WO2012005763A1 - Utilisation de populations de cellules progénitrices de type myéloïde pour traiter des tumeurs - Google Patents

Utilisation de populations de cellules progénitrices de type myéloïde pour traiter des tumeurs Download PDF

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WO2012005763A1
WO2012005763A1 PCT/US2011/001183 US2011001183W WO2012005763A1 WO 2012005763 A1 WO2012005763 A1 WO 2012005763A1 US 2011001183 W US2011001183 W US 2011001183W WO 2012005763 A1 WO2012005763 A1 WO 2012005763A1
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cells
tumor
subject
myeloid
human
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Faith Barnett
Martin Friedlander
Valentina Marchetti
Lea Scheppke
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The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/47—Brain; Nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma

Definitions

  • the present invention generally relates to methods and compositions for inhibiting tumor growth and treating cancer.
  • a tumor is an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, also called a neoplasm.
  • Tumors may be either benign (not cancerous) or malignant (i.e., cancer).
  • Various types of solid tumors can become cancerous which often lead to death.
  • brain tumors are an especially deadly form of cancer.
  • Brain tumors can be present either in the brain itself (neurons, glial cells (astrocytes, oligodendrocytes, ependymal cells), lymphatic tissue, blood vessels), in the cranial nerves (myelin-producing Schwann cells), in the brain envelopes (meninges), skull, pituitary and pineal gland, or spread from cancers primarily located in other organs (metastatic tumors).
  • glioma The most common type of brain tumor is glioma.
  • Gliomas are neuroectodermal tumors of neuroglial origin, and include astrocytoma, oligodendroglioma and ependymoma which are derived from astrocytes, oligodendrocytes and ependymal cells respectively.
  • tumor metastasis often results in recurrence of cancer in the form of the formation and growth of at least one additional or second tumor.
  • Radiotherapeutic and chemotherapeutic agents can be toxic to normal tissues at the dose levels administered, and often create life-threatening side effects in the patient.
  • These cancer therapies can often have high failure/remission rates which can result in death of the patient.
  • the present invention provides methods of treating a tumor in a subject.
  • the methods entail administering to the subject suffering from a tumor an effective amount of a population of myeloid-like progenitor cells.
  • the myeloid-like progenitor cells are isolated from the subject to be treated.
  • the subject to be treated is a human.
  • a human subject afflicted with a solid tumor can be treated with a population of human CD14 + cells or human monocytes.
  • the human subject can be treated with human CD14 + cells isolated from a human blood sample of the subject or CD33 HI monocytes isolated from bone marrow of the subject.
  • the cells in the employed population of human cells express the indicated antigen (e.g., CD14 for the blood derived cells and/or CD33 for the bone marrow derived cells).
  • the therapeutic cell population is administered via central injection to the subject.
  • the cells are stimulated with the indicated antigen (e.g., CD14 for the blood derived cells and/or CD33 for the bone marrow derived cells).
  • LPS lipopolysaccharides
  • Some preferred methods of the invention are directed to treating brain tumors including, e.g., glioma, glioblastoma, ocular melanoma or a metastatic brain tumor. Some of the methods further comprise treating the tumor with sublethal radiation therapy prior to administration of the therapeutic cells.
  • the invention provides other methods for treating a solid tumor in a subject.
  • the methods involve administering to the subject afflicted with a solid tumor an effective amount of a population of myeloid-like progenitor cells which have been transfected or transduced with a gene that operably encodes an anti-tumor agent.
  • the myeloid-like progenitor cells are human CD14 + cells or rodent CD44 HI cells.
  • the administered myeloid-like progenitor cells are monocytes (e.g., human CD33 HI monocytes isolated from bone marrow).
  • the anti-tumor agent can be an antiangiogenic agent, a cytotoxic agent or a tumor suppressor.
  • the gene encoding the anti-tumor agent is transfected into the cells via an expression vector.
  • the therapeutic cells are transduced with a lentiviral expression vector.
  • the engineered cells are injected centrally to the subject.
  • the subject to be treated is a human, and the myeloid-like progenitor cells are human CD14 + cells isolated from the subject.
  • the invention provides methods for delivering an anti-tumor agent to a solid tumor in a subject. These methods entail constructing a vector expressing the anti-tumor agent, transfecting or transducing the vector into a population of myeloid-like progenitor cells, and admimstering the transfected or transduced cells to the subject. Some of these methods employ myeloid-like progenitor cells which are human CD14 + cells or rodent CD44 HI cells. Some other methods employ myeloid-like progenitor cells which are human monocytes (e.g., CD33 HI monocytes isolated from bone marrow). Preferably, the cells are isolated from the subject afflicted with the solid tumor.
  • the anti-tumor agent to be delivered with the methods can be an antiangiogenic agent, a cytotoxic agent or a tumor suppressor.
  • the vector for expressing the anti-tumor agent is a lentiviral vector.
  • the transfected or transduced cells are injected centrally to the tumor in the subject.
  • Figure 1 shows that rodent Lin " myeloid-like cells migrate to tumor.
  • Figure 2 shows that mouse CD44 HI in combination with CD44 LO cells selectively kill sublethally irradiated tumor cells in culture.
  • Figure 3 shows presence of lentivirus infected Lin " cells in 21 day tumor. The image was taken 16 days post-transduction of the cells with the vector and 15 days post- injection.
  • FIGs 4A-4C show that human cord blood CD14 + cells transduced with a GFP- expressing adenoviral vector (Ad5 F16 eGFP) colocalized to 9L tumor cells in culture (4 A), reduced growth of tumor cells (4B), and down-regulated expression of proliferating cell nuclear antigen (PCNA) (4C). Shown in Fig. 4C is a reduction in PCNA expression (by implication, a decrease in DNA synthesis) resulted from addition of CD 14 cells in tumor group exposed to lOgy, but not 7.5gy.
  • a GFP- expressing adenoviral vector Ad5 F16 eGFP
  • PCNA proliferating cell nuclear antigen
  • Figure 5 shows that Ad5 F16 eGFP transduced human CD14 + cells, when pre- stimulated with LPS, killed lOgy irradiated 9L tumor cells in vitro.
  • Figure 6 shows that Ad5 F16 eGFP transduced human CD14 + cells, when pre- stimulated with LPS, secreted high levels of pro inflammatory cytokines.
  • Figure 7 shows presence in SCID mice of Ad5 F16 eGFP transduced human CD14 + cells co-injected with tumor cells. Shown in the figure are eGFP cells 7 days after injections.
  • Figure 8 shows anti-tumor activities of different cell populations from bone marrow.
