Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/22—Immunosuppressive or immunotolerising
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/418—Antigens related to induction of tolerance to non-self
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • 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
  • C12N5/0646—Natural killers cells [NK], NKT cells
• • 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
  • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
• • 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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/05—Adjuvants

Definitions

• Placental perfusate comprises a collection of placental cells obtained by passage of a perfusion solution through the placental vasculature, and collection of the perfusion fluid from the vasculature, from the maternal surface of the placenta, or both.
• Methods of perfusing mammalian placentas are described, e.g., in U.S. Patent No. 7,045,146 and U.S. Patent No. 7,255,879.
• the population of placental cells obtained by perfusion is heterogeneous, comprising hematopoietic (CD34 + ) cells, nucleated cells such as granulocytes, monocytes and macrophages, a small percentage (less than 1%) tissue culture substrate-adherent placental stem cells, and natural killer cells.
• hematopoietic (CD34 + ) cells comprising hematopoietic (CD34 + ) cells, nucleated cells such as granulocytes, monocytes and macrophages, a small percentage (less than 1%) tissue culture substrate-adherent placental stem cells, and natural killer cells.
• NK Natural killer cells are cytotoxic lymphocytes that constitute a major component of the innate immune system.
• NK cells play a role in the host rejection of tumors. Because cancer cells have reduced or no class I MHC expression, they can become targets of NK cells. Accumulating clinical data suggest that haploidentical transplantation of human NK cells isolated from PBMC or bone marrow mediate potent anti-leukemia effects without possessing detectable graft-versus-host disease (GVHD). See Ruggeri et al., Science 295:2097-2100 (2002)). Natural killer cells can become activated by cells lacking, or displaying reduced levels of, major histocompatibility complex (MHC) proteins.
• MHC major histocompatibility complex
• NK cells and LAK cells have been used in both ex vivo therapy and in vivo treatment of patients having advanced cancer, with some success against bone marrow related diseases, such as leukemia; breast cancer; and certain types of lymphoma.
• LAK cell treatment requires that the patient first receive IL-2, followed by leukopheresis and then an ex vivo incubation and culture of the harvested autologous blood cells in the presence of IL-2 for a few days.
• the LAK cells must be reinfused along with relatively high doses of IL-2 to complete the therapy. This purging treatment is expensive and can cause serious side effects. These include fluid retention, pulmonary edema, drop in blood pressure, and high fever.
• said placental perfusate is perfusate that has been passed through placental vasculature, e.g., only through placental vasculature.
• said placental perfusate has been passed through the placental vasculature and collected from the maternal face of the placenta.
• all, or substantially all ⁇ e.g., greater than 90%, 95%, 98% or 99%) of cells in said placental perfusate are fetal cells.
• the placental perfusate comprises fetal and maternal cells.
• a method of suppressing the proliferation of a tumor cell or plurality of tumor cells comprising contacting the tumor cell or plurality of tumor cells with a plurality of placental perfusate cells.
• said plurality of placental perfusate cells are, or comprise, total nucleated cells from placental perfusate.
• said placental perfusate or placental perfusate cells e.g., total nucleated cells from placental perfusate, have been treated to remove at least one type of cell.
• said contacting takes place in vitro.
• said contacting takes place in vivo.
• said in vivo contacting takes place in a mammal, e.g., a human.
• said placental perfusate cells have been treated to enrich for at least one type of cell, e.g., CD56 + cells.
• said placental perfusate cells are CD56 + placental cells.
• the CD56 + cells are CD56 CD16 natural killer cells, e.g., placental intermediate natural killer (PINK) cells, e.g., obtained from placental perfusate cells or placental cells obtained by mechanical or enzymatic disruption of placental tissue.
• said CD56 + cells are selected by CD56-conjugated microbeads.
• said CD56 + cells comprise cells that exhibit detectably lower expression of
• PINK cells are CD3 .
• at least 50% of the cells in said placental perfusate cells are said CD56 + cells.
• the tumor cell is a primary ductal carcinoma cell, a leukemia cell, an acute T cell leukemia cell, a chronic myeloid lymphoma (CML) cell, an acute myelogenous leukemia cell, a chronic myelogenous leukemia (CML) cell, a lung carcinoma cell, a colon adenocarcinoma cell, a histiocytic lymphoma cell, multiple myeloma cell, a retinoblastoma cell, a colorectal carcinoma cell or a colorectal adenocarcinoma cell.
• CML chronic myeloid lymphoma
• CML chronic myelogenous leukemia
• said contacting is contacting in vitro. In another embodiment, said contacting is contacting in vivo, e.g., in a mammal, e.g., a human.
• a method of suppressing the proliferation of a tumor cell or plurality of tumor cells comprising contacting the tumor cell or plurality of tumor cells with a plurality of natural killer cells from placenta, e.g., PINK cells.
• the natural killer cells from placenta are natural killer cells obtained from placental perfusate.
• the natural killer cells are natural killer cells obtained by physical disruption and/or enzymatic digestion of placental tissue.
• the natural killer cells are CD56 CD16 natural killer cells, e.g., PINK cells.
• said natural killer cells are selected, e.g., from placental perfusate cells or cells obtained by physical disruption and/or enzymatic digestion of placental tissue, by CD56- conjugated microbeads.
• the natural killer cells are CD3 .
• the plurality of natural killer cells is at least 80% of the cells in a population of cells that comprises the natural killer cells.
• said contacting takes place in vitro.
• said contacting takes place in vivo.
• said in vivo contacting takes place in a mammal, e.g., a human.
• said plurality of natural killer cells comprises cells that exhibit detectably lower expression of NKG2D, NKp46 or CD94 than an equivalent number of CD56 CD16 + natural killer cells.
• said plurality of natural killer cells e.g., PINK cells, expresses one or more of the microRNAs hsa- miPv-100, hsa-miPv-127, hsa-miR-211, hsa-miR-302c, hsa-miR-326, hsa-miR-337, hsa-miR-497, hsa-miR-512-3p, hsa-miR-515-5p, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a, hsa-miR-518e, hsa-miR-519d,
• said plurality of natural killer cells are contacted with an immunomodulatory compound in an amount and for a time sufficient for said plurality of natural killer cells to express detectably more granzyme B than an equivalent number of said natural killer cells not contacted with said immunomodulatory compound.
• said immunomodulatory compound is lenalidomide or pomalidomide.
• said plurality of natural killer cells are contacted with an immunomodulatory compound in an amount and for a time sufficient for said natural killer cells to exhibit detectably more cytotoxicity towards said tumor cells than an equivalent number of said natural killer cells not contacted with said immunomodulatory compound, e.g., lenalidomide or pomalidomide.
• said plurality of natural killer cells e.g., PINK cells, express one or more of BAX, CCL5, CCR5, CSF2, FAS, GUSB, IL2RA, or TNFRSF18 at a higher level than an equivalent number of said natural killer cells not contacted with said immunomodulatory compound.
• said plurality of natural killer cells express one or more of ACTB, BAX, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, ILIA, IL2RA, IL8, IL10, LTA, PRF1, PTGS2, SKI, and TBX21 at a higher level than an equivalent number of said natural killer cells not contacted with said immunomodulatory compound.
• the natural killer cells from placenta are combined with natural killer cells from another source, e.g., placental blood and/or umbilical cord blood, e.g., to form combined natural killer cells.
• natural killer cells from another source e.g., placental blood and/or umbilical cord blood, e.g., to form combined natural killer cells.
• the combined natural killer cells are natural killer cells from a mixed source, e.g., placenta (e.g., placental perfusate) and umbilical cord blood.
• a mixed source e.g., placenta (e.g., placental perfusate) and umbilical cord blood.
• combined natural killer cells may be obtained from matched umbilical cord blood and human placental perfusate, e.g., a donor matched source wherein placental perfusate is obtained from the same placenta as the cord blood.
• Natural killer cells from both are isolated separately or at the same time, and combined.
• natural killer cells are isolated from
• Combined natural killer cells may also be referred to herein as "pNK" cells.
• the natural killer cells from placenta are combined with natural killer cells from another source in a ratio of about 100: 1, 95:5, 90: 10, 85: 15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45: 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75:1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20:1, 15: 1, 10: 1, 5: 1, 1 : 1, 1 :5, 1 : 10, 1 : 15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, 1 :50, 1
• the combined natural killer cells are obtained from a combination of cells from placenta (e.g., from placental perfusate) and cells from another source, e.g., umbilical cord blood.
• the cells from placenta and another source are combined prior to isolation of combined natural killer cells.
• natural killer cells are isolated from placenta and, separately, natural killer cells are isolated from the other source, and then the natural killer cells from the two sources are combined to form the combined natural killer cells.
• the combined natural killer cells are not cultured. In other embodiments, the combined natural killer cells are cultured.
• the combined natural killer cells comprise a detectably greater amount of KIR2DL2/3+, NKp30+ , 2B4+, and/or CD94+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In certain embodiments, the combined natural killer cells comprise a detectably lower amount of NKp46+ natural killer cells than an equivalent number of natural killer cells from peripheral blood.
• the combined natural killer cells comprise: a detectably higher number of CD3 CD56 CD16 natural killer cells than an equivalent number of natural killer cells from peripheral blood; a detectably lower number of CD3 CD56 CD16 + natural killer cells than an equivalent number of natural killer cells from peripheral blood; a detectably higher number of CD3 CD56 KIR2DL2/L3 + natural killer cells than an equivalent number of natural killer cells from peripheral blood; a detectably lower number of CD3 CD56 NKp46 + natural killer cells than an equivalent number of natural killer cells from peripheral blood; a detectably higher number of CD3 CD56 NKp30 + natural killer cells than an equivalent number of natural killer cells from peripheral blood; a detectably higher number of CD3 CD56 2B4 + natural killer cells than an equivalent number of natural killer cells from peripheral blood; or a detectably higher number of CD3 CD56 CD94 + natural killer cells than an equivalent number of natural killer cells from peripheral blood.
• the combined natural killer cells are cultured and comprise: a detectably lower number of CD3 CD56 KIR2DL2/L3 + natural killer cells than an equivalent number of natural killer cells from peripheral blood; a detectably higher number of CD3 CD56 NKp46 + natural killer cells than an equivalent number of natural killer cells from peripheral blood; a detectably higher number of CD3 CD56 NKp44 + natural killer cells than an equivalent number of natural killer cells from peripheral blood; a detectably higher number of CD3 CD56 NKp30 + natural killer cells than an equivalent number of natural killer cells from peripheral blood.
• the combined natural killer cells express one or more of miRNAs hsa-miR-155, hsa-miR-337, hsa-miR-422a, hsa-miR-549 and hsa-miR-618 at a detectably greater amount than an equivalent number of natural killer cells from peripheral blood.
• the tumor cell is a solid tumor cell.
• the tumor cell is a liquid tumor cell, e.g., a blood tumor cell.
• the tumor cell is a primary ductal carcinoma cell, a leukemia cell, an acute T cell leukemia cell, a chronic myeloid lymphoma (CML) cell, an acute myelogenous leukemia cell, a chronic myelogenous leukemia (CML) cell, a lung carcinoma cell, a colon adenocarcinoma cell, a histiocytic lymphoma cell, multiple myeloma cell, a retinoblastoma cell, a colorectal carcinoma cell or a colorectal adenocarcinoma cell.
• CML chronic myeloid lymphoma
• CML chronic myelogenous leukemia
• a composition comprising isolated placental CD56 + , CD 16 natural killer cells, e.g., PINK cells.
• said placental natural killer cells are isolated from placental perfusate.
• said placental natural killer cells are isolated from placenta by physical disruption and/or enzymatic digestion of placental tissue.
• said natural killer cells comprise at least 50% of cells in the composition.
• said natural killer cells comprise at least 80% of cells in the composition.
• said composition comprises isolated CD56 + , CD16 + natural killer cells.
• said CD56 , CD 16 natural killer cells are from a different individual than said CD56 , CD 16 natural killer cells.
• said isolated CD56 + , CD 16 natural killer cells are from a single individual.
• said isolated CD56 + , CD 16 natural killer cells comprise natural killer cells from at least two different individuals.
• said placental natural killer cells, e.g., said PINK cells are expanded.
• the composition comprises placental natural killer cells and natural killer cells from another source.
• said other source is cord blood and/or umbilical cord blood.
• said other source is peripheral blood.
• the natural killer cells from placenta are combined with natural killer cells from another source in a ratio of about 100: 1, 95:5, 90: 10, 85: 15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45: 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100: 1, 95:1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50:1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 15: 1, 10: 1, 5:1, 1 : 1, 1 :5, 1 : 10, 1 : 15, 1 :20, 1 :25, 1
• the composition comprises isolated placental perfusate.
• said placental perfusate is from the same individual as said natural killer cells.
• said placental perfusate comprises placental perfusate from a different individual than said natural killer cells.
• all, or substantially all (e.g., greater than 90%, 95%, 98% or 99%) of cells in said placental perfusate are fetal cells.
• the placental perfusate comprises fetal and maternal cells.
• the fetal cells in said placental perfusate comprise less than about 90%, 80%, 70%, 60% or 50% of the cells in said perfusate.
• said perfusate is obtained by passage of a 0.9% NaCl solution through the placental vasculature.
• said perfusate comprises a culture medium.
• said perfusate has been treated to remove a plurality of erythrocytes.
• the composition comprises placental perfusate cells.
• said placental perfusate cells are from the same individual as said natural killer cells.
• said placental perfusate cells are from a different individual than said natural killer cells.
• the composition comprises isolated placental perfusate and isolated placental perfusate cells, wherein said isolated perfusate and said isolated placental perfusate cells are from different individuals.
• said placental perfusate comprises placental perfusate from at least two individuals.
• said isolated placental perfusate cells are from at least two individuals.
• the composition can additionally comprise isolated PINK cells, wherein the PINK cells are from a different individual than said placental perfusate or said perfusate cells.
• a method of isolating placental natural killer cells comprising obtaining a plurality of placental cells, and isolating natural killer cells from said plurality of placental cells.
