in any of the sections (Fig. 2). Immunohistochemical staining for SDF-I was positive in both adenomas (Fig. 3). The staining was widespread, though patchy. The expression pattern seemed to predominate in the epithelial layers of the adenoma rather than in stroma or blood vessels of the lamina propria..

Table 1. Adenoma chromosome analysis.

[000376] The findings indicate that bone marrow derived cells can incorporate into solid tumor neoplasms of the gastrointestinal tract. As described by our group previously, these cells may represent spontaneous fusion events between the HSC progeny and a differentiated cell. However, in this study, Y chromosome bearing cells were only of the diploid XY karyotype. Confocal microscopy of donor-derived epithelial cells found no XXY or XXXY karyotype, strongly suggesting that fusion has not played a major role. It has been suggested that cells which arise as fusion products may undergo "reduction division", dividing back into diploid cells. While that might still be an explanation for these findings, it should be noted that in models where fusion events have been described, most, if not all, of the fused cells persist in the tissues, without complete "resolution." A total of 40 Y positive colonic epithelial cells were scored with no evidence of hyperdiploidy. In the liver, where cell fusion has been demonstrated in severe disease stress states, it has been postulated that 28% of donor-derived hepatocytes are due to reduction division, resulting in diploid daughter cells. Based on the probability of binomial distribution, the chance that we would find 40 out of 40 diploid donor-derived colon cells amidst abackground fusion resolution rate of 28% is one in IxIO²². Thus, direct differentiation, rather then absolutely complete and perfect resolution of every fusion event, is the most likely explanation of our current findings.

[000377] Given that adenomas were found one month after hematopoietic cell transplantation, it is likely that the patients harbored these neoplastic lesions prior to transplant. The initial oncogenic switch likely first occurred in the transplant recipients. However, it is remarkable that incorporation of donor-derived bone marrow cells was detected as soon as 30 days post- transplant. The donor-derived colon cells were predominantly found in the basal stratus of the adenoma epithelial layer, which may indicate their recent emergence from the underlying stromal layer. Only one other study investigates donor contribution to a secondary malignancy after hematopoietic cell transplantation. In this study, a child with hepatitis and severe aplastic anemia received a liver transplant followed by an allogeneic bone marrow transplant (from different donors) and subsequently developed metastatic renal cell carcinoma eight months after bone marrow transplantation. Using PCR on laser dissected tumor cells, the renal cell carcinoma showed the same ABO blood group allele as the bone marrow transplant donor. However, degree of chimerism and cell fusion were not evaluated. In contrast, we capitalized on the gender mismatch status of the recipient and donor, were able to quantify chimerism using a combination of IHC and FISH, and employed confocal microscopy to assess cell fusion events. [000378] When considering how bone marrow derived cells incorporate into colonic adenomas, two possibilities should be regarded. First, bone marrow derived cells may migrate to neoplasias due to upregulation of tumor growth factors, which also act as inflammatory cytokines and chemokines, such as SDF-I. Up- or down-regulating SDF-I has profound effects on recruiting circulating bone marrow stem/progenitor cells for engraftment as nonneoplastic, end-organ epithelia. The data showing SDF-I in both of these tumors would support its involvement in recruitment of cells to neoplasms, as well. [000379] In these post-transplant cases, another consideration is the process of GVHD of the colon where donor immune cells infiltrate recipient colonic epithelium due to alloantigen recognition. After recruitment into the colonic epithelium, either by chemokine attraction or alloantigen detection, the bone marrow cells seem to have undergone naturalization through subjection to local chemical and physical factors influencing differentiation, morphology, and cell-cell junctions. A second consideration is that recipient adenoma cells may have been engulfed by roving donor-derived bone marrow cells. An apoptotic adenoma cell could have been phagocytosed by a donor scavenger cell. In this situation the fusion of nuclear material should result in a multiplicity of sex chromosomes (XXY or XXXY). However, as noted, the donor adenoma cells in this study demonstrated no extra X chromosomes. [000380] These results have strong implications for oncology, as well as stem cell biology. The findings, herein, demonstrating that human bone marrow cells have the ability to incorporate into human adenomas suggest that a transplantable hematopoietic cell responds to tumoral growth cues, migrates into the neoplastic environment, and activates tissue-specific differentiative genetic prograrnming. If this process is present for epithelial tissues in other organs as well, it may answer long-standing questions raised by clonality studies designed to determine if benign tumors are neoplastic or hyperplastic. In many such studies, (e.g., thyroid and parathyroid adenomas), tumors expected to be neoplastic and, therefore, monoclonal, have often been found to be polyclonal. This paradoxical result has remained an unresolved source of diagnostic confusion. Our current findings may suggest an explanation for these inconsistencies.