  • Figure 9 shows that different ratio of tumor cells to monocytes led to differing antitumor activities.
  • Figure 10 shows that both CD54 positive and CD54 negative lymphocytes, as well as the granulocyte population, are negative effector cells.
  • Figure 11 shows results of treatment of implanted gliosarcoma in rats with un- fractionated bone marrow cells.
  • Figure 12 shows in vivo anti-tumor activities of CD44 hi and CD44 low cell populations against melanoma in mice.
  • Figure 13 shows FACS profile of cell populations isolated from human bone marrow.
  • the present invention relates to methods of killing tumor cells, inhibiting tumor growth and treating cancers with a population of isolated or engineered myeloid-like progenitor cells.
  • the invention is predicated in part on the discovery of the present inventors that myeloid-like progenitor cell populations such as human CD14 + cells or rodent Lin- myeloid like cells are capable of both tumor-homing and/or tumor-killing.
  • myeloid-like progenitor cell populations such as human CD14 + cells or rodent Lin- myeloid like cells are capable of both tumor-homing and/or tumor-killing.
  • bone marrow derived rodent Lin- progenitor cells upon injection into mice brains, can localize and proliferate within brain tumors in the mice.
  • the observed tumor-homing activity of the Lin- cells is primarily attributable to a CD44 HI subset of cells.
  • CD44 LO cells generally do not localize and home to the tumors.
  • the CD44 HI cells under appropriate conditions can exhibit anti-tumor activity. It was found that they were able to mediate phagocytosis or killing of various tumors that have been sublethally irradiated.
  • rodent myeloid-like progenitor cells a population of blood derived human CD14 + cells was shown to be able to target and kill tumor cells in vitro. In vivo, the human cells were similarly able to localize to injected tumor cells.
  • the inventors further determined that monocytes, not lymphocytes or
  • granulocytes, from rodent bone marrow are the effector cell population with anti-tumor activities, and were able to isolate pure populations of the effector cells.
  • the inventors observed profound in vivo anti-glioma activity of isolated rat monocytes against syngeneic 9L glioma cells. Also, anti-melanoma activity was observed with mouse bone marrow cell populations. Similarly fractionated cell populations were also obtained from human bone marrow.
  • the inventors observed in vivo activity of whole bone marrow in reducing tumor size and improving survival.
  • sublethal tumor cell irradiation is not necessary to elicit anti-tumor activity of the effector cells. The sublethal irradiation was used to promote the activation of the immune cells.
  • the effector cells exhibited potent anti-tumor activity without sublethal irradiation of the tumor cells.
  • the present invention provides methods of using myeloid-like progenitor cell populations (e.g., human CD14 + cells from blood or human CD33 HI monocytes from bone marrow) to treat various types of tumors, e.g., brain tumors.
  • the cells can be used directly to kill tumor cells, e.g., tumors that have been subject to sub-lethal UV or radiation treatment or other types of sub-lethal stressors.
  • the isolated cells can also be engineered to express an anti-tumor agent such as an antiangiogenic agent or a cytotoxic polypeptide (e.g., by using an adenoviral or adeno-associated viral vector).
  • the engineered myeloid-like progenitor cells can be used to specifically deliver therapeutic agents to tumors and to treat tumors in subjects in need of treatment.
  • Cells engineered to stably express an anti-tumor agent e.g., via a lentivirus vector that integrates into the genome of the cells
  • the isolated or engineered myeloid-like progenitor cells are highly migratory. They can target tumors at the site of administration (e.g., site of local injection) as well as at distant tumor satellites.
  • the cells can be introduced centrally or locally into the subject, e.g., at tumor resection cavity or through an implanted catheter. Because the cells specifically target to tumors, toxicity is greatly reduced since normal cells are spared. In addition, when the cells are autologous to the subject to be treated, there is also reduced risk of any undesirable immune response.
  • Hematopoietic stem cells are stem cells that are capable of developing into various blood cell types e.g., B cells, T cells, granulocytes, platelets, and erythrocytes.
  • the lineage surface antigens are a group of cell-surface proteins that are markers of mature blood cell lineages, including CD2, CD3, CD1 1, CD1 la, Mac-1
  • CDl lb:CD18 CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, CD45RA, murine Ly-6G, murine TER-119, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leukocyte antigen DR (HLA-DR), and CD235a (Glycophorin A).
  • Hematopoietic stem cells that do not express significant levels of these antigens are commonly referred to as lineage negative (Lin .).
  • Human hematopoietic stem cells commonly express other surface antigens such as CD31 , CD34, CD117 (c-kit) and/or CD133.
  • Murine hematopoietic stem cells commonly express other surface antigens such as CD34, CD117 (c-kit), Thy-1, and/or Sca-1.
  • the cells that circulate in the bloodstream are generally divided into three types: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets or thrombocytes.
  • Leukocytes include granulocytes (polymorphonuclear leukocytes) and agranulocytes (mononuclear leucocytes).
  • Granulocytes are leukocytes characterized by the presence of differently staining granules in their cytoplasm when viewed under light microscopy. There are three types of granulocytes: neutrophils, basophils, and eosinophils.
  • Agranulocytes are leukocytes characterized by the apparent absence of granules in their cytoplasm. Although the name implies a lack of granules, these cells do contain non-specific azurophilic granules, which are lysosomes. Agranulocytes include
  • lymphocytes lymphocytes, monocytes, and macrophages.
  • Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. They constitute between three to eight percent of the leukocytes in the blood. In the tissues monocytes mature into different types of macrophages at different anatomical locations. Monocytes have two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (aprox. 8-12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Monocytes are usually identified in stained smears by their large bilobate nucleus.
  • myeloid-like progenitor cells refers to a population of cells which is isolated from rodent (e.g., mouse or rat) bone marrow or blood and contains a majority of cells that express CD44 antigen (CD44 HI cells). It also encompasses human analogue to rodent CD44 HI cells, e.g., human CD14 + cells which can be isolated from human blood (e.g., cord blood or peripheral blood) or bone marrow. The term also includes monocyte populations isolated from bone marrow (e.g., human CD33 HI monocytes). Typically, the rodent CD44 HI cell population contains at least about 50 percent of cells that express CD44.
  • the cell population typically contains at least about 50% cells that express CD 14 or CD33, with preferably at least about 75%, 85%, 90%, 95% or 99% of the cells expressing the antigen.
  • the CD44 HI rodent myeloid-like progenitor cells of the invention are also defined by being lineage negative (Lin ) in addition to expressing CD44.
  • lineage negative bone marrow cells can be divided into two subpopulations: CD44 H1 cells and CD44 LO cells.