• a method of isolating natural killer cells from a mixed source such as placenta (e.g. , placental perfusate) and umbilical cord blood, comprising obtaining a plurality of placental cells and umbilical cord blood cells, and isolating natural killer cells from said plurality of placental cells and umbilical cord blood cells.
• the placental cells are, or comprise, placental perfusate cells, e.g., total nucleated cells from placental perfusate.
• said plurality of placental cells are, or comprise, placental cells obtained by mechanical and/or enzymatic digestion of placental tissue.
• said isolating is performed using one or more antibodies.
• said one or more antibodies comprises one or more of antibodies to CD3, CD16 or CD56.
• said isolating comprises isolating CD56 + cells from CD56 cells in said plurality of placental cells.
• said isolating comprises isolating CD56 + , CD 16 placental cells from placental cells that are CD56 or CD 16 .
• said isolating comprises isolating CD56 + , CD 16 , CD3 placental cells from placental cells that are CD56 , CD16 + , or CD3 + .
• said method of isolating placental natural killer cells results in a population of placental cells that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% CD56 + , CD 16 natural killer cells.
• the placental perfusate cells have been expanded in culture.
• the placental perfusate cells and cells from another source have been expanded in culture.
• natural killer cells obtained from placenta e.g., placental perfusate
• a mixed source e.g., placenta and umbilical cord blood
• the cells have been expanded for at least, about, or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days.
• said cells have been expanded in the presence of a feeder layer and/or in the presence of at least one cytokine.
• said feeder layer comprises K562 cells or peripheral blood mononuclear cells (PBMCs), or a combination of both.
• said at least one cytokine is interleukin-2.
• a method of treating an individual having cancer comprising administering to said individual a therapeutically effective amount of placental perfusate, placental perfusate cells, placental intermediate natural killer cells, combined natural killer cells (e.g., natural killer cells obtained from placenta and another source such as umbilical cord blood), or combinations thereof, as described herein.
• said individual has a solid tumor.
• the individual has a blood cancer.
• said individual has primary ductal carcinoma, a leukemia, acute T cell leukemia, chronic myeloid lymphoma (CML), acute myelogenous leukemia, chronic myelogenous leukemia (CML), lung carcinoma, colon adenocarcinoma, histiocytic lymphoma, multiple myeloma, retinoblastoma, colorectal carcinoma, or colorectal adenocarcinoma.
• CML chronic myeloid lymphoma
• CML chronic myelogenous leukemia
• lung carcinoma colon adenocarcinoma
• histiocytic lymphoma multiple myeloma
• retinoblastoma retinoblastoma
• colorectal carcinoma colorectal adenocarcinoma.
• a method of treating an individual having graft-versus-host disease develops after an allogeneic bone marrow transplant.
• said graft-versus-host disease develops after a solid organ transplant.
• said graft-versus-host disease develops after a composite tissue allograft.
• said graft-versus-host disease is reduced in grade by at least one step by said administering.
• said graft-versus-host disease does not progress beyond grade II within 100 days after transplantation as a result of said administering.
• said graft-versus-host disease does not progress beyond grade I within 100 days after transplantation as a result of said administering.
• composite natural killer cells are natural killer cells, e.g., from matched umbilical cord blood and human placental perfusate, wherein placental perfusate is obtained from the same placenta as the cord blood. Natural killer cells from both are isolated separately or at the same time, and combined.
• PINK and PINK cells refer to placental intermediate natural killer cells that are obtained from human placenta, e.g., human placental perfusate or placental tissue that has been mechanically and/or enzymatically disrupted.
• the cells are CD56 + and CD16 , e.g., as determined by flow cytometry, e.g., fluorescence-activated cell sorting using antibodies to CD56 and CD 16.
• PINK cells are not obtained solely from cord blood or peripheral blood.
• placental perfusate means perfusion solution that has been passed through at least part of a placenta, e.g., a human placenta, e.g., through the placental vasculature, including a plurality of cells collected by the perfusion solution during passage through the placenta.
• placental perfusate cells means nucleated cells, e.g., total nucleated cells, isolated from, or isolatable from, placental perfusate.
• tumor cell suppression includes slowing the growth of a population of tumor cells, e.g., by killing one or more of the tumor cells in said population of tumor cells, for example, by contacting the population of tumor cells with PINK cells, a population of cells comprising PINK cells, combined natural killer cells, a population of cells comprising combined natural killer cells, human placental perfusate, or the like.
• FIG. 1 shows flow cytometry results using anti-CD3 antibodies and anti-CD56 antibodies for cells selected by CD56 microbeads from human placental perfusate (HPP). The majority of the isolated cells are CD56 + CD3 ⁇ .
• FIG. 2 depicts production of cytokines by PINK cells and/or tumor cells after 24 hour culture.
• FIG. 2A depicts secretion of interferon gamma (IFNy) by placental perfusate-derived intermediate natural killer cells (PINK) cells alone or in the presence of KG- la tumor cells.
• IFNy interferon gamma
• PINK cells and KG- la cells were cultured alone or in combination at a ratio of 1 : 1.
• Y axis picograms of IFNy produced by the cultures.
• FIG. 2B depicts secretion of granulocyte- macrophage colony stimulating factor (GM-CSF) by PINK cells alone or in the presence of KG- la tumor cells.
• GM-CSF granulocyte- macrophage colony stimulating factor
• FIG. 3 depicts cytotoxicity of PINK cells to KG- la tumor cells in 24 hour co-culture at a ratio of 1 : 1, 5: 1, 10: 1 or 20:1 PINK cells to tumor cells.
• X axis ratio of PINK cells to tumor cells.
• Y axis percentage of dead tumor cells compared to tumor cells without PINK cells.
• FIG. 4 depicts cytotoxicity of placental natural killer cells and peripheral blood (PB) NK cells cultured for 21 days towards K562 cells. Error bars stand for standard deviation of 4 units of cultured placental NK cells or 3 units of cultured peripheral blood NK cells.
• FIG. 5 depicts cytotoxicity of whole human placental perfusate, as obtained from the placenta, to KG- 1 a tumor cells in 24 hour co-culture at a ratio of 1 : 1 , 5: 1, 10: 1 or 20 : 1 or 100:1 HPP cells to tumor cells.
• X axis ratio of HPP cells to tumor cells.
• Y axis percentage of dead tumor cells compared to tumor cells without HPP cells.
• FIG. 6 depicts cytotoxicity of whole human placental perfusate, as obtained from the placenta, and umbilical cord blood, to KG- la tumor cells in 48 hour co-culture in serial dilutions of 100: 1, 50: 1, 25: 1, 12.5: 1, 6.25: 1, 3.12: 1, 1.56:1 or 0.78:1 HPP cells or UCB cells to tumor cells.
• X axis ratio of HPP cells or umbilical cord cells to tumor cells.
• Y axis percentage of dead tumor cells after 48 hours culture time compared to tumor cells without HPP cells or umbilical cord cells.
• FIG. 7 depicts cytotoxicity of whole human placental perfusate, as obtained from the placenta to KG-la tumor cells in 48 hour co-culture in serial dilutions of 100: 1, 50: 1, 25: 1, 12.5:1, 6.25: 1, 3.12: 1, 1.56: 1 or 0.78: 1 HPP cells to tumor cells.
• Perfusate was either used as collected, or stimulated for 24 hours with 100 U/mL or 1000 U/mL interleukin-2 (IL-2).
• X axis ratio of HPP cells to tumor cells.
• Y axis percentage of dead tumor cells after 48 hours culture time compared to tumor cells without HPP cells.
• FIG. 8 depicts the cytotoxic effect of human placental perfusate towards a panel of tumor cell lines after culture with HPP or UCB cells at a 50: 1 ratio to the tumor cells.
• FIG 8A co- culture for 24 hours.
• FIG. 8B co-culture for 48 hours.
• X axis tumor cell line tested.
• Y axis percentage of dead tumor cells after co-culture, compared to the number of tumor cells in the absence of tumor cells.
• FIG. 9 depicts IFNy production by HPP cells co-cultured with KG-la tumor cells at different ratios of HPP cells to tumor cells.
• X axis Experimental conditions, including ratio of HPP cells to tumor cells
• Y axis IFNy levels per milliliter after 24 hours co-culture..
• FIG. 10 Production of IFNy by HPP or UCB cells in co-culture with a panel of tumor cells.
• HPP or UCB cells were co-cultured at a ratio of 50: 1 with tumor cell lines for 24 hours (FIG. 10A) or 48 hours (FIG. 10B).
• IFNy levels were determined by Luminex assay (HCYTO- 60K-03, Millipore).
• X axis tumor cell line tested.
• Y axis picograms of IFNy produced by HPP or UCB cells, compared to picograms of IFNy produced in the absence of tumor cells.
• FIG. 11 depicts the reduction in tumor size upon administration of 2 x 10 7 human placental perfusate (HPP) cells to mice having KG-1 cell tumors approximately 332 mm 3 in volume.
• Intra-tumor - HPP cells were injected directly into the subcutaneous tumor site.
• IV - HPP cells administered intravenously.
• Control - vehicle administration only. Tumor volumes in mm 3 .
• FIG. 12 depicts a comparison of NK cells from donor-matched human placenta-derived stem cell and umbilical cord blood (CB) units.
• A Immunophenotypic characterization of NK cells from donor-matched human placenta-derived stem cell and CB units; the two-sample t-test was used to determine if population means are equal in human placenta-derived stem cells and CB.
• B Cytotoxicity of expanded NK cells from donor-matched human placenta-derived stem cell and CB units against K562 cells.
• FIG. 13 depicts phenotype characterization of placenta-derived NK cells (pNK) cells in comparison with peripheral blood (PB) NK cells.
• A Flow cytometric identification of NK cells from donor matched human placenta-derived stem cell and UCB (referred herein as "Combo unit") (pNK) and NK cells from PB (PB NK).
• CD56+CD3- gated NK cells expressed a repertoire of receptors important for regulating NK-cell activity: including CD 16, KIR3DL1, NKG2D, KIR2DL2/L3, NKp46, CD94, CD226, NKp44, NKp30 and 2B4.
• B Percentage of CD56+CD3- pNK cells from Combo unit prior to NK cell isolation.
• C 90% CD56+CD3- NK cells from Combo unit was achieved after NK cell isolation.
• FIG. 14 depicts cell cycle analysis of expanded pNK cells.
• A Representative APC- BrdU/7-AAD cell cycle analysis of ex vivo expanded pNK at different time points as indicated.
• FIG. 15 depicts fold expansion, phenotype and functional characterization of ex vivo expanded pNK cells.
• A Fold expansion and cell viability of Day 21 expanded pNK cells.
• B Phenotype characterization of Day 21 expanded pNK cells.
• C Cytotoxicity of expanded pNK cells against K562 at different time points as indicated in comparison with Day 21 expanded PB NK cells.
• FIG. 16 depicts cytotoxicity of ex vivo expanded Day 21 pNK cells against a wide range of tumor cell lines. Cytotoxicity of ex vivo expanded pNK cells against a wide range of tumor cell lines at E:T ratio of 10: 1, 5: 1, 2: 1 and 1 : 1 as indicated.
• placental perfusate, placental perfusate cells, and/or natural killer cells obtained therefore, such as placental perfusate-derived intermediate natural killer (“PINK”) cells or combined natural killer cells, to suppress the growth or proliferation of a tumor cell or plurality of tumor cells.
• PINK placental perfusate-derived intermediate natural killer
• NK cells natural killer cells
• populations of NK cells isolated from placental perfusate, e.g., human placental perfusate, or isolated from placental perfusate combined with cells from another source, e.g., umbilical cord blood (UCB), or isolated from placental tissue that has been disrupted mechanically and/or enzymatically, methods of obtaining the NK cells, and methods of using the cells.
• populations of cells e.g., populations of placental cells, comprising natural killer cells. Methods of obtaining placental perfusate, and obtaining cells from placental perfusate, are described in Section 5.1, below.
• Placental perfusate-derived natural killer cells e.g., placental intermediate natural killer (PINK) cells
• PINK placental intermediate natural killer
• Natural killer cells from placental perfusate cells and cells from another source such as umbilical cord blood, and methods of obtaining the combined natural killer cells, are described in Section 5.4, below.
• compositions comprising and methods of preserving and using the placental perfusate, placental perfusate-derived cells, placental perfusate-derived natural killer cells, e.g., intermediate natural killer cells, and combined natural killer cells, to suppress the proliferation of tumor cells, are described in Sections 5.5 to 5.7, below.
• the placental perfusate, perfusate cells and placental perfusate-derived natural killer cells provided herein can be collected by perfusion of a mammalian, e.g., human post-partum placenta using a placental cell collection composition.
• Perfusate can be collected from the placenta by perfusion of the placenta with any physiologically-acceptable solution, e.g., a saline solution, culture medium, or a more complex cell collection composition.
• a cell collection composition suitable for perfusing a placenta, and for the collection and preservation of perfusate cells, e.g., total nucleated placental perfusate cells or PINK cells, is described in detail in related U.S.
• the cell collection composition can comprise any physiologically-acceptable solution suitable for the collection and/or culture of stem cells, for example, a saline solution (e.g., phosphate-buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl. etc.), a culture medium (e.g., DMEM, H.DMEM, etc.), and the like.
• a saline solution e.g., phosphate-buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl. etc.
• a culture medium e.g., DMEM, H.DMEM, etc.
• the cell collection composition can comprise one or more components that tend to preserve placental cells, that is, prevent the placental cells from dying, or delay the death of the placental cells, reduce the number of placental cells in a population of cells that die, or the like, from the time of collection to the time of culturing.
• Such components can be, e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesium sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin- releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.); a necrosis inhibitor (e.g., 2-(lH-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); a TNF-a inhibitor; and/or an oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bro
• the cell collection composition can comprise one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, a hyaluronidase, an R ase, or a DNase, or the like.
• tissue-degrading enzymes include, but are not limited to, collagenases (e.g., collagenase I, II, III or IV, a collagenase from Clostridium histolyticum, etc); dispase, thermolysin, elastase, trypsin, LIB ERASE, hyaluronidase, and the like.