[000381] The physiologic role, if any, of circulating cell engraftment into a developing tumor is of course an important question. One may speculate that the incorporation of a non-mutated cell, displaying normal "self antigens, might interact with the immune system in some beneficial fashion. Given that these two, small adenomas are early lesions in the colonic neoplasia sequence, one might expect that a "normal" functioning of benign epithelium, such as up-regulation of SDF-I , may be better maintained than in overtly malignant and more poorly differentiated neoplasms. Both of these possibilities are amenable to investigation in animal models of neoplasia. Such studies, beginning with investigation of the kinetics of engraftment as tumors grow, are currently underway. Given the incorporation potential of these cells, our findings also suggest that human bone marrow cells may have the potential to serve as a therapeutic source of readily harvestable cells for targeting malignant environments. These cells might then, for example, be useful as vectors for gene therapy of tumors. Example 2: Bone Marrow Can Be A Primary Source Of Cancer

[000382] Bone marrow derived cells (BMDCs) can remodel remote tissues. In particular, transplantation studies in animals and humans have demonstrated that BMDCs contribute to epithelial layers of a variety of tissues such as the gastrointestinal tract. The origin of these epithelial cells could be from a single hematopoietic stem cell (HSC), though other contributing marrow-derived cells may also play a role in this process. Moreover, BMDC engraftment in the gastrointestinal tract occurs by direct differentiation without evidence of cell fusion in mice. The ability of bone marrow cells to remodel distant organs occurs at very low levels in everyday physiology. However, contribution from marrow is enhanced in settings of injury or disease, which is likely related to the homing effects of inflammation. [000383] Although epithelial cancers are believed to arise from tissue-resident stem cells, a recent murine study demonstrated that BMDCs may contribute to cancer arising from the stomach lining (Houghton, J., et al. (2004). Science 306, 1568-1571). Transplantation studies performed in mice with chronic gastritis due to bacterial infection show that resultant gastric carcinomas were derived from marrow origin.

[000384] Several crucial questions about BMDC contribution to cancer abound, such as whether BMDCs can be a source of epithelial cancers in general, which cell in the bone marrow is the primary source of epithelial cancer, what inflammatory cues are important in BMDC homing to sites of cancer development, and whether BMDC contribution to cancer occurs in humans, bringing into question the clinical relevancy.

[000385] We investigated the cellular origin of cancers found post-transplant in women having received hematopoietic cell transplantation. Specifically, cases of colonic adenoma, basal cell skin carcinoma, and squamous cell carcinoma of the lung were found post- transplant. Given the findings of BMDC plasticity and contribution to cancer in settings of chronic inflammation, we questioned whether second cancers after hematopoietic cell transplantation are from donor bone marrow origin. Here we show, using a combination of _ immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) analyzed with confocal microscopy, that second cancers after hematopoietic cell transplantation in humans can originate from donor bone marrow origin, without evidence for cell-cell fusion. [000386] Furthermore, we use APC mutant mice to demonstrate that BMDCs can also contribute to neoplasias arising in the small bowel and colon. Given the plasticity potential of the hematopoietic stem cell (HSC) we further questioned whether this acts as a primary source of epithelial cancer. Here we used a lung cancer model employing single and secondary HSC transplantation to demonstrate that lung cancer can arise from donor HSC. [000387] Because the chemokine, stromal derived factor 1 (SDF-I), is a potent chemoattractant of hematopoietic stem cells and is widely expressed in many tissues during development and injury we hypothesized that SDF-I chemoattraction may be at least partly responsible for the homing and migration of BMDCs into these neoplastic environments. Here, we show that, indeed, SDF-I is upregulated in the epithelial cancers where BMDC incorporation is found.