  • the CD44 HI cells are primarily myeloid in origin while the CD44 LO cells are largely lymphoid in origin (see, e.g., Ritter et al., J. Clin. Invest. 116:3266-76, 2006).
  • the rodent myeloid-like progenitor cells used in the practice of the invention is a population of CD44 HI cells.
  • the Lin " CD44 + myeloid-like cells is used herein
  • myeloid-like progenitor cells are isolated from bone marrow, they are also termed myeloid- like bone marrow (MLBM) cells.
  • MLBM myeloid- like bone marrow
  • the bone marrow derived myeloid-like progenitor cells include monocytes.
  • the myeloid-like progenitor cells to be used in the practice of the invention can be isolated in accordance with methods that have been known in the art (Ritter et al., J. Clin. Invest. 116:3266-76, 2006) or the procedures described in the Examples below for isolating human CD14 + cells.
  • cells from a sample of bone marrow, peripheral blood or umbilical cord blood can be first selected to isolate lineage negative cells. This can be accomplished by negative selection with antibodies against one or more of the lineage markers described above.
  • the isolated Lin " cells can then be further subject to positive selection for CD44 + cells. This can be performed with an antibody specific for CD44 (hyaluronic acid receptor).
  • the Lin " CD44 + cells may be further selected for expression of other surface markers that are indicative of myeloid origin.
  • the various selection steps may also be performed by other means commonly practiced in the art, e.g., flow cytometry.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant (i.e., cancer) or benign, and all pre-cancerous and cancerous cells and tissues.
  • Solid tumors are tumors which have a physical swelling or lesion formed by an abnormal growth of cells. Examples of solid tumors include brain tumor, melanoma, breast cancer, lung cancer, liver cancer, colon cancer, renal cancer, pancreatic cancer, esophageal cancer, bladder cancer, prostate cancer, head and neck cancer, gastric cancer, and ovarian cancer.
  • a brain tumor refers to any tumor that grows in the brain, which can be cancerous or non-cancerous (benign). It is defined as any intracranial tumor created by abnormal and uncontrolled cell division that normally presents in the brain itself (neurons, glial cells (astrocytes, oligodendrocytes, ependymal cells), lymphatic tissue, blood vessels), in the cranial nerves (myelin-producing Schwann cells), in the brain envelopes (meninges), skull, pituitary and pineal gland, or that spreads from cancers primarily located in other organs (metastatic tumors).
  • brain tumors are commonly located in the posterior cranial fossa in children and in the anterior two-thirds of the cerebral hemispheres in adults, although they can affect any part of the brain.
  • Examples of brain tumors include, but are not limited to, glioma, glioblastoma, medulloblastoma, astrocytoma, and other primitive neuroectoderma.
  • Retroviruses are enveloped viruses that belong to the viral family Retroviridae.
  • the virus itself stores its nucleic acid, in the form of a +mRNA (including the 5 '-cap and 3'- PolyA inside the virion) genome and serves as a means of delivery of that genome into host cells it targets as an obligate parasite, and constitutes the infection.
  • the virus replicates by using a viral reverse transcriptase enzyme to transcribe its RNA into DNA.
  • the DNA is then integrated into the host's genome by an integrase enzyme.
  • the retroviral DNA replicates as part of the host genome, and is referred to as a provirus.
  • Retroviruses include the genus of Alpharetrovirus (e.g., avian leukosis virus), the genus of Betaretrovirus; (e.g., mouse mammary tumor virus), the genus of Gammaretrovirus (e.g., murine leukemia virus or MLV), the genus of Deltaretrovirus (e.g., bovine leukemia virus and human T-lymphotropic virus), the genus of Epsilonretrovirus (e.g., Walleye dermal sarcoma virus), and the genus of Lentivirus.
  • Alpharetrovirus e.g., avian leukosis virus
  • Betaretrovirus e.g., mouse mammary tumor virus
  • Gammaretrovirus e.g., murine leukemia virus or MLV
  • Deltaretrovirus e.g., bovine leukemia virus and human T-lymphotropic virus
  • Epsilonretrovirus e.g., Walleye dermal
  • Lentivirus is a genus of viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector.
  • lentiviruses include human immunodeficiency viruses (HIV- 1 and HIV-2), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Additional examples include BLV, EIAV and CEV.
  • operably linked when referring to a polynucleotide, means a linkage of polynucleotide elements in a functional relationship.
  • a polynucleotide element is "operably linked” to another polynucleotide element when the two are placed into a functional relationship.
  • a promoter or enhancer is operably linked to a coding sequence if it controls or affects the transcription of the coding sequence.
  • Operably linked means that the linked polynucleotide elements are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • subject and “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals.
  • Animals include all vertebrates, e.g., mammals and non-mammals, such as dogs, cats, sheeps, cows, pigs, rabbits, chickens, and etc.
  • Preferred subjects for practicing the therapeutic methods of the present invention are human.
  • Subjects in need of treatment include patients already suffering from a cancer or a tumor, as well as those who are at risk of or prone to developing such a condition or disease.
  • Sublethal irradiation refers to radiation at a dose level that, when applied to a cell (e.g., a tumor cell), does not cause the cell to die but instead allows the cell to repair damages itself.
  • a dose range from 0.25 to 2 sievert may be considered sublethal.
  • a sublethal dose may also be expressed as an absorbed dose measured in gray (Gy). For example, an absorbed dose of less than 8 Gy, less than 6 Gy, or less than 5 Gy may be considered sublethal.
  • the exact dose level for a specific subject should be determined based on many other factors, like age, sex, health etc.
  • substantially pure or substantially purity when referring to an isolated cell population means the percentage of a given cell (target cell) in the population is significantly higher than that found in a natural environment (e.g., in a tissue or a blood stream of a subject).
  • percentage of the target cell (e.g., Lin " and/or CD44 + cells) in a substantially pure cell population is at least about 50%, preferably at least about 60%, 70%, 75%, and more preferably at least about 80%, 85%, 90% or 95% of total cells in the cell population.
  • treating includes (i) preventing a pathologic condition (e.g., tumor) from occurring (e.g. prophylaxis); (ii) inhibiting the pathologic condition (e.g., tumor growth) or arresting its development; and (iii) relieving symptoms associated with the pathologic condition.
  • pathologic condition e.g., tumor
  • treatment includes the administration of a cell population of the invention and/or other therapeutic compositions or agents to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease described herein, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • Treatment further refers to any indicia of success in the treatment or amelioration or prevention of tumor growth or cancer development, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient.