• the cell collection composition can comprise a bacteriocidally or bacteriostatically effective amount of an antibiotic.
• the antibiotic is a macro lide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc.
• the antibiotic is active against Gram(+) and/or Gram(-) bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus, and the like.
• the cell collection composition can also comprise one or more of the following compounds: adenosine (about 1 mM to about 50 mM); D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50 mM); a macromolecule of molecular weight greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain endothelial integrity and cellular viability (e.g.
• a synthetic or naturally occurring colloid a polysaccharide such as dextran or a polyethylene glycol present at about 25 g/1 to about 100 g/1, or about 40 g/1 to about 60 g/1); an antioxidant (e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present at about 25 ⁇ to about 100 ⁇ ); a reducing agent (e.g., N-acetylcysteine present at about 0.1 mM to about 5 mM); an agent that prevents calcium entry into cells (e.g., verapamil present at about 2 ⁇ to about 25 ⁇ );
• an antioxidant e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present at about 25 ⁇ to about 100 ⁇
• a reducing agent e.g., N-acetylcysteine present at about 0.1 m
• nitroglycerin e.g., about 0.05 g/L to about 0.2 g/L
• an anticoagulant in one embodiment, present in an amount sufficient to help prevent clotting of residual blood (e.g., heparin or hirudin present at a concentration of about 1000 units/1 to about 100,000 units/1); or an amiloride containing compound (e.g., amiloride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride present at about 1.0 ⁇ to about 5 ⁇ ).
• an amiloride containing compound e.g., amiloride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride present at about 1.0 ⁇ to about 5 ⁇ ).
• a human placenta is recovered shortly after its expulsion after birth.
• the placenta is recovered from a patient after informed consent and after a complete medical history of the patient is taken and is associated with the placenta.
• the medical history continues after delivery.
• Such a medical history can be used to coordinate subsequent use of the placenta or the cells harvested therefrom.
• human placental cells can be used, in light of the medical history, for personalized medicine for the infant associated with the placenta, or for parents, siblings or other relatives of the infant.
• the umbilical cord blood and placental blood Prior to recovery of perfusate, the umbilical cord blood and placental blood are removed. In certain embodiments, after delivery, the cord blood in the placenta is recovered.
• the placenta can be subjected to a conventional cord blood recovery process. Typically a needle or cannula is used, with the aid of gravity, to exsanguinate the placenta (see, e.g., Anderson, U.S. Patent No. 5,372,581; Hessel et ah, U.S. Patent No. 5,415,665).
• the needle or cannula is usually placed in the umbilical vein and the placenta can be gently massaged to aid in draining cord blood from the placenta.
• cord blood recovery may be performed commercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell.
• the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.
• a placenta is transported from the delivery or birthing room to another location, e.g., a laboratory, for recovery of cord blood and collection of perfusate.
• the placenta is preferably transported in a sterile, thermally insulated transport device (maintaining the temperature of the placenta between 20-28°C), for example, by placing the placenta, with clamped proximal umbilical cord, in a sterile zip-lock plastic bag, which is then placed in an insulated container.
• the placenta is transported in a cord blood collection kit substantially as described in U.S. Patent No. 7,147,626.
• the placenta is delivered to the laboratory four to twenty-four hours following delivery.
• the proximal umbilical cord is clamped, preferably within 4-5 cm (centimeter) of the insertion into the placental disc prior to cord blood recovery.
• the proximal umbilical cord is clamped after cord blood recovery but prior to further processing of the placenta.
• the placenta prior to collection of the perfusate, can be stored under sterile conditions and at either room temperature or at a temperature of 5 to 25°C (centigrade).
• the placenta may be stored for a period of longer than forty eight hours, and preferably for a period of four to twenty- four hours prior to perfusing the placenta to remove any residual cord blood.
• the placenta is preferably stored in an anticoagulant solution at a temperature of 5°C to 25°C
• Suitable anticoagulant solutions are well known in the art.
• a solution of heparin or warfarin sodium can be used.
• the anticoagulant solution comprises a solution of heparin (e.g., 1% w/w in 1 : 1000 solution).
• the exsanguinated placenta is preferably stored for no more than 36 hours before placental perfusate is collected.
• Perfusate can be obtained by passage of perfusion solution, e.g., saline solution, culture medium or cell collection compositions described above, through the placental vasculature.
• perfusion solution e.g., saline solution, culture medium or cell collection compositions described above
• a mammalian placenta is perfused by passage of perfusion solution through either or both of the umbilical artery and umbilical vein.
• the flow of perfusion solution through the placenta may be accomplished using, e.g., gravity flow into the placenta.
• the perfusion solution is forced through the placenta using a pump, e.g., a peristaltic pump.
• the umbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula, that is connected to a sterile connection apparatus, such as sterile tubing.
• a sterile connection apparatus such as sterile tubing.
• the sterile connection apparatus is connected to a perfusion manifold.
• the placenta is preferably oriented in such a manner that the umbilical artery and umbilical vein are located at the highest point of the placenta.
• the placenta can be perfused by passage of a perfusion solution through the placental vasculature, or through the placental vasculature and surrounding tissue.
• the umbilical artery and the umbilical vein are connected simultaneously to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution.
• the perfusion solution is passed into the umbilical vein and artery.
• the perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation.
• the perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the maternal uterine wall.
• the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins, that is, is passed through only the placental vasculature (fetal tissue).
• the umbilical artery and the umbilical vein are connected simultaneously, e.g., to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution.
• the perfusion solution is passed into the umbilical vein and artery.
• the perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation.
• the perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the maternal uterine wall.
• Placental cells that are collected by this method which can be referred to as a "pan” method, are typically a mixture of fetal and maternal cells.
• the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins.
• Placental cells collected by this method which can be referred to as a "closed circuit" method, are typically almost exclusively fetal.
• the closed circuit perfusion method can, in one embodiment, be performed as follows.
• a post-partum placenta is obtained within about 48 hours after birth.
• the umbilical cord is clamped and cut above the clamp.
• the umbilical cord can be discarded, or can processed to recover, e.g., umbilical cord stem cells, and/or to process the umbilical cord membrane for the production of a biomaterial.
• the amniotic membrane can be retained during perfusion, or can be separated from the chorion, e.g., using blunt dissection with the fingers.
• amniotic membrane If the amniotic membrane is separated from the chorion prior to perfusion, it can be, e.g., discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce, e.g., an amniotic membrane biomaterial, e.g., the biomaterial described in U.S. Application Publication No.
• the umbilical cord vessels are exposed, e.g., by partially cutting the umbilical cord membrane to expose a cross-section of the cord.
• the vessels are identified, and opened, e.g., by advancing a closed alligator clamp through the cut end of each vessel.
• the apparatus e.g., plastic tubing connected to a perfusion device or peristaltic pump, is then inserted into each of the placental arteries.
• the pump can be any pump suitable for the purpose, e.g., a peristaltic pump.
• Plastic tubing connected to a sterile collection reservoir, e.g., a blood bag such as a 250 mL collection bag, is then inserted into the placental vein.
• a sterile collection reservoir e.g., a blood bag such as a 250 mL collection bag
• the tubing connected to the pump is inserted into the placental vein, and tubes to a collection reservoir(s) are inserted into one or both of the placental arteries.
• the placenta is then perfused with a volume of perfusion solution, e.g., about 750 ml of perfusion solution. Cells in the perfusate are then collected, e.g., by centrifugation.
• the proximal umbilical cord is clamped during perfusion, and more preferably, is clamped within 4-5 cm (centimeter) of the cord's insertion into the placental disc.
• exsanguination process is generally colored with residual red blood cells of the cord blood and/or placental blood.
• the perfusion fluid becomes more colorless as perfusion proceeds and the residual cord blood cells are washed out of the placenta.
• perfusion fluid Generally from 30 to 100 mL of perfusion fluid is adequate to initially flush blood from the placenta, but more or less perfusion fluid may be used depending on the observed results.
• the volume of perfusion liquid used to perfuse the placenta may vary depending upon the number of placental cells to be collected, the size of the placenta, the number of collections to be made from a single placenta, etc.
• the volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL.
• the placenta is perfused with 700-800 mL of perfusion liquid following exsanguination.
• the placenta can be perfused a plurality of times over the course of several hours or several days. Where the placenta is to be perfused a plurality of times, it may be maintained or cultured under aseptic conditions in a container or other suitable vessel, and perfused with a cell collection composition, or a standard perfusion solution ⁇ e.g., a normal saline solution such as phosphate buffered saline ("PBS") with or without an anticoagulant (e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g., ⁇ - mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 ⁇ g/ml), penicillin (e.g., at 40U/ml), amphotericin B (e.g., at 0.5 ⁇ g/ml).
• PBS phosphate buffered saline
• an isolated placenta is maintained or cultured for a period of time without collecting the perfusate, such that the placenta is maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days before perfusion and collection of perfusate.
• the perfused placenta can be maintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a second time with, e.g., 700-800 mL perfusion fluid.
• the placenta can be perfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours.
• perfusion of the placenta and collection of perfusion solution e.g., placental cell collection composition, is repeated until the number of recovered nucleated cells falls below 100 cells/ml.
• the perfusates at different time points can be further processed individually to recover time-dependent populations of cells, e.g., total nucleated cells. Perfusates from different time points can also be pooled.
• placental perfusate from a single placental perfusion comprises about 100 million to about 500 million nucleated cells.
• the placental perfusate or perfusate cells comprise stem cells.
• the placental perfusate or perfusate cells comprise CD34 + cells, e.g., hematopoietic stem or progenitor cells.
• Such cells can, in a more specific embodiment, comprise CD34 CD45 stem or progenitor cells, CD34 + CD45 + stem or progenitor cells, myeloid progenitors, lymphoid progenitors, and/or erythroid progenitors.
• placental perfusate as collected from the placenta and depleted of erythrocytes, or perfusate cells isolated from such perfusate comprise about 6-7% natural killer cells (CD3 , CD56 + ); about 21-22% T cells (CD3 + ); about 6-7% B cells (CD19 + ); about 1-2%) endothelial progenitor cells (CD34 + , CD31 + ); about 2-3% neural progenitor cells (nestin + ); about 1-5% (e.g., 2-5%) hematopoietic progenitor cells (CD34 + ); and about 0.5-1.5%) adherent placental stem cells (e.g., CD34 , CD117 " , CD105 + and CD44 + ), as determined, e.g. by flow cytometry, e.g., by FACS analysis.
• adherent placental stem cells e.g., CD34 , CD117 " , CD105 + and CD44 +
• said at least one cytokine is
• an isolated population of cells e.g., placental cells, comprising PINK cells.
• the isolated population of cells is total nucleated cells from placental perfusate, e.g., placental perfusate cells, comprising autologous, isolated PINK cells.
• the population of cells is an isolated population of cells produced by CD56-microbead isolation of cells from placental perfusate.
• the population comprises at least about 50%, 55%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least about 99% PINK cells.
• the post-partum placenta comprises tissue and cells from the fetus and from the mother placental perfusate, depending upon the method of collection, can comprise fetal cells only, or a substantial majority of fetal cells ⁇ e.g., greater than about 90%>, 95%, 98% or 99%), or can comprise a mixture of fetal and maternal cells ⁇ e.g., the fetal cells comprise less than about 90%), 80%), 70%), 60%), or 50% of the total nucleated cells of the perfusate).
• PINK cells that also serve as a cell proliferation and functions as a cell proliferation.
• PINK cells that also serve as a cell proliferation and functions as a cell proliferation.
• a method of isolating placental natural killer cells comprising obtaining a plurality of placental cells, and isolating natural killer cells from said plurality of placental cells.
• the placental cells are, or comprise, placental perfusate cells, e.g., total nucleated cells from placental perfusate.
• said plurality of placental cells are, or comprise, placental cells obtained by mechanical and/or enzymatic digestion of placental tissue.
• said isolating is performed using one or more antibodies.
• said one or more antibodies comprises one or more of antibodies to CD3, CD16 or CD56.
• matched units indicates that the NK cells are obtained from placental perfusate cells, and umbilical cord blood cells, wherein the umbilical cord blood cells are obtained from umbilical cord blood from the placenta from which the placental perfusate is obtained, i.e., the placental perfusate cells and umbilical cord blood cells, and thus the natural killer cells from each, are from the same individual.
• such matched units are referred to as "donor matched" units.
• the combined natural killer cells comprise natural killer cells from placental perfusate and natural killer cells from umbilical cord blood in, e.g., a ratio of about 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3, 9:2, 10:1, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 100:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45: 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100:1, 95:1, 90:1, 85:1,80:1,75:1,70:1,65:1,60:1,55:1,50:1,45:1,40:1,35:1,30:1,25:1,
• the combined natural killer cells comprise a detectably lower number of CD3 CD56 KIR2DL2/L3 + natural killer cells than an equivalent number of natural killer cells from peripheral blood. In another specific embodiment, the combined natural killer cells comprise a detectably higher number of CD3 CD56 + NKp44 + natural killer cells than an equivalent number of natural killer cells from peripheral blood. In a specific embodiment, the combined natural killer cells comprise a detectably higher number of CD3 CD56 NKp30 natural killer cells than an equivalent number of natural killer cells from peripheral blood.
• the combined natural killer cells comprise a detectably lower amount of NKp46+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In certain specific embodiments, the combined natural killer cells comprise less than 5%, 10%, 15%, 20%, or 25% NKp46+ natural killer cells.
• the combined natural killer cells do not detectably express miRNAs hsa-let-7b, hsa-miR-146b, hsa-miR-19b, hsa-miR-24, hsa-miR-347, hsa-miR-381 , hsa-miR-517c and hsa-miR-631.
• miRNAs hsa-let-7b hsa-miR-146b, hsa-miR-19b, hsa-miR-24, hsa-miR-347, hsa-miR-381 , hsa-miR-517c and hsa-miR-631.