[000388] Tissue analysis: Slides were analyzed using a Leica laser scanning spectral confocal microscope (Leica Microsystems, Bannockburn, IL). DAB staining for tissue specific antigens and characteristic cellular morphology were used to specifically classify cells. Paraffin-embedded adenoma blocks were sectioned and immunohistochemically stained with specific antibodies to identify epithelial neoplastic tissues (cytokeratin) and leukocytes (CD45). Basal cell skin cancer appeared in rests below the epidermis. Neoplastic adenoma cells appeared elongated and large, with an epithelial orientation and staining positive with anti-cytokeratin antisera and periodic acid Schiff (PAS) staining. Leukocytes appeared small and round with positive anti-CD45 antisera staining. Y-chromosome signal was nuclear, punctate and green. X chromosome signal was similarly nuclear and punctate, but red. A total of 1000 nonoverlapping neoplastic epithelial cells (usually 25 - 30 per slide) with distinct nuclei and absence of CD45 staining were examined in each case for X- and Y- signals.

Table 2. Patient Characteristics

[000389] Bone Marrow Contributes to Colonic Adenoma in Humans: Two women were found to have neoplasias involving their colon (Table 2). The colorectal adenomas were found shortly after hematopoietic cell transplantation and also demonstrated donor marrow incorporation. The donor cells lost their hematopoietic surface protein, CD45 (leukocyte common antigen), and adopted surface protein expression typical of surrounding colonic epithelial cells (cytokeratin and mucin) (Figure 1A-1F). Transdifferentiated BMDCs were predominately located in the basal strata of adenoma epithelia, suggesting recent immigration. In total, over 1000 adenoma epithelial cells were evaluated, demonstrating 1 - 4% of epithelial cells per patient originating from donor BMDCs. Confocal microscopy performed to enumerate X and Y chromosomes within each donor cell nucleus showed no evidence of a fusion sex chromosome karyotype (XXY or XXXY) (Figure 2). [000390] Bone Marrow As a Primary Source of Skin Cancer in Humans: Of the three women, one women developed basal cell skin carcinoma of the forehead prior to and after hematopoietic cell transplantation (Table 2). She is a multiparous women and was found to have a basal cell carcinoma of strictly female origin prior to transplantation (Figure 1 A-IF). For her acute myelogenous leukemia she received allogeneic hematopoietic cell transplantation from her brother. Her post-transplant course was complicated by acute and chronic graft- versus-host-disease of the skin. Approximately four years after transplant she developed another basal carcinoma involving the forehead. Biopsy of the lesion post- transplant demonstrates that the cancer is of 100% male origin (Figure IA- IF). [000391] We hypothesized that the powerful chemoattractant, stromal derived factor 1 (SDF-I), may be at least partly responsible for the homing and migration of bone marrow cells to the neoplastic environment . To test this hypothesis, adenomas were immunohistochemically stained for SDF-I and demonstrated intense expression, which was predominately located in the epithelial layers of the adenomas rather than in stroma or blood vessels of the lamina propria (Figure 4). These results suggest that SDF-I is an important signaling molecule in the recruitment of circulating marrow stem/progenitor cells into sites of epithelial neoplasia. In the neoplastic adenoma environment, SDF-I may be liberated as a signal of cellular distress. BMDCs, endowed with potent proliferative and differentiation capacities, may then respond to this injury cue, migrate into the epithelium, and then through influence of other local chemical and physical factors undergo colonic epithelial transdifferentiation.