  • Detailed procedures for the treatment or amelioration of a tumor or symptoms thereof can be based on objective or subjective parameters, including the results of an examination by a physician.
  • transfection refers to the introduction of foreign DNA into cells.
  • a cell has been "transformed” or “transfected” by exogenous or heterologous polynucleotide when such polynucleotide has been introduced inside the cell.
  • Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co- precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment) and the like.
  • transduction When a viral vector is introduced into a bacterial host cell, the process is termed "transduction.”
  • stable transfection or "stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell.
  • stable transfectant refers to a cell that has stably integrated foreign DNA into the genomic DNA.
  • transient transfection or “transiently transfected” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell.
  • the foreign DNA persists in the nucleus of the transfected cell for a period of time (e.g., several days).
  • transient transfectant refers to cells that have taken up foreign DNA but have failed to integrate this DNA.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment.
  • Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as "expression vectors".
  • a retrovirus e.g., a lentivirus
  • a vector derived from the virus means that genome of the vector comprises components from the virus as a backbone.
  • the viral particle generated from the vector as a whole contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Usually these will include the gag and pol proteins derived from the virus.
  • the viral particles are capable of infecting and transducing non-dividing cells.
  • Recombinant retroviral particles are able to deliver a selected exogenous gene or polynucleotide sequence such as therapeutically active genes, to the genome of a target cell.
  • Mveloid-like progenitor cell populations [0052] The present invention provides methods of employing myeloid-like (ML) progenitor cell populations to treat tumors.
  • cell populations include human CD14 + cell populations and rodent CD44 HI cell populations.
  • human myeloid-like progenitor cells are used.
  • the human myeloid-like cell population can be isolated from a blood sample (e.g., cord blood or peripheral blood) or a bone marrow sample from a human subject.
  • a population of rodent myeloid- like progenitor cells is used.
  • the rodent myeloid-like cell population contains a majority of cells that express CD44 (CD44 HI ).
  • the rodent cell population can also be characterized by the lack of significant levels of lineage surface antigens (Lin) on their cell surfaces.
  • the cell population suitable for use in the invention should contain mammalian cells in which at least about 50% of the cells express the respective surface marker (CD44 for rodent cells and CD 14 for human cells).
  • the employed cell population contains at least about 60%, 70%, 80%, 90%, 95%, 99% or more of cells that express the marker.
  • the myeloid-like progenitor cell populations can be obtained from bone marrow or a blood sample from a mammalian subject (e.g., a rodent or a human subject) as described herein or taught in the art.
  • the cells are isolated from the same subject in need of treatment of a tumor.
  • human CD 14+ cell populations can be obtained in accordance with the procedures described in the Examples below.
  • Rodent myeloid-like progenitor cell populations can be obtained from a subject in accordance with procedures disclosed herein or well known in the art. See, e.g., WO 06/104609; and Ritter et al., J. Clin. Invest. 116:3266-76, 2006.
  • a bone marrow can be obtained postnatally from juvenile and adult subjects.
  • the lineage negative progenitor cells from bone marrow of a rodent can be isolated by the following standard procedures: (a) extracting bone marrow from an adult mammal; (b) separating a plurality of monocytes from the bone marrow; (c) labeling the monocytes with biotin- conjugated lineage panel antibodies to one or more lineage surface antigens, preferably lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD1 1 , CD1 la, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G (murine), TER-119 (murine), CD45RA, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leukocyte antigen DR (
  • the bone marrow or blood cells can also be positively selected for CD44 expression (for rodent cells) or CD 14 expression (for human cells) to obtain the myeloid-like progenitor cell populations suitable for the present invention.
  • positive selection for CD44 expression can be performed with, e.g., using an antibody specific for the CD44 antigen (anti-CD44), and then selecting cells that immunoreact with the antibody.
  • the antibody can be removed from the cells by methods that are well known in the art.
  • the cells can be selected by using flow cytometry, using antibodies bound to or coated on beads followed by filtration, or using other separation methods that are well known in the art.
  • a population of human myeloid-like progenitor cells (CD14 + cells) is used.
  • the human myeloid-like progenitor cells that express the respective surface marker can also be isolated from a blood sample from a human subject, e.g., umbilical cord blood. It can also be obtained from other samples such as peripheral blood and bone marrow. Regardless of the manner by which the cell population is obtained, at least about 25% or about 40% of the cells in the population express the surface marker CD 14. With some selection scheme, a majority of the selected cells (e.g., at least 50%, 75% or 90%) express the surface.
  • the isolated cell populations Prior to administration to a subject, the isolated cell populations can be maintained in an appropriate culture medium such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the CD14 + human cells were isolated from human cord blood.
  • red blood cells in the blood sample e.g., peripheral blood or cord blood
  • lysed e.g., with ammonium chloride
  • Erythrocytes and granulocytes present in thee blood were also removed, e.g., via centrifugation.
  • the other cells in the sample were subject to fluorescence-activated cell sorting to separate monocytes from lymphocytes based on differences in cell size and granularity.
  • FACS based sorting the human myeloid-like cell populations can also be obtained by density centrifugation. It was found that that the isolated human myeloid-like cell population contains CD14 + /CD33 + progenitor cells with purities of 80% - 85%.
  • a rodent myeloid like cell population containing cells with high expression of CD44 (CD44 HI ) is used.
  • CD44 HI myeloid cells can be prepared and obtained in accordance with methods described in the art, e.g., Ritter et al., J. Clin. Invest. 116:3266-76, 2006.
  • the CD44 HI subpopulation of isolated Lin " CD44 + myeloid like progenitor cells typically contains cells in which at least about 50% of the cells express CD44. More preferably, at least about 65%, 75% or 90% of the cells in the CD44 HI subpopulation express CD44.
  • the CD44 HI myeloid like cell population is further selected for lack of expression of one or more of the following cell markers: Term 19, CD45RB220, and CD3e. It is known that bone marrow cells that do not express CD44 (CD44 LO cells) generally express one or more of these markers. Thus, the CD44 HI myeloid-like cell population to be used in the present invention can be isolated by a method involving further negative cell-marker selection.
  • the method involve contacting a plurality of bone marrow cells, peripheral blood cells or umbilical cord cells with one or more antibodies specific for Terl 19, CD45RB220, and CD3e, removing cells from the plurality of bone marrow cells that immunoreact with Terl 19, CD45RB220, and CD3e antibodies, and recovering myeloid- like bone marrow cells that are depleted in Terl 19, CD45RB220, and CD3e-expressing cells. With such negative selection, a myeloid-like cell population can be recovered in which greater than 90 percent of the cells express CD44.
  • Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.