• the combined natural killer cells have not been cultured or expanded.
• the combined natural killer cells have been expanded, e.g., for 7 days, 10 days, 14 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more.
• the combined natural killer cells are expanded for more than 14 days.
• the combined natural killer cells are expanded for less than 28 days.
• the combined natural killer cells are expanded for 21 days.
• expansion is ex vivo.
• expansion of combined natural killer cells is in the presence of feeder cells.
• expanded combined natural killer cells induce interferon
• the PINK cells when PINK cells are combined with a plurality of placental perfusate cells and/or combined natural killer cells, the PINK cells generally comprise about, greater than about, or fewer than about, 50%>, 45%, 40%>, 35%, 30%>, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells.
• the combined natural killer cells when combined natural killer cells are combined with PINK cells and/or placental perfusate cells, the combined natural killer cells generally comprise about, greater than about, or fewer than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells.
• any of the above combinations is, in turn, combined with umbilical cord blood or nucleated cells from umbilical cord blood.
• Perfusate from multiple perfusions of the same placenta can similarly be pooled.
• placental perfusate cells, and placenta-derived intermediate natural killer cells that are obtained from two or more sources, e.g., two or more placentas, and pooled.
• Such pooled cells can comprise approximately equal numbers of cells from the two or more sources, or different numbers of cells from one or more of the pooled sources.
• the relative numbers of cells from each source can be selected based on, e.g., the number of one or more specific cell types in the cells to be pooled, e.g., the number of CD34 + cells, the number of CD56 + cells, etc.
• Pools can comprise, e.g., placental perfusate supplemented with placental perfusate cells; placental perfusate supplemented with placenta-derived intermediate natural killer (PINK) cells; placental perfusate supplemented with both placental perfusate cells and PINK cells; placental perfusate cells supplemented with placental perfusate; placental perfusate cells supplemented with PINK cells; placental perfusate cells supplemented with both placental perfusate and PINK cells; PINK cells supplemented with placental perfusate; PINK cells supplemented with placental perfusate cells; or PINK cells supplemented with both placental perfusate cells and placental perfusate.
• PINK intermediate natural killer
• an aliquot or sample number of cells is contacted with a known number of tumor cells under conditions in which the tumor cells would otherwise proliferate, and the rate of proliferation of the tumor cells in the presence of placental perfusate, perfusate cells, placental natural killer cells, or combinations thereof, over time ⁇ e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or longer) is compared to the proliferation of an equivalent number of the tumor cells in the absence of perfusate, perfusate cells, placental natural killer cells, or combinations thereof.
• the potency of the placental perfusate, placental perfusate cells and/or PINK cells, or combinations or pools of the same can be expressed, e.g., as the number of cells or volume of solution required to suppress tumor cell growth, e.g., by about 10%, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, or the like.
• placental perfusate, placental perfusate cells, and PINK cells are provided as pharmaceutical grade administrable units. Such units can be provided in discrete volumes, e.g., 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, 500 mL, or the like.
• the combinations comprise about, at least about, or at most about 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 or more such natural killer cells per milliliter, or 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , 1 x 10 11 or more cells per unit.
• the CD56 CD 16 natural killer cells can be used as isolated from a natural source, or can be expanded prior to inclusion in one of the above combinations or pools.
• the CD56 CD16 + NK cells can be autologous (that is, obtained from the same individual as the placental perfusate, placental perfusate cells and/or PINK cells; or obtained from a recipient) or homologous (that is, derived from an individual different from the placental perfusate, placental perfusate cells and/or PINK cells; or from an individual that is not recipient).
• each unit is labeled to specify volume, number of cells, type of cells, whether the unit has been enriched for a particular type of cell, and/or potency of a given number of cells in the unit, or a given number of milliliters of the unit, causes a measurable suppression of proliferation of a particular type or types of tumor cell.
• compositions comprising placental intermediate natural killer cells, alone or in combination with placental perfusate cells and/or placental perfusate.
• a composition comprising isolated CD56 , CD 16 natural killer cells, wherein said natural killer cells are isolated from placental perfusate, and wherein said natural killer cells comprise at least 50% of cells in the composition.
• said natural killer cells comprise at least 80% of cells in the composition.
• said composition comprises isolated CD56 + , CD16 + natural killer cells.
• said CD56 + , CD16 + natural killer cells are from a different individual than said CD56 + , CD 16 natural killer cells.
• said natural killer cells are from a single individual.
• said isolated natural killer cells comprise natural killer cells from at least two different individuals.
• the composition comprises isolated placental perfusate.
• said placental perfusate is from the same individual as said natural killer cells.
• said placental perfusate comprises placental perfusate from a different individual than said natural killer cells.
• the natural killer cells are from a single individual.
• said isolated natural killer cells comprise natural killer cells from at least two different individuals.
• the composition comprises isolated placental perfusate.
• said placental perfusate is from the same individual as said natural killer cells.
• said placental perfusate comprises placental perfusate from a different individual than said natural killer cells.
• the natural killer cells are from a single individual.
• said isolated natural killer cells comprise natural killer cells from at least two different individuals.
• the composition comprises isolated placental perfusate.
• said placental perfusate is from the same individual as said natural killer cells.
• compositions Comprising Adherent Placental Stem Cells
• the placental perfusate, plurality of placental perfusate cells, plurality of combined natural killer cells, and/or plurality of PINK cells, or a combination or pool of any of the foregoing can be supplemented with, e.g., 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 or more cells per milliliter, or 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , 1 x 10 11 or more adherent
• Adherent placental stem cells are, however, morphologically distinguishable from fibroblasts cultured under the same conditions, as the placental stem cells exhibit a greater number of such processes than do fibroblasts. Morphologically, placental stem cells are also distinguishable from hematopoietic stem cells, which generally assume a more rounded, or cobblestone, morphology in culture.
• Adherent placental stem cells generally express the markers CD73, CD105,
• the adherent placental stem cells can express CD200 and HLA-G, a fetal-specific marker, they can be distinguished from mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells, which express neither CD200 nor HLA-G.
• the lack of expression of CD34, CD38 and/or CD45 identifies the adherent placental stem cells as non-hematopoietic stem cells.
• adherent placental stem cells are CD73 + , CD105 + ,
• said stem cells detectably suppress cancer cell proliferation or tumor growth.
• said adherent stem cells are HLA-G + .
• said adherent stem cells are CD34 , CD38 or CD45 .
• said adherent stem cells are CD34 , CD38 and CD45 .
• said adherent stem cells are CD34 , CD38 , CD45 , and HLA-G .
• said adherent placental stem cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
• the adherent placental stem cells are CD200 + , OCT-4 + , wherein said stem cells detectably suppress cancer cell proliferation or tumor growth.
• said adherent stem cells are CD73 + and CD105 + .
• said adherent stem cells are HLA-G + .
• said adherent stem cells are CD34 , CD38 and CD45 .
• said adherent stem cells are CD34 , CD38 , CD45 , CD73 + , CD105 + and HLA-G + .
• the adherent placental stem cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
• adherent placental stem cells are CD73 + , CD105 + and
• the adherent placental stem cells are CD73 + , CD105 + stem cells, wherein said stem cells produce one or more embryoid-like bodies under conditions that allow formation of embryoid-like bodies, and wherein said adherent stem cells detectably suppress cancer cell proliferation or tumor growth.
• said adherent stem cells are also CD34 , CD38 or CD45 .
• said adherent stem cells are also CD34 , CD38 and CD45 .
• said adherent stem cells are also OCT-4 + .
• said adherent stem cells are also OCT-4 , CD34 , CD38 and CD45 .
• At least 10%, at least 20%>, at least 30%>, at least 40%>, at least 50% at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said isolated placental cells are OCT4 + stem cells.
• said stem cells are CD73 + and CD105 + .
• said stem cells are CD34 , CD38 , or CD45 .
• said stem cells are CD200 + .
• said stem cells are CD73 + , CD105 + , CD200 + , CD34 , CD38 , and CD45 .
• said population has been expanded, for example, passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.
• the adherent placental cells express ABC-p (a placenta-specific ABC transporter protein; see, e.g., AUikmets et al, Cancer Res. 58(23):5337-9 (1998)).
• ABC-p a placenta-specific ABC transporter protein
• adherent placental stem cells are CD29 + , CD44 + ,
• the adherent placental stem cells constitutively secrete IL-6, IL-8 and monocyte chemoattractant protein (MCP-1).
• MCP-1 monocyte chemoattractant protein
• Each of the above -referenced placental stem cells can comprise placental stem cells obtained and isolated directly from a mammalian placenta, or placental stem cells that have been cultured and passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30 or more times, or a combination thereof.
• Tumor cell suppressive pluralities of the adherent placental stem cells described above can comprise about, at least, or no more than, 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , 1 x 10 11 or more adherent placental stem cells.
• compositions Comprising Placental Stem Cell Conditioned Media
• a tumor-suppressive composition comprising
• PINK cells combined natural killer cells, and/or placental perfusate, and additionally
• conditioned medium Adherent placental stem cells, placental perfusate cells, combined natural killer cells, and/or placental intermediate natural killer cells can be used to produce conditioned medium that is tumor cell suppressive, that is, medium comprising one or more biomolecules secreted or excreted by the stem cells that have a detectable tumor cell suppressive effect on a plurality of one or more types of immune cells.
• the conditioned medium comprises medium in which placental cells (e.g., stem cells, placental perfusate cells, PINK cells) or combined natural killer cells have grown for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more days.
• the conditioned medium comprises medium in which the cells have grown to at least 30%, 40%>, 50%), 60%), 70%), 80%), 90%) confluence, or up to 100% confluence.
• Such conditioned medium can be used to support the culture of a separate population of placental cells, or cells of another kind.
• the conditioned medium provided herein comprises medium in which adherent placental stem cells and non-placental stem cells have been cultured.
• Such conditioned medium can be combined with any of, or any combination of, placental perfusate, placental perfusate cells, combined natural killer cells and/or placental intermediate natural killer cells to form a tumor cell suppressive composition.
• the composition comprises less than half conditioned medium by volume, e.g., about, or less than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%) by volume.
• a composition comprising culture medium from a culture of placental stem cells, wherein said placental stem cells (a) adhere to a substrate; (b) express CD200 and HLA-G, or express CD73, CD 105, and CD200, or express CD200 and OCT-4, or express CD73, CD 105, and HLA-G, or express CD73 and CD 105 and facilitate the formation of one or more embryoid-like bodies in a population of placental cells that comprise the placental stem cells, when said population is cultured under conditions that allow formation of embryoid-like bodies, or express OCT-4 and facilitate the formation of one or more embryoid-like bodies in a population of placental cells that comprise the placental stem cells when said population is cultured under conditions that allow formation of embryoid-like bodies; and (c) detectably suppress the growth or proliferation of a tumor cell or population of tumor cells.
• the composition further comprises a plurality of said placental stem cells.
• the composition comprises a plurality of non-placental cells.
• said non-placental cells comprise CD34+ cells, e.g., hematopoietic progenitor cells, such as peripheral blood hematopoietic progenitor cells, cord blood hematopoietic progenitor cells, or placental blood hematopoietic progenitor cells.
• the non-placental cells can also comprise other stem cells, such as mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells.
• the non-placental cells can also be one or more types of adult cells or cell lines.
• the composition comprises an anti-proliferative agent, e.g., an anti-MIP-l or anti- ⁇ - ⁇ antibody.
• placental cell-conditioned culture medium or supernatant is obtained from a plurality of placental stem cells co-cultured with a plurality of tumor cells at a ratio of about 1 : 1, about 2: 1, about 3: 1, about 4: 1 , or about 5 : 1 placental stem cells to tumor cells.
• the conditioned culture medium or supernatant can be obtained from a culture comprising about 1 x 10 5 placental stem cells, about 1 x 10 6 placental stem cells, about 1 x 10 7 placental stem cells, or about 1 x 10 8 placental stem cells, or more.
• the conditioned culture medium or supernatant is obtained from a co-culture comprising about 1 x 10 5 to about 5 x 10 5 placental stem cells and about 1 x 10 5 tumor cells; about 1 x 10 6 to about 5 x 10 6 placental stem cells and about 1 x 10 6 tumor cells; about 1 x 10 7 to about 5 x 10 7 placental stem cells and about 1 x 10 7 tumor cells; or about 1 x 10 8 to about 5 x 10 8 placental stem cells and about 1 x 10 8 tumor cells.
• Conditioned medium can be condensed to prepare an administrable pharmaceutical-grade product.
• conditioned medium can be condensed to about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or more by removal of water, e.g., by evaporation, lyophilization, or the like.
• 200 mL conditioned medium from about 70 million placental stem cells can be condensed to a volume of about 180 mL, 160 mL, 140 mL, 120 mL, 100 mL, 80 mL, 60 mL, 40 mL, 20 mL or less.
• the conditioned medium can also be substantially dried, e.g., to a powder, e.g., by evaporation, lyophilization or the like.
• Placental perfusate, placental cells, e.g., perfusate cells or PINK cells, and combined natural killer cells can be preserved, that is, placed under conditions that allow for long-term storage, or under conditions that inhibit cell death by, e.g., apoptosis or necrosis.
• Placental perfusate can be produced by passage of a placental cell composition through at least a part of the placenta, e.g., through the placental vasculature.
• the placental cell collection composition comprises one or more compounds that act to preserve the cells contained within the perfusate.
• a placental cell collection composition is described in Section 5.1, above, e.g., a composition comprising an apoptosis inhibitor, necrosis inhibitor and/or an oxygen-carrying perfluorocarbon, as described in related U.S. Application Publication No. US 2007-0190042, published August 16, 2007.
• the placental cell collection composition, passed through the placenta or placental tissue is the placental perfusate useful in the methods described in Section 5.4, below.
• placental perfusate and/or placental cells are collected from a mammalian, e.g., human, post-partum placenta by contacting the cells with a placental cell collection composition comprising an inhibitor of apoptosis and an oxygen-carrying
• the inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis in the population of stem cells, as compared to a population of stem cells not contacted with the inhibitor of apoptosis.