[000392] hi each case, graft- versus-host disease of the target tissue was present prior to neoplasia development. The risk of developing a new cancer after bone marrow transplantation is estimated to be up to eight times higher than aged-matched controls. Predisposing risk factors such as radiation, chemotherapy, and use of immunosuppressants have been recognized. However, another factor in the post-transplant setting may be that multipotent donor marrow cells incorporate into inflamed epithelia such as the skin and gut, and then undergo pathologic plasticity changes resulting in a new cancer. [000393] A final consideration is that recipient neoplastic cells may have been engulfed by donor-derived bone marrow cells, such as monocytes or macrophages. In this situation the fusion of nuclear material should result in a multiplicity of sex chromosomes (XXY or XXXY). However, as noted, the donor neoplastic cells of all three women in this study demonstrated no extra X chromosomes. It has been suggested that cells which arise as fusion products may undergo "reduction division", dividing back into diploid cells. While that might still be an explanation for these findings, it should be noted that in models where fusion events have been described, most, if not all, of the fused cells persist in the tissues, without complete "resolution." We scored a total of 40 Y positive colonic epithelial cells with no evidence of hyperdiploidy. In the liver, where cell fusion has been demonstrated in severe disease stress states, it has been postulated that 28% of donor-derived hepatocytes are due to reduction division, resulting in diploid daughter cells. Based on the probability of binomial distribution, the chance that we would find 40 out of 40 diploid donor-derived colon cells amidst a background fusion resolution rate of 28% is one in IxIO²². Thus, direct differentiation, rather then absolutely complete and perfect resolution of every fusion event is the most likely explanation of our current findings.

[000394] These results have strong implications for oncology, as well as stem cell biology. Our findings - demonstrating that human bone marrow cells can be a source of neoplasia - suggest that a transplantable hematopoietic cell responds to inflammatory cues (such as SDF- 1), activates tissue-specific differentiative genetic programming, and is susceptible to further neoplastic changes. Future studies are aimed at studying other epithelial cancers (e.g., lung, head and neck, esophageal, gastric, cervical) to determine what threshold of mitogen conditions (e.g., infection, inflammation, trauma) are necessary and sufficient to induce (1) hematopoietic cell homing and migration, and (2) neoplastic differentiation. However, in the meantime it appears that human BMDCs much like their murine counterparts may play a role in epithelial cancer growth and development.

Other Embodiments

[000395] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims

Claims

What is claimed is:

1. A composition comprising: isolated and purified cells, a vector encoding an anti-tumor molecule, wherein the isolated cells are transformed with the vector.

2. The composition of claim 1, wherein the isolated cell is a stem cell.

3. The composition of claim 1 , wherein the vector further comprises oligonucleotides encoding ligands specific for cell surface antigens.

4. The composition of claim 3, wherein the isolated cell expresses the ligands on its surface.

5. The composition of claim 1, wherein the isolated stem cell expresses molecules cytolytic for tumors.

6. The composition of claim 1 , wherein the isolated stem cell is targeted to a specific tumor location in vivo.

7. The composition of claim 1, wherein the isolated cell is used to provide therapy to a patient suffering from or susceptible to tumors.

8. A method for tracking cells in vivo, the method comprising the steps of:

(a) isolating and purifying stem cells from a subject;

(b) providing a chemically heteroconjugated bispecific antibody with a binding site specific for a stem cell antigen and a binding site specific for a target antigen in a patient; and,

(c) arming the stem cells with the bispecific antibody under conditions wherein;

(i) the bispecific antibody binds to the stem cells via the c-kit ligand; and

(ii) the second antigenic binding site of the bispecific antibody is free to bind to the target antigen; and

(iii) binding of a labeled antibody to the Fc region of the bispecific antibody; or (iv) fluorescently labeling the bispecific antibody whereby a secondary labeled antibody is not required; and

(d) reinfusing the armed and labeled stem cells into a patient;

(e) tracking the armed and labeled stem cells by extracting blood and/or tissue samples from the patient at different time intervals;

(f) identifying the armed and labeled cells by phenotyping the cells using flow cytometry cell sorting; and

(g) identifying the armed and labeled cells by immunohistochemical staining to detect the primary antibody on the cells in target tissues.