  • a beam of light usually laser light
  • a number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (SSC) and one or more fluorescent detectors).
  • Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source.
  • This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to derive various types of information about the physical and chemical structure of each individual particle.
  • FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).
  • a flow cytometer is similar to a microscope, except that instead of producing an image of the cell, flow cytometry offers high-throughput automated quantification of set parameters.
  • a flow cytometer has 5 main components: a flow cell - liquid stream, a light source (e.g., laser), a detector and Analogue to Digital Conversion (ADC) system which generate FSC and SSC as well as fluorescence signals, an amplification system, and a computer for analysis of the signals.
  • a light source e.g., laser
  • ADC Analogue to Digital Conversion
  • the data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two dimensional dot plots or even in three dimensions.
  • the regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates".
  • Specific gating protocols exist for diagnostic and clinical purposes especially in relation to haematology.
  • the plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally.
  • Fluorescence-activated cell sorting is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
  • the cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter.
  • a vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet.
  • the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured.
  • An electrical charging ring is placed just at the point where the stream breaks into droplets.
  • a charge is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream.
  • the charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems the charge is applied directly to the stream and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.
  • FACS can be carried out on a BD FACSAria Cell-Sorting System (BD Biosciences, San Jose, CA) using a series of gates. No antibodies or other selection agents are used in the sorting. Dead cells and debris can be first gated out by drawing a region that includes only viable white blood cells. Thereafter, doublets or aggregated cells can be removed with secondary and tertiary gates that interrogate forward scatter width (FSC-W) vs. forward scatter area (FSC-A) and side scatter width (SSC-W) vs. side scatter area (SSC-A), respectively.
  • FSC-W forward scatter width
  • FSC-A forward scatter area
  • SSC-W side scatter width
  • the invention can also employ isolated cells that have been subsequently modified.
  • the isolated cells can be engineered to express an anti-tumor agent.
  • the anti-tumor agent can include, e.g., antiangiogenic agents, cytotoxic agents or proteins encoded by tumor suppressor genes.
  • the isolated myeloid like cell populations are stably or transiently transfected with a vector harboring a polynucleotide that encodes the therapeutically useful agent. Examples of genes encoding such agents and methods for their transfection into myeloid like progenitor cells have been described in the art. See, e.g., PCT publication WO 04/098499.
  • the cells are engineered with a vector that allows the anti-tumor agent to be stably expressed.
  • the transfected cells can include any gene which is therapeutically useful for treatment of tumors.
  • the transfected cells from the myeloid-like cell population of the present invention harbors a gene operably encoding an antiangiogenic agent.
  • antiangiogenic agents include fragments derived from the carboxyl-terminal fragment of tryptophan tRNA synthetase (T2-TrpRS), angiopoietin 2, endostatin, angiostatin, PEX, IL-12, IFN-a, prolactin and thrombospondin TSP-1 and TSP-2.
  • the transfected cells are engineered to express a cytotoxic protein or peptide.
  • the cytotoxic protein or peptide can be one that is naturally occurring or synthetic in nature.
  • naturally occurring cytotoxic peptides that can be expressed by the transfected cells include venoms (e.g., bee venom, scorpion toxins, and jumper ant venom) and peptides involved in autocytotoxicity (e.g., amyloid polypeptide IAPP).
  • Synthetic peptides that possess cytotoxic activity similar to that of the naturally occurring cytotoxic peptides are also known in the art. Examples of such peptides are disclosed in, e.g., Chen et al., FEBS Lett 236: 462-466, 1988; and Cornut et al., FEBS Lett 349: 29-33, 1994.
  • the myeloid like cells can also be engineered to express a cytotoxic protein or polypeptide drug.
  • cytotoxic protein or polypeptide drug examples include, e.g., IL-2, GM-CSF, TNFa, gelonin, momordin, Diphtheria toxin, and Pseudomonas exotoxin.
  • any other antitumor agents suitable for recombinant expression in the myeloid like progenitor cells can also be used in the invention.
  • the myeloid like cells can be transfected with a gene encoding tumor suppressor gene p53, p21 and BRCA2. It was shown that transfecting p53 into human breast cancer cell lines has led to restored growth suppression in the cells (Casey et al., Oncogene 6: 1791-7, 1991).
  • the myeloid like cells can also be transfected with an exogenous gene encoding an enzyme for anti-tumor activity.
  • the gene can encode a cyclin-dependent kinase (CDK) or a cytosine deaminase. It was shown that restoration of the function of a wild-type cyclin-dependent kinase, pl6INK4, by transfection with a pl6I K4-expressing vector reduced colony formation by some human cancer cell lines (Okamoto, Proc. Natl. Acad. Sci. U.S.A. 91 : 1 1045-9, 1994).
  • the exogenous gene encoding the therapeutic agent can be transfected into the myeloid-like progenitor cells (e.g., human CD14 + cells or rodent CD44 HI cells) in an appropriate vector.
  • the employed vector should allow introduction of the exogenous gene into the cells and also subsequent expression (transient or stable) of the gene once transfected into the cells.
  • Various vectors are suitable for this aspect of the invention.
  • retroviral vectors and corresponding packaging cell lines well known in the art can be employed.
  • Particularly suitable for the present invention are lentiviral vectors.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers.
  • Retroviral vectors are comprised of cw-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis -acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HTV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739, 1992; Johann et al., J.
  • Vectors suitable for the present invention can also be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques can be found in common molecular biology references such as Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), D. Goeddel, ed., Gene Expression Technology, Methods in Enzymology series, Vol. 185, Academic Press, San Diego, Calif. (1991), and Innis, et al. PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, Calif. (1990).
  • a transcriptional regulatory region capable of driving gene expression in the target cell.
  • the transcriptional regulatory region can comprise a promoter, enhancer, silencer or repressor element and is functionally associated with a nucleic acid of the present invention.
  • the transcriptional regulatory region drives high level gene expression in the target cell.
  • Transcriptional regulatory regions suitable for use in the present invention include but are not limited to the human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polyomavirus promoter, the albumin promoter, PG and the a-actin promoter coupled to the CMV enhancer.
  • CMV human cytomegalovirus
  • producer cell line or packaging cell line for transfecting retroviral vectors and producing viral particles are also known in the art.
  • the producer cell to be used in the invention needs not to be derived from the same species as that of the target cell (e.g., human target cell).
  • producer or packaging cell lines suitable for the present invention include cell lines derived from human (e.g., HEK 292 cell), monkey (e.g., COS-1 cell), mouse (e.g., NIH 3T3 cell) or other species (e.g., canine). Additional examples of retroviral vectors and compatible packaging cell lines for producing recombinant retroviruses in gene transfers are reported in, e.g., Markowitz et al., Virol.