• the placenta can be perfused with the placental cell collection composition, and placental cells, e.g., total nucleated placental cells, are isolated therefrom.
• the inhibitor of apoptosis is a caspase inhibitor.
• said inhibitor of apoptosis is a JNK inhibitor.
• said JNK inhibitor does not modulate differentiation or proliferation of said stem cells.
• the placental cell collection composition comprises said inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in separate phases.
• the placental cell collection composition comprises said inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in an emulsion.
• the placental cell collection composition additionally comprises an emulsifier, e.g., lecithin.
• said apoptosis inhibitor and said perfluorocarbon are between about 0°C and about 25°C at the time of contacting the placental cells.
• said apoptosis inhibitor and said perfluorocarbon are between about 2°C and 10°C, or between about 2°C and about 5°C, at the time of contacting the placental cells.
• said contacting is performed during transport of said population of stem cells.
• said contacting is performed during freezing and thawing of said population of stem cells.
• placental perfusate and/or placental cells can be collected and preserved by contacting the perfusate and/or cells with an inhibitor of apoptosis and an organ-preserving compound, wherein said inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis of the cells, as compared to perfusate or placental cells not contacted with the inhibitor of apoptosis.
• the organ-preserving compound is UW solution
• said organ-preserving composition is hydroxyethyl starch, lactobionic acid, raffinose, or a combination thereof.
• the placental cell collection composition additionally comprises an oxygen-carrying perfluorocarbon, either in two phases or as an emulsion.
• placental perfusate and/or placental cells are contacted with a placental cell collection composition comprising an apoptosis inhibitor and oxygen-carrying perfluorocarbon, organ-preserving compound, or combination thereof, during perfusion.
• placental cells are contacted with said stem cell collection compound after collection by perfusion.
• placental perfusate a placental cell, or population of placental cells
• a hypoxic condition is a concentration of oxygen that is less than normal blood oxygen concentration.
• said population of placental cells is exposed to said hypoxic condition for less than two hours during said preservation.
• said population of placental cells is exposed to said hypoxic condition for less than one hour, or less than thirty minutes, or is not exposed to a hypoxic condition, during collection, enrichment or isolation. In another specific embodiment, said population of placental cells is not exposed to shear stress during collection, enrichment or isolation.
• cryopreservation medium includes, but is not limited to, culture medium including, e.g., growth medium, or cell freezing medium, for example commercially available cell freezing medium, e.g., C2695, C2639 or C6039 (Sigma).
• Cryopreservation medium preferably comprises DMSO (dimethylsulfoxide), at a concentration of, e.g., about 10% (v/v).
• Cryopreservation medium may comprise additional agents, for example, methylcellulose and/or glycerol.
• Cells are preferably cooled at about l°C/min during cryopreservation.
• a preferred cryopreservation temperature is about -80°C to about -180°C, preferably about -125°C to about -140°C.
• Cryopreserved cells can be transferred to liquid nitrogen prior to thawing for use. In some embodiments, for example, once the ampoules have reached about -90°C, they are transferred to a liquid nitrogen storage area.
• Cryopreserved cells preferably are thawed at a temperature of about 25°C to about 40°C, preferably to a temperature of about 37°C.
• a method of suppressing the proliferation of a tumor cell, or plurality of tumor cells comprising contacting the tumor cell or plurality of tumor cells with placental perfusate, placental perfusate cells, combined natural killer cells, and/or PINK cells, such that the proliferation of the tumor cell or plurality of tumor cells is detectably reduced compared to a tumor cell or plurality of tumor cells of the same type not contacted with the placental perfusate, perfusate cells, combined natural killer cells, and/or PINK cells.
• contacting in one embodiment, encompasses direct physical, e.g., cell-cell, contact between placental perfusate, placental perfusate cells, placental natural killer cells, e.g., placental intermediate natural killer cells, and/or combined natural killer cells; and a tumor cell or plurality of tumor cells.
• "contacting" encompasses presence in the same physical space, e.g., placental perfusate, placental perfusate cells, placental natural killer cells, e.g., placental intermediate natural killer cells, and/or combined natural killer cells are placed in the same container e.g., culture dish, multiwell place) as a tumor cell or plurality of tumor cells.
• "contacting" placental perfusate, placental perfusate cells, combined natural killer cells or placental intermediate natural killer cells, and a tumor cell or plurality of tumor cells is accomplished, e.g., by injecting or infusing the placental perfusate or cells, e.g., placental perfusate cells, combined natural killer cells or placental intermediate natural killer cells into an individual, e.g., a human comprising a tumor cell or plurality of tumor cells, e.g., a cancer patient.
• placental perfusate is used in any amount that results in a detectable therapeutic benefit to an individual comprising a tumor cell or plurality of tumor cells, e.g., a cancer patient.
• placental perfusate cells, placental intermediate natural killer cells, and/or combined natural killer cells are used in any amount that results in a detectable therapeutic benefit to an individual comprising a tumor cell or plurality of tumor cells.
• a method of suppressing the proliferation of a tumor cell, or plurality of tumor cells comprising contacting the tumor cell or plurality of tumor cells with placental perfusate, placental perfusate cells, a placenta-derived intermediate natural killer cell, or plurality of PINK cells, and/or combined natural killer cells within an individual such that said contacting is detectably or demonstrably therapeutically beneficial to said individual.
• therapeutic benefits include, but are not limited to, e.g., reduction in the size of a tumor; lessening or cessation of expansion of a tumor; reduction in the number of cancer cells in a tissue sample, e.g., a blood sample, per unit volume; the clinical improvement in any symptom of the particular cancer said individual has, the lessening or cessation of worsening of any symptom of the particular cancer the individual has, etc.
• placental perfusate cells e.g., nucleated cells from placental perfusate, combined natural killer cells, and/or placental intermediate natural killer cells are used in any amount or number that results in a detectable therapeutic benefit to an individual comprising a tumor cell or plurality of tumor cells, e.g., a cancer patient.
• Placental perfusate cells, combined natural killer cells and/or placental natural killer cells, e.g., placental intermediate natural killer cells can be administered to such an individual by numbers of cells, e.g., said individual can be administered at about, at least about, or at most about, 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , or 5 x 10 10 placental perfusate cells, combined natural killer cells and/or placenta-derived intermediate natural killer cells.
• placental perfusate cells, combined natural killer cells, and/or placenta-derived intermediate natural killer cells can be administered to such an individual by numbers of cells, e.g., said individual can be administered at about, at least about, or at most about, 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , or 5 x 10 10 placental perfusate cells, combined natural killer cells, and/or placenta-derived intermediate natural killer cells per kilogram of the individual.
• Placental perfusate cells, combined natural killer cells and/or placenta-derived intermediate natural killer cells can be administered to such an individual according to an approximate ratio between placental perfusate cells, combined natural killer cells and/or placental natural killer cells, e.g., placental
• placental perfusate cells, combined natural killer cells and/or placental natural killer cells, e.g., placental intermediate natural killer cells can be administered to said individual in a ratio of about, at least about or at most about 1 : 1, 1 :1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1, 20: 1, 25:1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60:1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1 or 100: 1 to the number of tumor cells in the individual.
• the number of tumor cells in such an individual can be estimated, e.g., by counting the number of tumor cells in a sample of tissue from the individual, e.g., blood sample, biopsy, or the like. In specific embodiments, e.g., for solid tumors, said counting is performed in combination with imaging of the tumor or tumors to obtain an approximate tumor volume.
• a method for the suppression of the proliferation of a tumor cell or plurality of tumor cells using combinations of placental perfusate, placental perfusate cells, combined natural killer cells, and/or placenta-derived intermediate natural killer cells.
• a method of suppressing the proliferation of a tumor cell or plurality of tumor cells comprising contacting said tumor cell or tumor cells with placental perfusate supplemented with a plurality of placental perfusate cells, combined natural killer cells or PINK cells; placental perfusate cells supplemented with placental perfusate or a plurality of combined natural killer cells or PINK cells; PINK cells or combined natural killer cells supplemented with placental perfusate and placental perfusate cells, a plurality of PINK cells and/or a plurality of combined natural killer cells; a plurality of combined natural killer cells and a plurality of placental perfusate cells; placental perfusate supplemented with combined natural killer cells; or a combination of all of placental perfusate, placental perfusate cells, combined natural killer cells, and PINK cells.
• the proliferation of a tumor cell or plurality of tumor cells is suppressed by placental perfusate supplemented with a plurality of placental perfusate cells, combined natural killer cells and/or a plurality of placental intermediate natural killer cells.
• each milliliter of placental perfusate is supplemented with about 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 or more placental perfusate cells, combined natural killer cells or placental intermediate natural killer cells.
• placental perfusate e.g., one unit ⁇ i.e., the collection from a single placenta), or about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mL of perfusate, is supplemented with about 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x
• the proliferation of a tumor cell or plurality of tumor cells is suppressed by a plurality of placental perfusate cells supplemented with placental perfusate, combined natural killer cells, and/or placental intermediate natural killer cells.
• a plurality of placental perfusate cells supplemented with placental perfusate, combined natural killer cells, and/or placental intermediate natural killer cells.
• the proliferation of a tumor cell or plurality of tumor cells is suppressed by a plurality of placental intermediate natural killer cells or combined natural killer cells supplemented by placental perfusate, placental perfusate cells, and/or combined natural killer cells.
• about 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 or more placental perfusate cells and/or combined natural killer cells per milliliter, or 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , 1 x 10 11 or more placental intermediate natural killer cells and/or combined natural killer cells are supplemented with about, or at least about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60
• the proliferation of a tumor cell or plurality of tumor cells is suppressed by contacting the tumor cell or tumor cells with placental perfusate, perfusate cells, PINK cells, and/or combined natural killer cells supplemented with adherent placental stem cells.
• the placental perfusate, perfusate cells, combined natural killer cells or PINK cells are supplemented with about 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 or more adherent placental stem cells per milliliter, or 1 x 10 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , J x 10 10 , 1 x 10 11 or more adherent placental stem cells.
• the proliferation of a tumor cell or plurality of tumor cells is suppressed by contacting the tumor cell or tumor cells with placental perfusate, perfusate cells, combined natural killer cells, and/or PINK cells supplemented with adherent placental stem cell- conditioned medium, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.1, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mL of stem cell-conditioned culture medium per unit of perfusate, perfusate cells, combined natural killer cells, and/or PINK cells, or per 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 cells.
• adherent placental stem cell- conditioned medium e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.1, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mL of stem cell-conditioned culture medium per unit of perfusate,
• the placental perfusate, placental perfusate cells, placental natural killer cells, e.g., PINK cells, combined natural killer cells, and combinations and pools comprising the same are used as initially obtained, that is, perfusate as obtained during perfusion, placental perfusate cells as isolated from such perfusate, combined natural killer cells from such perfusate and matched umbilical cord blood, or PINK cells isolated from such perfusate or such placental perfusate cells.
• the placental perfusate, placental perfusate cells, PINK cells, combined natural killer cells and combinations and pools of the same are processed prior to use.
• placental perfusate can be used in its raw, unprocessed form as collected from the placenta. Placental perfusate can also be processed prior to use, e.g., by the negative selection of one or more types of cells, reduction in volume by dehydration;
• populations of perfusate cells can be used as initially isolated from placental perfusate, e.g., as total nucleated cells from placental perfusate, or can be processed, e.g., to remove one or more cell types ⁇ e.g., erythrocytes).
• PINK cells can be used as initially isolated from placental perfusate, e.g., using CD56 microbeads, or can be processed, e.g., to remove one or more non-killer cell types.
• a method of suppressing the proliferation of a tumor cell or tumor cells comprising contacting the tumor cell or tumor cells with placental perfusate, placental perfusate cells, PINK cells, combined natural killer cells, or pools or combinations comprising the same, wherein said placental perfusate, placental perfusate cells, PINK cells, combined natural killer cells, or pools or combinations comprising the same have been contacted with interleukin-2 (IL-2) for a period of time prior to said contacting.
• said period of time is about, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48 prior to said contacting.
• the perfusate, perfusate cells, PINK cells, combined natural killer cells, or pools and/or combinations of the same can be administered once to an individual having cancer, or an individual having tumor cells during a course of anticancer therapy, or can be administered multiple times, e.g., once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, or once every 1, 2, 3, 4, 5, 6 or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks during therapy.
• the perfusate, perfusate cells, PINK cells, combined natural killer cells, and pools and/or combinations of the same can be administered without regard to whether the perfusate, perfusate cells, PINK cells, combined natural killer cells, pools and/or combinations of the same have been administered to a person having cancer, or having tumor cells, in the past.
• the methods provided herein encompasses the administration to a person having cancer or having tumor cells any combination of placental perfusate, perfusate cells, PINK cells, combined natural killer cells, pools and/or combinations comprising the same.
• the tumor cells are blood cancer cells.
• the tumor cells are primary ductal carcinoma cells, leukemia cells, acute T cell leukemia cells, chronic myeloid lymphoma (CML) cells, acute myelogenous leukemia cells, chronic myelogenous leukemia (CML) cells, lung carcinoma cells, colon adenocarcinoma cells, histiocytic lymphoma cells, multiple myeloma cell, retinoblastoma cells, colorectal carcinoma cells or colorectal adenocarcinoma cells.
• CML chronic myeloid lymphoma
• CML chronic myelogenous leukemia
• CML chronic myelogenous leukemia
• lung carcinoma cells colon adenocarcinoma cells
• histiocytic lymphoma cells multiple myeloma cell
• retinoblastoma cells colorectal carcinoma cells or colorectal adenocarcinoma cells.
• the placental perfusate, perfusate cells, PINK cells, combined natural killer cells, pools, and/or combinations comprising the same can be part of an anticancer therapy regimen that includes one or more other anticancer agents.
• anticancer agents are well-known in the art.