9. The method of claim 8, wherein the bispecific antibody is specific for c-kit ligand of stem cells and tumor antigens.

10. The method of claim 8, wherein the bispecific antibody is directly fluorescently labeled.

11. The method of claim 8, wherein the armed and labeled stem cells home to, and bind to the target tissue antigens.

12. The method of claim 8, wherein the stem cells accumulate at the target antigen site.

13. The method of claim 8, wherein samples are taken from a patient at different time intervals after reinfusion of the stem cells to track the location of the armed and labeled cells.

14. The method of claim 8, wherein the numbers of armed and labeled cells at a particular time interval and/or in vivo location are quantitatively assessed by comparing the number of armed and labeled cells that were reinfused with the number of armed and labeled cells present in a sample at the particular time interval and/or in vivo location by phenotyping the cellular population using flow cytometry. 15. The method of claim 8, wherein blood samples and target tissue samples taken from a patient at a particular time interval and quantitatively assessed using flow cytometry, is indicative of in vivo homing progress of armed and labeled stem cells to target tissues.

16. A method of treating a patient suffering from cancer, comprising the steps of:

(a) isolating cells from a patient suffering from cancer and/or from HLA- matched or partially HLA-matched subject;

(b) modifying a stem cell by arming the cell with an antibody or introducing a vector encoding a desired molecule; and

(c) reinfusing the stem cells into a patient.

17. The method of claim 16, wherein the isolated cells are bone marrow cells.

18. The method of claim 16, wherein the isolated cells are hematopoietic stem cells.

19. The method of claim 16, wherein the isolated cells are erythroid stem cells.

20. The method of claim 16, wherein the isolated cells are cells of the immune system.

21. The method of claim 16, wherein the antibody binds to a specific ligand of a desired cell.

23. A method of treating a patient suffering from cancer, comprising the steps of:

(b) modifying a stem cell by arming the cell with an antibody or introducing a vector encoding a desired molecule;

(c) reinfusing the stem cells into a patient; and

(d) administering an anti-inflammatory compound, thereby, treating a patient suffering from cancer.

- Ill - 24. The method of claim 23, wherein the vector expresses ant-inflammatory agents that inhibit cell migration to the tumor.

25. The method of claim 24, wherein the anti-inflammatory agents are directed to stromal derived factor 1 (SDF-I), MCP-I, MIP-Ia, MIP-IjS, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, α4 integrins, oδ integrins, beta defensins 3 and 4.

26. The method of claim 23, wherein the anti-inflammatory compounds are administered, prior, in conjunction with, and/or after the stem cells.

27. The method of claim 23, wherein the anti-inflammatory compounds are antibodies specific for stromal derived factor 1 (SDF-I), MCP-I, MIP- lα, MIP- IjS, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, α4 integrins, oδ integrins, beta defensins 3 and 4.

28. The method of claim 23, wherein the anti-inflammatory compounds are: 5- aminosalicylates, glucocorticoids, thioguanine derivatives, methotrexate (MTX), cyclosporine, antibiotics, and infliximab.

29. The method of claim 23, wherein the chronic inflammation is caused by bacterial, viral or parasitic infection.

30. The method of claim 23, wherein the chronic inflammation is caused by exposure to a carcinogen.

31. A method of treating a chronic inflammatory condition comprising administering to a patient having a chronic inflammatory condition stem cells expressing at least one protein which is an anti-inflammatory agent.

32. The method of claim 31 , comprising the steps of: (a) obtaining stem cells from the patient; (b) transfecting, infecting or transducing the stem cells with an antiinflammatory agent; and

(c) administering the transfected, infected or transduced stem cells expressing the anti-inflammatory nucleic acid to the patient in an amount sufficient to treat the chronic inflammatory condition.

33. The method of claim 31, wherein the anti-inflammatory agent inhibits cell migration to the inflammatory in vivo site.

34. The method of claim 31 , wherein the nucleic acid expressing an antiinflammatory agent inhibits cellular interactions between a cell expressing markers comprising stromal derived factor 1 (SDF-I), MCP-I, MIP- lα, MIP- 1/3, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, aA integrins, cδ integrins, beta defensins 3 and 4 and ligands thereof.

35. The method of claim 31 , wherein the anti-inflammatory agent expressed by the vector is an antibody which binds to said markers.