  • retroviral vectors and packing cell lines used for gene transfer in the art can be obtained commercially.
  • a number of retroviral vectors and compatible packing cell lines are available from Clontech (Mountain View, CA).
  • lentiviral based vectors include, e.g., pLVX-Puro, pLVX-IRES-Neo, pLVX-IRES-Hyg, and pLVX-IRES-Puro.
  • Corresponding packaging cell lines are also available, e.g., Lenti-X 293T cell line.
  • other retroviral based vectors and packaging systems are also commercially available.
  • MMLV based vectors pQCXIN, pQCXIQ and pQCXIH include MMLV based vectors pQCXIN, pQCXIQ and pQCXIH, and compatible producer cell lines such as HEK 293 based packaging cell lines GP2-293, EcoPack 2-293 and AmphoPack 293, as well as NIH/3T3-based packaging cell line RetroPack PT67. Any of these and other retroviral vectors and producer cell lines can be employed in the practice of the present invention.
  • recombinant retroviruses expressing a gene encoding a therapeutic agent can be readily used to infect cells in a Lin " CD44 + progenitor cell population.
  • Methods for infecting primary cells with a recombinant retrovirus are well known in the art.
  • the recombinant viruses can be transfected into the cells in accordance with methods well known in the art of gene therapy (see, e.g., Mulligan et al., Hum. Gene Ther. 5:543-563, 1993).
  • the engineered cells can be administered or implanted into a subject afflicted with a tumor.
  • the Lin " CD44 + cells to be transfected with the recombinant virus are isolated from a subject to be treated.
  • the transfected cells are not autologous to the subject to whom the cells are ultimately administered.
  • the present invention provides methods of inhibiting tumor growth and promoting tumor regression (e.g., killing tumor cells) in a subject.
  • an isolated myeloid-like progenitor cell population or its engineered variant that harbors an expression vector operably encoding an anti-tumor agent is administered to the subject.
  • the isolated or engineered myeloid-like progenitor cell population is directly administered to a subject in treatment.
  • the cell population is stimulated or activated prior to being administered to the subject.
  • the cell population e.g., a human CD14 + cell population
  • LPS lipopolysaccharide
  • TLR4 toll-like receptor 4
  • the cell populations to be used in the therapeutic methods of the invention can be treated with a TLR4 ligand, e.g., LPS, monophosphoryl lipid A (MPLA), myeloid related protein 8 (MRP8) or myeloid related protein 14 (MRP 14).
  • MPLA monophosphoryl lipid A
  • MRP8 myeloid related protein 8
  • MRP 14 myeloid related protein 14
  • Activation of the myeloid-like progenitor cell populations with a TLR4 ligand can be readily performed in accordance with methods well known in the art or specifically exemplified herein. See, e.g., Umland et al., J. Leu. Biol. 75:671-679, 2004; Chang et al., J. Dent. Res. 84:994, 2005; and Crisostomo et al., Am. J. Physiol. Cell. Physiol. 294:C675-C682, 2008.
  • the myeloid-like progenitor cells are administered to the subject by injection at a site that is within or close to a solid tumor.
  • the invention provides methods for delivering an anti-tumor agent to a solid tumor in a subject.
  • a vector expressing an anti-tumor agent as disclosed herein is first constructed.
  • the vector e.g., a lentivirus based vector
  • the cells can then be administered to a subject afflicted with a tumor, e.g., via local or central injection at a site close to or distant from the tumor.
  • the myeloid-like progenitor cell population is preferably isolated from the subject in need of treatment of a tumor.
  • the cells from the myeloid-like cell population are typically administered (e.g., via injection) in a physiologically tolerable medium, such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the isolated cells, or their engineered form as disclosed herein, should be administered to the subject in a number sufficient to inhibit the growth and promote the regression of a tumor in the subject.
  • the subject to be treated can be one already suffering from the development of a tumor.
  • the subject can be one that is known to be predisposed to develop a tumor (i.e., through genetic predisposition).
  • the isolated myeloid-like cell population can be stored after isolation, and can then be injected prophylactically during early stages of a later developed tumor.
  • administration of therapeutic cell populations of the invention for treating tumors is carried out by local or central injection of the cells into the subject (i.e., as opposed to systemic administration such as peripheral administration).
  • the site of injection can be within the tumor, close to the tumor or distant from the tumor.
  • the cells can be injected at a site that is in the opposite hemisphere from the tumor or that is in the same hemisphere but posterior to the tumor.
  • Various types of tumors are suitable for treatment with methods and
  • compositions of the present invention include both benign and malignant tumors.
  • the methods of the invention are directed to killing tumor cells in a malignant tumor.
  • solid tumors that are suitable for treatment with methods of the invention include brain tumors, melanoma (e.g., ocular melanoma), gastrointestinal tumors, breast cancer, lung cancer and ovarian cancer.
  • the methods of the invention are directed to treating and killing cells in primary brain tumors and other vascular malignancies.
  • the isolated or further engineered myeloid- like progenitor cells described herein can be employed to treat subjects suffering from brain tumors such as glioma, glioblastoma, medulloblastoma, astrocytoma, other primitive neuroectoderma.
  • the number of myeloid-like progenitor cells to be administered to a subject afflicted with a tumor should be sufficient for arresting tumor growth and/or for promoting tumor regression in the subject.
  • the sufficient number of cells can be determined based on the type of tumor, its size and stage of development, the route of administration, the age of the specific subject to be treated and other factors that will be readily apparent to one of ordinary skill in the art of treating retinal diseases. As a general guidance for
  • the cells may be administered in a single dose or by multiple dose administration over a period of time, as determined by the clinician in charge of the treatment. Also, the number of cells and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low number of cells may be administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high number of cells at relatively short intervals may be required until progression of the tumor is reduced or terminated, and preferably until the subject shows partial or complete regression of the tumor. Thereafter, the subject can be administered a prophylactic regime.
  • the therapeutic regimen of the invention can be used alone in the treatment of a tumor in a subject.
  • the treatment may also employed in combination with other known antitumor drugs or treatment methods.
  • the cell therapy disclosed herein can be used together with surgery, chemotherapies or radiation therapies that have been routinely practiced in the art for the treatment of tumors.
  • subjects who have been undergoing surgical procedures to remove a tumor can be administered a population of myeloid-like progenitor cells to kill residual tumor cells and to prevent recurrence or metastasis.
  • subjects suffering from cancers can be simultaneously treated with a known chemotherapy regimen and the cell therapy of the invention.