• Specific anticancer agents that may be administered to an individual having cancer, in addition to the perfusate, perfusate cells, PINK cells, combined natural killer cells, pools and/or combinations of the same, include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;
• caracemide caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor); chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride;
• decitabine dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;
• doxorubicin doxorubicin hydrochloride
• droloxifene droloxifene citrate
• dromostanolone propionate duazomycin
• edatrexate eflomithine hydrochloride
• elsamitrucin enloplatin
• estramustine estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate;
• etoprine fadrozole hydrochloride
• fenretinide floxuridine
• fludarabine phosphate fluorouracil
• flurocitabine fosquidone
• fostriecin sodium gemcitabine
• gemcitabine gemcitabine
• irinotecan irinotecan
• irinotecan hydrochloride lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol;
• mitindomide mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide;
• hydrochloride temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard;
• vapreotide uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;
• vinrosidine sulfate vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicin
• anti-cancer drugs include, but are not limited to: 20-epi-l,25
• adozelesin aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;
• angiogenesis inhibitors antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense
• oligonucleotides oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;
• apurinic acid ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;
• bicalutamide bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate;
• bropirimine bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3;
• CARN 700 cartilage derived inhibitor
• carzelesin casein kinase inhibitors (ICOS)
• combretastatin A4 combretastatin analogue
• conagenin crambescidin 816
• crisnatol
• cryptophycin 8 cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;
• dehydrodidenmin B deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;
• eflornithine emene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; trasrabine;
• fenretinide filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
• fluorodaunorunicin hydrochloride forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
• glutathione inhibitors glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC®), imiquimod; immunostimulant peptides; insulin- like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;
• irsogladine isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;
• lamellarin-N triacetate lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin;
• letrozole leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic
• disaccharide peptide lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase;
• metoclopramide MIF inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; Erbitux, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent; mycaperoxide B;
• mycobacterial cell wall extract myriaporone; N-acetyldinaline; N-substituted benzamides;
• nemorubicin neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen (GENASENSE®); 0 6 -benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;
• ormaplatin osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;
• parabactin parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;
• phosphatase inhibitors picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine
• rubiginone Bl ruboxyl; safmgol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol;
• somatomedin binding protein sonermin; sparfosic acid; spicamycin D; spiromustine;
• splenopentin spongistatin 1; squalamine; stipiamide; stromelysin inhibitors; sulfmosine;
• tallimustine tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;
• tellurapyrylium tellurapyrylium; telomerase inhibitors; temoporfm; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
• tyrphostins UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor;
• urokinase receptor antagonists vapreotide; variolin B; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
• a method of treating an individual comprising administering to the individual a therapeutically effective amount of placental perfusate, placental perfusate cells, PINK cells, combined natural killer cells, or pools or combinations comprising the same, wherein said placental perfusate, placental perfusate cells, PINK cells, combined natural killer cells, or pools or combinations comprising the same that have been contacted with interleukin-2 (IL-2) for a period of time prior to said contacting, wherein the therapeutically effective amount is an amount sufficient to cause a detectable improvement in one or more symptoms of GVHD, or sufficient to detectably reduce the onset of one or more symptoms of GVHD.
• said period of time is about, at least, or at most 1, 2, 3, 4, 5,
• Isolated natural killer cells e.g., PINK cells or combined natural killer cells, as described elsewhere herein, can be treated with an immunomodulatory compound, e.g., contacted with an immunomodulatory compound, to enhance the antitumor activity of the cell.
• an immunomodulatory compound e.g., contacted with an immunomodulatory compound
• a method of increasing the cytotoxicity of a natural killer cell to a tumor cell comprising contacting the natural killer cell with an immunomodulatory compound for a time and in a concentration sufficient for the natural killer cell to demonstrate increased cytotoxicity towards a tumor cell compared to a natural killer cell not contacted with the immunomodulatory compound.
• a method of increasing the expression of granzyme B in a natural killer cell comprising contacting the natural killer cell with an immunomodulatory compound for a time and in a concentration sufficient for the natural killer cell to demonstrate increased expression of granzyme B compared to a natural killer cell not contacted with the immunomodulatory compound.
• the immunomodulatory compound can be any compound described below, e.g., lenalidomide or pomalidomide.
• Also provided herein is a method of increasing the cytotoxicity of a population of natural killer cells, e.g., PINK cells or combined natural killer cells, to a plurality of tumor cells comprising contacting the population of natural killer cells with an immunomodulatory compound for a time and in a concentration sufficient for the population of natural killer cells to demonstrate detectably increased cytotoxicity towards said plurality of tumor cells compared to an equivalent number of natural killer cells not contacted with the immunomodulatory compound.
• a population of natural killer cells e.g., PINK cells or combined natural killer cells
• a method of increasing the expression of granzyme B in a population of natural killer cells comprising contacting the population of natural killer cells with an immunomodulatory compound for a time and in a concentration sufficient for the population of natural killer cells to express a detectably increased amount of granzyme B compared to an equivalent number of natural killer cells not contacted with the immunomodulatory compound.
• said population of natural killer cells is contained within placental perfusate cells, e.g., total nucleated cells from placental perfusate.
• the natural killer cells are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-oxide
• the natural killer cells are combined natural killer cells, i.e., natural killer cells from matched placental perfusate and umbilical cord blood.
• said plurality of natural killer cells e.g., PINK cells or combined natural killer cells, contacted with said immunomodulatory compound express one or more of BAX, CCL5, CCR5, CSF2, FAS, GUSB, IL2RA, or TNFRSF18 at a higher level than an equivalent number of said natural killer cells not contacted with said immunomodulatory compound.
• said plurality of natural killer cells e.g., PINK cells
• said immunomodulatory compound express one or more of ACTB, BAX, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, ILIA, IL2RA, IL8, IL10, LTA, PRF1, PTGS2, SKI, and TBX21 at a higher level than an equivalent number of said natural killer cells not contacted with said immunomodulatory compound.
• Also provided herein is a method of increasing the cytotoxicity of a population of human placental perfusate cells, e.g., total nucleated cells from placental perfusate, towards a plurality of tumor cells, comprising contacting the placental perfusate cells with an
• a method of increasing the expression of granzyme B in a population of placental perfusate cells comprising contacting the population of placental perfusate cells with an immunomodulatory compound for a time and in a concentration sufficient for the population of placental perfusate cells to express a detectably increased amount of granzyme B compared to an equivalent number of placental perfusate cells not contacted with the immunomodulatory compound.
• Immunomodulatory compounds can either be commercially purchased or prepared according to the methods described in the patents or patent publications referred to herein, all of which are incorporated by reference. Further, optically pure compositions can be asymmetrically synthesized or resolved using known resolving agents or chiral columns as well as other standard synthetic organic chemistry techniques. Immunomodulatory compounds may be racemic, stereomerically enriched or stereomerically pure, and may encompass
• immunomodulatory compounds encompass small organic molecules that markedly inhibit TNF-a, LPS induced monocyte IL- ⁇ ⁇ and IL-12, and partially inhibit IL-6 production.
• the immunomodulatory compounds are lenalidomide, pomalidomide or thalidomide.
• immunomodulatory compounds include, but are not limited to, cyano and carboxy derivatives of substituted styrenes such as those disclosed in U.S. patent no. 5,929, 1 17; l-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and l ,3-dioxo-2-(2,6- dioxo-3-fluoropiperidine-3-yl) isoindolines such as those described in U.S. patent nos.
• patent no. 6,458,810 a class of non-polypeptide cyclic amides disclosed in U.S. patent nos.
• the immunomodulatory compounds are 1-oxo-and 1 ,3 dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines substituted with amino in the benzo ring as described in U.S. Patent no. 5,635,517, which is incorporated herein by reference in its entirety. These compounds have the structure I:
• immunomodulatory compounds include, but are not limited to:
• each of R 1 , R 2 , R 3 , and R 4 independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R 2 , R 3 , and R 4 is -NHR 5 and the remaining of R 1 , R 2 , R 3 , and R 4 are hydrogen;
• R 5 is hydrogen or alkyl of 1 to 8 carbon atoms
• R 1 is hydrogen or methyl.
• enantiomerically pure forms e.g. optically pure (R) or (S) enantiomers
• R 1 is H, (Ci-Cs )alkyl, (C 3 -C 7 )cycloalkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (Co-C 4 )alkyl-(Ci-C 6 )heterocycloalkyl, (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl, C(0)R 3 , C(S)R 3 , C(0)OR 4 , (Ci-C 8 )alkyl-N(R 6 ) 2 , (Ci-C 8 )alkyl-OR 5 , (Ci-C 8 )alkyl-C(0)OR 5 , C(0)NHR 3 , C(S)NHR 3 , C(0)NR 3 R 3' , C(S)NR 3 R 3' or (Ci-C 8 )alkyl-0(CO)R
• R 2 is H, F, benzyl, (C C 8 )alkyl, (C 2 -C 8 )alkenyl, or (C 2 -C 8 )alkynyl;
• R 3 and R 3' are independently (Ci-C 8 )alkyl, (C 3 -C 7 )cycloalkyl, (C 2 -C 8 )alkenyl, (C 2 - C 8 )alkynyl, benzyl, aryl, (Co-C 4 )alkyl-(Ci-C6)heterocycloalkyl, (Co-C 4 )alkyl-(C 2 -C5)heteroaryl, (C 0 -C 8 )alkyl-N(R 6 ) 2 , (Ci-C 8 )alkyl-OR 5 , (Ci-C 8 )alkyl-C(0)OR 5 , (Ci-C 8 )alkyl-0(CO)R 5 , or C(0)OR 5 ;
• R 4 is (Ci-C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, (Ci-C 4 )alkyl-OR 5 , benzyl, aryl, (C 0 - C 4 )alkyl-(Ci-C6)heterocycloalkyl, or (Co-C 4 )alkyl-(C 2 -C5)heteroaryl;
• R 5 is (Ci-C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, or (C 2 -Cs)heteroaryl; each occurrence of R 6 is independently H, (d-C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 2 -Cs)heteroaryl, or (Co-C 8 )alkyl-C(0)0-R 5 or the R 6 groups can join to form a heterocycloalkyl group;
• n 0 or 1 ;
• R 1 is (C 3 -C 7 )cycloalkyl, (C 2 - C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (Co-C 4 )alkyl-(Ci-C6)heterocycloalkyl, (Co-C 4 )alkyl- (C 2 -C 5 )heteroaryl, C(0)R 3 , C(0)OR 4 , (Ci-C 8 )alkyl-N(R 6 ) 2 , (Ci-C 8 )alkyl-OR 5 , (Ci-C 8 )alkyl- C(0)OR 5 , C(S)NHR 3 , or (Ci-C 8 )alkyl-0(CO)R 5 ;
• R 2 is H or (Ci-C 8 )alkyl
• R 3 is (Ci-C 8 )alkyl, (C 3 -C 7 )cycloalkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 0 - C 4 )alkyl-(Ci -C 6 )heterocycloalkyl, (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl, (C 5 -C 8 )alkyl-N(R 6 ) 2 ; (C 0 - C 8 )alkyl-NH-C(0)0-R 5 ; (Ci-C 8 )alkyl-OR 5 , (Ci-C 8 )alkyl-C(0)OR 5 , (Ci-C 8 )alkyl-0(CO)R 5 , or C(0)OR 5 ; and the other variables have the same definitions.
• R 2 is H or (Ci-C 4 )alkyl.
• R 1 is (Ci-C 8 )alkyl or benzyl.
• R 1 is H, (Ci-C 8 )alkyl, benzyl,
• Q is O or S, and each occurrence of R is independently H,(CiX 8 )alkyl, (C 3 _
• R 1 is C(0)R 3 .
• R 3 is (Co C4)alkyl-(C2 C5)heteroaryl
• heteroaryl is pyridyl, furyl, or thienyl.
• R 1 is C(0)OR 4 .
• the H of C(0)NHC(0) can be replaced with (Ci-C 4 )alkyl, aryl, or benzyl.
• R is H or CH 2 OCOR'
• each of R 1 , R 2 , R 3 , or R 4 independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R 2 , R 3 , or R 4 is nitro or -NHR 5 and the remaining of R 1 , R 2 , R 3 , or R 4 are hydrogen;
• R 5 is hydrogen or alkyl of 1 to 8 carbons
• R 6 hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;
• R' is R 7 -CHR 10 -N(R 8 R 9 );
• R 7 is m-phenylene or p-phenylene or -(C n H 2n )- in which n has a value of 0 to 4;
• each of R 8 and R 9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R 8 and R 9 taken together are tetramethylene, pentamethylene, hexamethylene, or -CH 2 CH 2 X 1 CH 2 CH 2 - in which Xj is -0-, -S-, or -NH-;
• R 10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl
• each of R 1 , R 2 , R 3 , or R 4 independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R 2 , R 3 , and R 4 is -NHR 5 and the remaining of R 1 , R 2 , R 3 , and R 4 are hydrogen;
• R 5 is hydrogen or alkyl of 1 to 8 carbon atoms
• R 6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;
• R 7 is m-phenylene or p-phenylene or -(C n H 2n )- in which n has a value of 0 to 4;
• each of R 8 and R 9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R 8 and R 9 taken together are tetramethylene, pentamethylene, hexamethylene, or - CH 2 CH 2 X 1 CH 2 CH 2 - in which X 1 is -0-, -S-, or -NH-;
• R 10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl.
• each of R 1 , R 2 , R 3 , and R 4 is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R 2 , R 3 , and R 4 is nitro or protected amino and the remaining of R 1 , R 2 , R 3 , and R 4 are hydrogen; and
• R 6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.
• each of R 1 , R 2 , R 3 , and R 4 independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R 2 , R 3 , and R 4 is -NHR 5 and the remaining of R 1 , R 2 , R 3 , and R 4 are hydrogen;
• R 5 is hydrogen, alkyl of 1 to 8 carbon atoms, or CO-R 7 -CH(R 10 )NR 8 R 9 in which each of R 7 , R 8 , R 9 , and R 10 is as herein defined;
• R 6 is alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.
• R 6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, chloro, or fluoro;
• R 7 is m-phenylene, p-phenylene or -(C n H 2n )- in which n has a value of 0 to 4;
• each of R 8 and R 9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R 8 and R 9 taken together are tetramethylene, pentamethylene, hexamethylene, or - CH 2 CH 2 X 1 CH 2 CH 2 - in which X 1 is -0-, -S- or -NH-; and
• R 10 is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl.
• the most preferred immunomodulatory compounds are 4-(amino)-2-(2,6-dioxo(3- piperidyl))-isoindoline-l ,3-dione and 3-(4-amino- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl)- piperidine-2,6-dione.
• the compounds can be obtained via standard, synthetic methods (see e.g., United States Patent No. 5,635,517, incorporated herein by reference). The compounds are available from Celgene Corporation, Warren, NJ.
• 4-(Amino)-2-(2,6-dioxo(3-piperidyl))- isoindoline-l,3-dione has the following chemical structure:
• the compound 3-(4-amino-l-oxo-l,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione has the following chemical structure:
• specific immunomodulatory compounds encompass polymorphic forms of 3-(4-amino-l-oxo-l,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione such as Form A, B, C, D, E, F, G and H, disclosed in U.S. publication no. US 2005/0096351 Al, which is incorporated herein by reference.
• Form A of 3-(4-amino-l-oxo-l,3 dihydro- isoindol-2-yl)-piperidene-2,6-dione is an unsolvated, crystalline material that can be obtained from non-aqueous solvent systems.
• Form A has an X-ray powder diffraction pattern comprising significant peaks at approximately 8, 14.5, 16, 17.5, 20.5, 24 and 26 degrees 2 ⁇ , and has a differential scanning calorimetry melting temperature maximum of about 270°C.
• Form A is weakly or not hygroscopic and appears to be the most thermodynamically stable anhydrous polymorph of 3-(4-amino-l-oxo-l,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione discovered thus far.
• Form B of 3-(4-amino-l-oxo-l,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione is a hemihydrated, crystalline material that can be obtained from various solvent systems, including, but not limited to, hexane, toluene, and water.
• Form B has an X-ray powder diffraction pattern comprising significant peaks at approximately 16, 18, 22 and 27 degrees 2 ⁇ , and has endotherms from DSC curve of about 146 and 268°C, which are identified dehydration and melting by hot stage microscopy experiments. Interconversion studies show that Form B converts to Form E in aqueous solvent systems, and converts to other forms in acetone and other anhydrous systems.
• Form C of 3-(4-amino-l-oxo-l,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione is a hemisolvated crystalline material that can be obtained from solvents such as, but not limited to, acetone.
• Form C has an X-ray powder diffraction pattern comprising significant peaks at approximately 15.5 and 25 degrees 2 ⁇ , and has a differential scanning calorimetry melting temperature maximum of about 269°C.
• Form C is not hygroscopic below about 85% RH, but can convert to Form B at higher relative humidities.
• Form D of 3-(4-amino-l-oxo-l,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione is a crystalline, solvated polymorph prepared from a mixture of acetonitrile and water.
• Form D has an X-ray powder diffraction pattern comprising significant peaks at approximately 27 and 28 degrees 2 ⁇ , and has a differential scanning calorimetry melting temperature maximum of about 270°C.
• Form D is either weakly or not hygroscopic, but will typically convert to Form B when stressed at higher relative humidities.
• Form E of 3-(4-amino- 1 -oxo- 1 ,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione is a dihydrated, crystalline material that can be obtained by slurrying 3-(4-amino-l-oxo-l,3 dihydro- isoindol-2-yl)-piperidene-2,6-dione in water and by a slow evaporation of 3-(4-amino-l-oxo-l,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione in a solvent system with a ratio of about 9: 1 acetone: water.
• Form E has an X-ray powder diffraction pattern comprising significant peaks at approximately 20, 24.5 and 29 degrees 2 ⁇ , and has a differential scanning calorimetry melting temperature maximum of about 269°C.
• Form E can convert to Form C in an acetone solvent system and to Form G in a THF solvent system. In aqueous solvent systems, Form E appears to be the most stable form. Desolvation experiments performed on Form E show that upon heating at about 125°C for about five minutes, Form E can convert to Form B. Upon heating at 175°C for about five minutes, Form B can convert to Form F.
• Form F of 3-(4-amino- 1 -oxo- 1 ,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione is an unsolvated, crystalline material that can be obtained from the dehydration of Form E.
• Form F has an X-ray powder diffraction pattern comprising significant peaks at approximately 19, 19.5 and 25 degrees 2 ⁇ , and has a differential scanning calorimetry melting temperature maximum of about 269°C.
• Form G of 3-(4-amino-l-oxo-l,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione is an unsolvated, crystalline material that can be obtained from slurrying forms B and E in a solvent such as, but not limited to, tetrahydrofuran (THF).
• Form G has an X-ray powder diffraction pattern comprising significant peaks at approximately 21, 23 and 24.5 degrees 2 ⁇ , and has a differential scanning calorimetry melting temperature maximum of about 267°C.
• Form H of 3-(4-amino-l-oxo-l,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione is a partially hydrated (about 0.25 moles) crystalline material that can be obtained by exposing Form E to 0 % relative humidity.
• Form H has an X-ray powder diffraction pattern comprising significant peaks at approximately 15, 26 and 31 degrees 2 ⁇ , and has a differential scanning calorimetry melting temperature maximum of about 269°C.
• immunomodulatory compounds usable in the methods provided herein include, but are not limited to, l-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and l,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such as those described in U.S. patent nos. 5,874,448 and 5,955,476, each of which is incorporated herein by reference.
• each of R 1 , R 2 , R 3 , and R 4 is hydrogen, halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino.
• each of R 1 , R 2 , R 3 , and R 4 independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms.
• Y is oxygen or H 2 ,
• a first of R 1 and R 2 is halo, alkyl, alkoxy, alkylamino, dialkylamino, cyano, or carbamoyl
• the second of R 1 and R 2 independently of the first, is hydrogen, halo, alkyl, alkoxy, alkylamino, dialkylamino, cyano, or carbamoyl
• R 3 is hydrogen, alkyl, or benzyl.
• R 1 and R 2 are halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl,
• the second of R 1 and R 2 independently of the first, is hydrogen, halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl, and
• R 3 is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl. Specific examples include, but are not limited to, l-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.
• R 1 and R 2 are halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl,
• the second of R 1 and R 2 independently of the first, is hydrogen, halo, alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms, alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl, and
• R 3 is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl.
• the carbon atom designated C* constitutes a center of chirality (when n is not zero and R 1 is not the same as R 2 ); one of X 1 and X 2 is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X 1 or X 2 is hydrogen; each of R 1 and R 2 independent of the other, is hydroxy or NH-Z; R 3 is hydrogen, alkyl of one to six carbons, halo, or haloalkyl; Z is hydrogen, aryl, alkyl of one to six carbons, formyl, or acyl of one to six carbons; and n has a value of 0, 1, or 2; provided that if X 1 is amino, and n is 1 or 2, then R 1 and R 2 are not both hydroxy; and the salts thereof.
• the carbon atom designated C* constitutes a center of chirality when n is not zero and R 1 is not R 2 ;
• one of X 1 and X 2 is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X 1 or X 2 is hydrogen; each of R 1 and R 2 independent of the other, is hydroxy or NH-Z;
• R 3 is alkyl of one to six carbons, halo, or hydrogen;
• Z is hydrogen, aryl or an alkyl or acyl of one to six carbons; and
• n has a value of 0, 1, or 2.
• the carbon atom designated C* constitutes a center of chirality when n is not zero and R 1 is not R 2 ;
• one of X 1 and X 2 is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X X 2 is hydrogen; each of R 1 and R 2 independent of the other, is hydroxy or NH-Z;
• R 3 is alkyl of one to six carbons, halo, or hydrogen;
• Z is hydrogen, aryl, or an alkyl or acyl of one to six carbons; and
• n has a value of 0, 1, or 2; and the salts thereof.
• Specific examples include, but are not limited to, 4-carbamoyl-4- ⁇ 4-[(furan-2-yl- methyl)-amino]-l ,3-dioxo-l ,3-dihydro-isoindol-2-yl ⁇ -butyric acid, 4-carbamoyl-2- ⁇ 4-[(furan-2- yl-methyl)-amino]- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl ⁇ -butyric acid, 2- (4-[(furan-2-yl-methyl)- amino]-l,3-dioxo-l,3-dihydro-isoindol-2-yl ⁇ -4-phenylcarbamoyl-butyric acid, and 2- ⁇ 4-[(furan- 2-yl-methyl)-amino]-l,3-dioxo-l,3-diox
• X 1 and X 2 are nitro, or NH-Z, and the other of X 1 or X 2 is hydrogen; each of R 1 and R 2 , independent of the other, is hydroxy or NH-Z;
• R 3 is alkyl of one to six carbons, halo, or hydrogen
• Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of one to six carbons;
• n has a value of 0, 1 , or 2;
• X 1 and X 2 are alkyl of one to six carbons; each of R 1 and R 2 , independent of the other, is hydroxy or NH-Z;
• R 3 is alkyl of one to six carbons, halo, or hydrogen
• Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of one to six carbons;
• n has a value of 0, 1 , or 2;
• Still other specific immunomodulatory compounds include, but are not limited to, isoindoline-l-one and isoindoline-l,3-dione substituted in the 2-position with 2,6-dioxo-3- hydroxypiperidin-5-yl described in U.S. patent no. 6,458,810, which is incorporated herein by reference.
• Representative compounds are of formula:
• X is -C(O)- or -CH 2 -;
• R 1 is alkyl of 1 to 8 carbon atoms or -NHR 3 ;
• R 2 is hydrogen, alkyl of 1 to 8 carbon atoms, or halogen
• R 3 is hydrogen
• alkyl of 1 to 8 carbon atoms unsubstituted or substituted with alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms,
• phenyl unsubstituted or substituted with alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms,
• benzyl unsubstituted or substituted with alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms, or -COR 4 in which
• R 4 is hydrogen, alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms,
• phenyl unsubstituted or substituted with alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms, or
• benzyl unsubstituted or substituted with alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbon atoms.
• optically pure compounds can be asymmetrically synthesized or resolved using known resolving agents or chiral columns as well as other standard synthetic organic chemistry techniques.
• Various immunomodulatory compounds contain one or more chiral centers, and can exist as racemic mixtures of enantiomers or mixtures of diastereomers. Encompassed is the use of stereomerically pure forms of such compounds, as well as the use of mixtures of those forms. For example, mixtures comprising equal or unequal amounts of the enantiomers of a particular immunomodulatory compounds may be used in methods and compositions provided herein. These isomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, e.g., Jacques, J., et ah, Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, S. H., et ah,
• the PINK cells, human placental perfusate cells, combined natural killer cells, populations of cells comprising such cells, or combinations thereof may be administered to an individual, e.g., an individual having tumor cells, e.g., a cancer patient, by any medically- acceptable route known in the art suitable to the administration of live cells.
• the cells provided herein may be surgically implanted, injected, infused, e.g., by way of a catheter or syringe, or otherwise administered directly or indirectly to the site in need of repair or augmentation.
• the cells are administered to an individual intravenously.
• the cells are administered to the individual at the site of a tumor, e.g., a solid tumor.
• the cells are administered to at least two, or all, tumor sites.
• the cells provided herein, or compositions comprising the cells are administered orally, nasally, intraarterially, parenterally, ophthalmically, intramuscularly, subcutaneously, intraperitoneally, intracerebrally, intraventricularly, intracerebroventricularly, intrathecally, intracisternally, intraspinally and/or perispinally.
• the cells are delivered via intracranial or intravertebral needles and/or catheters with or without pump devices.
• the PINK cells, human placental perfusate cells, combined natural killer cells, or combinations thereof, or cell populations comprising such cells can be administered to an individual in a composition, e.g., a matrix, hydrogel, scaffold, or the like that comprise the cells.
• a composition e.g., a matrix, hydrogel, scaffold, or the like that comprise the cells.
• the cells provided herein are seeded onto a natural matrix, e.g., a placental biomaterial such as an amniotic membrane material.
• a placental biomaterial such as an amniotic membrane material.
• an amniotic membrane material can be, e.g., amniotic membrane dissected directly from a mammalian placenta; fixed or heat-treated amniotic membrane, substantially dry (i.e., ⁇ 20% H 2 0) amniotic membrane, chorionic membrane, substantially dry chorionic membrane, substantially dry amniotic and chorionic membrane, and the like.
• Preferred placental biomaterials on which placental stem cells can be seeded are described in Hariri, U.S. Application Publication No.
• the PINK cells, human placental perfusate cells, combined natural killer cells, or combinations thereof, or cell populations comprising such cells are suspended in a hydrogel solution suitable for, e.g., injection.
• a hydrogel solution suitable for such compositions include self-assembling peptides, such as RAD 16.
• a hydrogel solution comprising the cells can be allowed to harden, for instance in a mold, to form a matrix having cells dispersed therein for implantation. The cells in such a matrix can also be cultured so that the cells are mitotically expanded prior to implantation.
• the hydrogel can be, for example, an organic polymer (natural or synthetic) that is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel.
• Hydrogel-forming materials include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block polymers such as polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively.
• the hydrogel or matrix of the invention is biodegradable.
• the formulation comprises an in situ polymerizable gel ⁇ see., e.g., U.S. Patent Application Publication 2002/0022676; Anseth et al., J. Control Release, 78(1-3): 199-209 (2002); Wang et al, Biomaterials, 24(22):3969-80 (2003).
• the polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof.
• aqueous solutions such as water, buffered salt solutions, or aqueous alcohol solutions
• polymers having acidic side groups that can be reacted with cations are poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly( vinyl acetate), and sulfonated polymers, such as sulfonated polystyrene.
• Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used.
• acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated
• the placental stem cells of the invention or co-cultures thereof can be seeded onto a three-dimensional framework or scaffold and implanted in vivo.