  • antineoplastic drugs and cytotoxic agents which can be readily utilized in combination with myeloid-like progenitor cells for treating tumors.
  • Antineoplastic drugs include classes of agents such as alkylating agents, antimetabolites, antimitotics and topoisomerase II inhibitors. Specific examples include actinomycin, anthracyclines (e.g., doxorubicin, daunorubicin, Valrubicine, Idarubicine and epirubicin) and other cytotoxic antibiotics (e.g., bleomycin, plicamycin and mitomycin).
  • anthracyclines e.g., doxorubicin, daunorubicin, Valrubicine, Idarubicine and epirubicin
  • other cytotoxic antibiotics e.g., bleomycin, plicamycin and mitomycin.
  • various chemotherapy regiment using two or more drugs can also be employed in combination with the cell therapy disclosed herein. Detailed information about such chemotherapy regimens is readily available from, e.g., National Comprehensive Cancer Network (Jen Kintown, Pennysylvania).
  • subjects in need of treatment for a tumor can be subject to the combination of a radiation therapy and the cell therapy disclosed herein.
  • the subject to be treated can be first administered with a radiotherapy regimen.
  • Radiotherapy is often used as the primary therapy for the treatment of malignant tumors.
  • Most common cancer types can be treated with radiotherapy in some way.
  • the amount of radiation used in radiation therapy is typically measured in gray (Gy), and varies depending on the type and stage of cancer being treated.
  • the typical dose for a solid epithelial tumor ranges from 60 to 80 Gy, while lymphoma tumors are treated with 20 to 40 Gy.
  • the doses are typically around 45 - 60 Gy in 1.8 - 2 Gy fractions. Many other factors are considered by radiation oncologists when selecting a dose, including whether the patient is receiving chemotherapy, whether radiation therapy is being administered before or after surgery, and the degree of success of surgery.
  • the total radiotherapy dose is fractionated (spread out over time) for several important reasons. Fractionation allows normal cells time to recover, while tumor cells are generally less efficient in repair between fractions. Fractionation also allows tumor cells that were in a relatively radio-resistant phase of the cell cycle during one treatment to cycle into a sensitive phase of the cycle before the next fraction is given.
  • a curative dose is used in a radiotherapy as the primary therapy for treating a tumor patient.
  • a subject may be administered a preventative or sublethal dose of radiation prior to or simultaneously with treatment with the myeloid-like progenitor cells disclosed herein.
  • mice we tested the homing of mouse bone marrow cells to intracerebrally established rat-RG2 brain tumors in SCID mice and also human U87 glioma in SCID mice. Results from the study confirmed that the cells home to the tumors. To show that the observed activity was not an artifact of the chimeric mouse brain tumor model, we observed a similar response in the mouse GL261 gliosarcoma model. In this model, mouse-GL261 gliosarcoma cells are implanted into the cerebrum of c57bl/6 mice. We found that mtratumorally-injected bone marrow cells from actin-GFP labeled mice (c57bl/6 background) home to the tumor cells.
  • Lin " myeloid-like progenitor cells can be divided into two subpopulations: CD44 HI and CD44 LO cells (Ritter et al., J. Clin. Invest. 116:3266-76, 2006). We therefore tested these two fractions to determine if they specifically localize within the tumor. We found that CD44 HI cells localize within the tumor. In addition, they replicate within the bed of the tumor as determined by Ki67 staining. Conversely, the CD44 LO cells generally do not localize to the tumor. As observed with Lin " cells, the CD44 HI subpopulation of cells also migrate from one hemisphere to the other to localize and proliferate within the tumor.
  • the CD44 LO cells do not home to the tumor.
  • CD44 HI and CD44 LO cell populations were further characterized in vivo and in vitro. It was found that the CD44 HI cells divided to a limited extent in vivo and in vitro as determined by Ki67 staining. CD44 cells are known to be mainly of myeloid lineage (Ritter et al., J. Clin. Invest. 116:3266-76, 2006). We observed that these cells express F4/80, a macrophage and dendritic cell marker, in the presence of established 9L tumors in vivo or when co-cultured with 9Ltumor cells in vitro. An extensive evaluation of cell markers for other cell lineages did not show any evidence of alternative differentiation of the CD44 HI cells.
  • CD1 lc also a dendritic and monocyte/macrophage marker.
  • CD45 we additionally observed some early staining of the cells in vivo with CD1 lb and a very scant occasional cell staining with CD45.
  • CD44 HI cells are avid phagocytes of tumor cells damaged by uv or gamma irradiated, but not undamaged tumor cells.
  • CD44 HI cells proliferate when cocultured with various tumor cells. Based on their surface markers, cell morphology and their phagocytic function, these cells are likely to be macrophage precursors. Within the context of the brain, these cells can be called microglial cells.
  • the CD44 LO population in mice is mainly erythroid and lymphoid lineage (Ritter et al., J. Clin. Invest. 1 16:3266-76, 2006).
  • the CD44 LO population of cells when implanted into brain tumors generally do not remain, and typically die off when co-cultured with 9L cells in vitro. Therefore, they cannot be characterized by immunohistochemistry.
  • CD44 HI cells We examined phagocytotic activity of CD44 HI cells on various tumor cells that have been sublethally irradicated. These tumor cells include rat gliosarcoma cell line 9L, mouse glioma cell line GL261, rat glioma cell line CNSl, rat glioma cell line RG2, and mouse B16 melanoma line. It was found that CD44 HI cells alone or in combination with CD44 LO cells appear to kill and remove the sublethally irradiated tumor cells. As shown in Figure 2, CD44 HI cells combined with CD44 LO cells were able to selectively kill 9L cells that have been irradiated at a dose of 4.5Gy.
  • CD44 HI cells need to be injected centrally in order to home to the tumors. This result could be due to the number of cells delivered peripherally, the relative immune privilege of the brain, or other factors. Signals expressed by the tumor may set up a local gradient that can direct a migrating cell from one hemisphere to the other, but either the signal is not released systemically or the cells cannot cross the blood brain barrier. Alternatively, it is possible that peripheral injection results in a dilution effect so that the injected cells are not at a sufficient concentration in circulation to establish in the tumor.
  • Example 5 Activities of rodent CD44* cells transduced with a lentiviral vector
  • a lentiviral based vector that expresses GFP.
  • Use of the lenti-GFP vector and its transduction into CD44 + cells from C57B1/6 mice were carried out as described in Miyoshi et al., Science 283:682-686, 1999.
  • the GFP-expressing FG12 lentiviral vector was efficiently transduced into CD44 HI and CD44 LO cells.
  • employing a similar vector we achieved a transduction efficiency of 60-80% in Lin + and Lin " cell fractions.
  • VEGFR-2 and CD 144 (R&D system).
  • the cells were transduced with a GFP-expressing adenoviral vector.
  • a GFP-expressing adenoviral vector Specifically, the freshly isolated human CD14 + cells from cord blood were incubated for 4-6 hr in Ml 99 (Invitrogen) and 20% FBS with AD5 F16 (Adenovirus expressing the fiber 16 and encoding eGFP (Ad5 F16 eGFP) at 5000 MOI. After the infection the cells were washed out from the virus and plated overnight. The percentage of GFP-expressing cells was analyzed by flow cytometry. We found that 70% of the CD14 + cells were efficiently transduced and expressed high level of the eGFP.
  • the human myeloid-like cells expressing the exogenous gene were then examined for their ability to home to tumor cells and control the growth of tumor cells in vitro.
  • the results indicate that the Ad5F16 eGFP CD14 + cells were able to target to 9L tumor cells in culture (Fig. 4A).
  • the transduced CD14 + cells reduced the growth of sub-lethally irradiated tumor cells in vitro (Fig. 4B).
  • the cells also inhibited the proliferation of 7.5gy and lOgy irradiated 9L tumors cells, as evidenced by 60% decrease in the expression level of proliferating cell nuclear antigen (PCNA), a marker of DNA synthesis in the nucleus (Fig. 4C).
  • PCNA proliferating cell nuclear antigen
  • the cells when stimulated with LPS, the cells killed tumor cells in vitro (Fig. 5).
  • the cells were found to secret high level of important proinflammatory cytokines such as IL- ⁇ ⁇ and IL-6 (Fig. 5 and Table 1).
  • the human CD44 + cells When in contact with 7.5gy and lOgy irradiated 9L tumor cells, the human CD44 + cells also up-regulated the expression of human IL-8 when in contact with 7.5gy and lOgy irradiated 9L tumor cells and activated pro inflammatory pathways (Table 2). Additional to the in vitro studies, we also examined the in vivo appearance and behavior of these transduced human cells.
  • CD14 + cells appear to localize to the tumor cells and also display a morphology that is similar to the rodent myeloid-like cells (CD44 + cells) described above (Fig. 7).
  • lymphocytes are the anti-tumor effector cells, and that lymphocytes (and less cleanly granulocytes) from both bone marrow and spleen did not have anti-tumor activity and could serve as negative control cells.
  • Bone Marrow derived cells consist of hematopoietic stem/progenitor cells and their differentiated progenies of blood cells (monocytes, granulocytes, lymphocytes and erythrocytes). We wanted to determine if a single cell population from the bone marrow was active against tumor cells or whether synergism between cell populations was necessary. Therefore we separated the bone marrow into its 3 constituent populations and tested these against whole bone marrow. Specifically, after histopaque purification, rat bone marrow contains 26% granulocytes, 50% lymphocytes and 23% monocytes. Several cell surface antigens were used to identify pure sub populations in the granulocyte, lymphocyte and monocyte gates.
  • CD54 also called ICAM-1
  • All monocytes and granulocytes were positive for CD1 lb.
  • the clone HIS48 stained all granulocytes and monocytes.
  • granulocytes, lymphocytes and monocytes were purified by FACS using antibodies against CD54 and HIS48 antigens.
  • the purified cell populations include double negative cells (CD54 " , HIS48 " ) or single positive for CD54 (CD54 + HIS48 " ) which are lymphocytes and confirmed by expression of CD3.
  • Purified double positive cells (CD54 + , HIS48 + ) were monocytes, and purified single positive cells for HIS48 (HIS48 + CD54 " ) were granulocytes. These sub- populations of bone marrow cells were then subject to further studies to investigate their anti-tumor activities, as described below.
  • the splenic cells were only 7% monocytes (CD54 + HIS48 + ) and 5.7% granulocytes (CD54 " HIS48 ") .
  • the majority of the cells were lymphocytes (CD54 + HIS48 ⁇ CD54 " fflS48 " , CD3 + ) and other cells.
  • lymphocytes CD54 + HIS48 ⁇ CD54 " fflS48 " , CD3 + .
  • Using an in vitro (in tissue culture) cell adhesion assay we found that this population had little if any anti-tumor activity. Therefore, the splenocytes with the splenic lymphocytes in particular, can serve as an additional negative control population.
  • CD44 hi and CD44 low cell populations contain monocytes, we are working to determine if the anti-tumor activity identified against melanoma in vivo is purely related to the monocytic fraction or whether there is in vivo synergism.
  • Example 9 Anti-tumor activities of cell populations of human bone marrow [00104]

Abstract

Cette invention concerne des méthodes de traitement de tumeurs. Les méthodes consistent à injecter ou implanter une population de cellules progénitrices de type myéloïde (par exemple des cellules CD14+ humaines ou des monocytes CD33HI humains) chez un sujet atteint par une tumeur. Les cellules isolées peuvent être génétiquement modifiées de façon additionnelle afin d'exprimer un agent antitumoral, par exemple un agent antiangiogénique ou un agent cytotoxique.
PCT/US2011/001183 2010-07-06 2011-07-06 Utilisation de populations de cellules progénitrices de type myéloïde pour traiter des tumeurs WO2012005763A1 (fr)

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US11628218B2 (en) 2020-11-04 2023-04-18 Myeloid Therapeutics, Inc. Engineered chimeric fusion protein compositions and methods of use thereof
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US11918604B2 (en) 2015-02-19 2024-03-05 Myeloid Therapeutics, Inc. Chimeric antigen receptor dendritic cell (CAR-DC) for treatment of cancer
US11918605B1 (en) 2015-02-19 2024-03-05 Myeloid Therapeutics, Inc. Chimeric antigen receptor dendritic cell (CAR-DC) for treatment of cancer
WO2017214830A1 (fr) * 2016-06-14 2017-12-21 石庆学 Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène pd-1, et ses applications
WO2017214829A1 (fr) * 2016-06-14 2017-12-21 石庆学 Vecteur d'expression de lentivirus pour favoriser spécifiquement l'expression élevée du gène pd-l1, et ses applications
WO2020095044A1 (fr) * 2018-11-06 2020-05-14 Macrophox Ltd Monocytes de ciblage du cancer
US11672874B2 (en) 2019-09-03 2023-06-13 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration
WO2021119538A1 (fr) * 2019-12-11 2021-06-17 Myeloid Therapeutics, Inc. Compositions de cellules thérapeutiques et procédés de production et méthodes d'utilisation associés
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