• a three-dimensional framework or scaffold can be implanted in combination with any one or more growth factors, cells, drugs or other components that stimulate tissue formation or otherwise enhance or improve the practice of the invention.
• Nonwoven mats examples include nonwoven mats, porous foams, or self assembling peptides.
• Nonwoven mats can be formed using fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (e.g., PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N.J.).
• Foams composed of, e.g., poly(8- caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed by processes such as freeze- drying, or lyophilization (see, e.g., U.S. Pat. No. 6,355,699), can also be used as scaffolds.
• Placental stem cells of the invention can also be seeded onto, or contacted with, a physiologically-acceptable ceramic material including, but not limited to, mono-, di-, tri-, alpha- tri-, beta-tri-, and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active glasses such as BIOGLASS ® , and mixtures thereof.
• a physiologically-acceptable ceramic material including, but not limited to, mono-, di-, tri-, alpha- tri-, beta-tri-, and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active glasses such as BIOGLASS ® , and mixtures thereof.
• Porous biocompatible ceramic materials currently commercially available include SURGIBONE ® (CanMedica Corp., Canada), ENDOBON ® (Merck Biomaterial France, France), CEROS ® (Mathys, AG, Bettlach, Switzerland), and mineralized collagen bone grafting products such as HEALOS TM (DePuy, Inc., Raynham, MA) and VITOSS ® , RHAKOSS TM , and CORTOSS ® (Orthovita, Malvern, Pa.).
• the framework can be a mixture, blend or composite of natural and/or synthetic materials.
• placental stem cells can be seeded onto, or contacted with, a felt, which can be, e.g., composed of a multifilament yarn made from a bioabsorbable material such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.
• a felt which can be, e.g., composed of a multifilament yarn made from a bioabsorbable material such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.
• the placental stem cells of the invention can, in another embodiment, be seeded onto foam scaffolds that may be composite structures.
• foam scaffolds can be molded into a useful shape, such as that of a portion of a specific structure in the body to be repaired, replaced or augmented.
• the framework is treated, e.g., with 0.1M acetic acid followed by incubation in polylysine, PBS, and/or collagen, prior to inoculation of the cells of the invention in order to enhance cell attachment.
• External surfaces of a matrix may be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma- coating the matrix, or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), a cellular matrix, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, and the like.
• proteins e.g., collagens, elastic fibers, reticular fibers
• glycoproteins e.g., glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulf
• the scaffold comprises, or is treated with, materials that render it non-thrombogenic. These treatments and materials may also promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of these materials and treatments include but are not limited to natural materials such as basement membrane proteins such as laminin and Type IV collagen, synthetic materials such as EPTFE, and segmented polyurethaneurea silicones, such as PURSPAN TM (The Polymer Technology Group, Inc., Berkeley, Calif.).
• the scaffold can also comprise anti-thrombotic agents such as heparin; the scaffolds can also be treated to alter the surface charge (e.g., coating with plasma) prior to seeding with placental stem cells.
• the present example demonstrates the isolation, culture and characterization of natural killer cells from human placental perfusate and umbilical cord blood.
• HPP human placental perfusate
• UB umbilical cord blood
• CB umbilical cord blood
• Isolation was conducted by magnetic bead selection (Miltenyi Biotec). The post partum placenta was exsanguinated and perfused with about 200 to about 750 mL of perfusion solution (0.9% NaCl injection solution USP Grade (Cat No. 68200-804, VWR). The unprocessed perfusate was collected and processed to remove erythrocytes.
• Mononuclear cells from HPP or UCB were washed one time with fluorescence- activated cell sorting (FACS) buffer (RPMI 1640, without phenol red, plus 5% FBS), then centrifuged at 1500 rpm for 6 minutes. The cell number was counted, and the cell pellet was resuspended in 80 of buffer per 10 7 total cells with 20 of CD3 Microbeads (Catalog No. 130-050-101, Miltenyi). The system was mixed well and incubated for 15 minutes at 4-8°C. 1-2 mL of buffer per 10 7 total cells was added, and the mixture was then centrifuged at 300g for 10 minutes. The supernatant was pipetted off completely.
• FACS fluorescence- activated cell sorting
• the cell pellet was resuspended up to 10 8 cells in 500 of buffer and prepared for magnetic separation.
• An LS column (Miltenyi Biotec) was placed in the magnetic field of a MIDIMACSTM cell separator (Miltenyi Biotec), 3 mL buffer was applied to rinse the column, and the cell/microbead suspension was applied to the column.
• Unlabeled CD3 cells which passed through the column and which would include natural killer cells, were collected, together with 2 x 3 mL washing buffer. The CD3 cells were counted, washed one time, then were stained with CD56 MicroBeads (Cat#: 130-050-401, Miltenyi), and separated/isolated using the same protocol as for the CD3 microbead separation described above.
• CD56+CD3- population was thus collected and ready for further analysis.
• the percentage range of natural killer cells was 3.52 to 11.6 (median 6.04, average 5.22) in HPP, and 1.06 to 8.44 in UCB (median: 3.42, average: 4.2).
• CD56 microbead selection of natural killer cells from HPP produced a population that was approximately 80% pure. See FIG. 1.
• the percentage range of CD56 + , CD 16 natural killer cells was 56.6 to 87.2 (median 74.2, average 65.5) from HPP
• the percentage range of CD56 + , CD 16 " natural killer cells from UCB was 53.7 to 96.6 (median 72.8).
• the percentage range of CD56 + , CD16 + natural killer cells was 12.8 to 43.3 (median 25.8, average 34.5) from HPP, and the percentage range of CD56 + , CD16 + natural killer cells from UCB was 3.4 to 46.3 (median 27.3, average 33.4).
• HPP and UCB cryopreserved units were thawed and diluted at 1 : 1 with Thaw media (RPMI Media 1640 (Catalog #22400, Gibco) plus 20% Fetal Bovine Serum-Heat Inactivated (Catalog #SH30070.03, Hyclone)) and centrifuged at 1500 rpm for 8 minutes. The supernatant was removed and ammonium chloride treatment was applied to further deplete erythrocytes; each unit was resuspended in
• the cell number was counted and the cell pellet was resuspended in 5xl0 7 live cells/ml in RoboSep Buffer (Catalog #20104, Stem Cell) plus 0.1 mg/mL DNAase I solution (Catalog #07900, Stem Cell) was added to the cell suspension, mixed gently by pipette and incubated 15 minutes at room temperature prior to isolation. Clumps were removed from the cell suspension by filtering with 40 ⁇ mesh nylon strainer (Catalog #352340, BD Falcon) before proceeding to isolation.
• RoboSep Buffer Catalog #20104, Stem Cell
• DNAase I solution Catalog #07900, Stem Cell
• Start Medium for natural killer cell culture was prepared based on a modification of a protocol described in Yssel et al., J. Immunol. Methods 72(l):219-227 (1984) and Litwin et al., J. Exp. Med. 178(4): 1321-1326 (1993).
• Start Medium includes IMDM (Invitrogen) with 10% FCS (Hyclone), containing the following reagents with final concentration of 35 ⁇ g/mL transferrin (Sigma- Aldrich), 5 ⁇ g/mL insulin (Sigma- Aldrich), 2 x 10 "5 M ethanolamine (Sigma- Aldrich), 1 ⁇ g/mL oleic acid (Sigma-Aldrich), 1 ⁇ g/mL linoleic acid (Sigma-Aldrich), 0.2 ⁇ g/mL palmitic acid (Sigma-Aldrich), 2.5 ⁇ g/mL BSA (Sigma-Aldrich) and 0.1 ⁇ g/mL
• CD56 + CD3 NK cells were resuspended at 2.5xl0 5 live cells/mL Start Medium plus 200 U/mL IL-2 (R&D Systems) in cell culture treated 24-well plate or T flask. Mitomycin C-treated allogenic PBMC and K562 cells (chronic myelogenous leukemia cell line) were both added to the Start Medium as feeder cells, to a final concentration of 1 x 10 6 per mL. NK cells were cultured for 5-6 days at 37°C in 5% C0 2 .
• NK cells were harvested at day 21.
• IMDM IMDM with 10% FCS, 2% Human AB serum, antibiotics, L-glutamine and 400 units of IL-2 per mL
• CD56 enriched natural killer cell population was stained with the following antibodies (BD Bioscience if not otherwise indicated) for immunophenotypic characterization: anti-CD56 conjugated to PE-Cy-7, anti-CD3 APC Cy7, anti-CD 16 FITC, anti-NKG2D APC, anti-NKp46 APC, anti-CD94 PE(R&D), anti-NKBl PE, and anti-KIR-NKAT2 PE.
• CD94, NKG2D and NKp46 are markers absent, or showing reduced expression, in NK cell progenitors but present on fully-differentiated NK cells. See Freud et al., "Evidence for Discrete States of Human Natural Killer Cell Differentiation //? Vivo," J. Exp. Med. 203(4): 1033-1043 (2006);
• Table 2 List of antibodies used in immunophenotypic characterization.
• NK cells can be divided into two main groups: CD56 + CD16 + NK cells, and CD56 CD16 cells.
• CD56 CD16 + NK cells have abundant cytolytic granules and high expression of CD 16, and are therefore capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC).
• CD56 + CD16 NK cells conversely, have very few cytolytic granules, low or no expression of CD 16, but are capable of producing cytokines and chemokines upon activation.
• NK cells display a diverse repertoire of activating and inhibitory receptors, including the killer immunoglobulin-like receptors (KIRs, e.g., KIR3DL1, and KIR2DL2/3), natural cytotoxicity receptors NCRs ⁇ e.g., NKp30, NKp44, and NKp46), killer cell lectin-like receptors (KLRs; e.g., CD94, NKG2D), 2B4 and CD226.
• KIRs killer immunoglobulin-like receptors
• NCRs ⁇ e.g., NKp30, NKp44, and NKp46
• KLRs killer cell lectin-like receptors
• Table 3 A Phenotypic characterization of CD3 CD56 + NK cells in 16 units of combined donor- matched umbilical cord blood and human placental perfusate (combo) and 13 units of peripheral blood (PB). The two-sample t-test is used to determine if population means are equal in placental and peripheral blood units.
• Tables 3B and 3C show the phenotypic characterization of CD3 CD56 CD 16 " and CD3 "
• CD56 CD16 NK cells in 16 units of combined donor-matched umbilical cord blood and human placental perfusate (combo) and 13 units of peripheral blood (PB) in a separate experiment.
• CD3-CD56+CD16-KIR3DL1 7.8 1.5 0.004
• CD3-CD56+CD16-2B4 3.7 0.5 0.078 Table 3C.
• CD56 CD 16 placental NK cells
• CD56 + CD16 peripheral blood NK cells
• NK cells Isolated or expanded NK cells were subjected to microRNA (miRNA) preparation using a MIRVANATM miRNA Isolation Kit (Ambion, Cat# 1560). NK cells (0.5 to 1.5 x 10 6 cells) were disrupted in a denaturing lysis buffer. Next, samples were subjected to acid-phenol+chloroform extraction to isolate RNA highly enriched for small RNA species. 100% ethanol was added to bring the samples to 25% ethanol. When this lysate/ethanol mixture was passed through a glass fiber filter, large RNAs were immobilized, and the small RNA species were collected in the filtrate.
• miRNA miRNA
• the ethanol concentration of the filtrate was then increased to 55%, and the mixture was passed through a second glass fiber filter where the small R As became immobilized. This R A was washed a few times, and eluted in a low ionic strength solution. The concentration and purity of the recovered small RNA was determined by measuring its absorbance at 260 and 280 nm.
• miRNAs found to be unique for PINK cells are shown in Table 5.
• NK receptors such as KIRs, NKG2D, NKp46, NKp44 and 2B4 were analyzed. Cytotoxicity assays were performed by labeling tumor cells (K562 cells) with PKH26 then co-culturing with PINK cells for 4 hours.
• KIR3DL1 killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail 1 , an inhibitory receptor
• KIR2DL2/L3 killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail 2 and long cytoplasmic tail 3; inhibitory receptors
• the proteoliposomes were then injected onto an LPITMFlow Cell (Nanoxis AB, Gothenburg, Sweden) using a standard pipette tip and allowed to immobilize for 1 hour. After immobilization, a series of washing steps were carried out and trypsin at 5 ⁇ g/mL (Princeton Separations, Adelphi, NJ) was injected directly onto the LPITM Flow Cell. The chip was incubated overnight at 37°C. Tryptic peptides were then eluted from the chip and then desalted using a Sep-Pak cartridge (Waters Corporation, Milford, MA).
• polysufoETHYL aspartamide SCX packing material Peptides were eluted from the SCX TOP- TIPTM using a step-gradient of ammonium formate buffer, pH 2.8 (10 mM-500 mM). Each SCX fraction was dried using a speed- vac system and reconstituted with 5% acetonitrile, 0.1% Formic Acid in preparation for downstream LC/MS analysis.
• LTQ Linear Ion Trap LC/MS/MS Analysis Each SCX fraction was separated on a 0.2 mm x 150 mm 3 ⁇ 200 A MAGIC C18 column (Michrom Bioresources, Inc., Auburn, CA) that was interfaced directly to an axial desolvation vacuum-assisted nanocapillary electrospray ionization (ADVANCE) source (Michrom Bioresources, Inc.) using a 180 min gradient (Buffer A: Water, 0.1% Formic Acid; Buffer B: Acetonitrile, 0.1% Formic Acid).
• the ADVANCE source achieves a sensitivity that is comparable to traditional nanoESI while operating at a considerably higher flow rate of 3 ⁇ / ⁇ .
• TPP Trans-Proteomic Pipeline
• the analysis revealed the identification of 8 membrane proteins from cultured placental NK cells that were unique with respect to membrane proteins identified from peripheral blood NK cells. See Table 7. Further, 8 membrane proteins were identified from peripheral blood NK cells that were unique with respect to cultured placental NK cells. See Table 7. Only 10 membrane proteins identified were found to be shared among both cultured placental NK cells and peripheral blood NK cells.
• PINK cells from HPP are cytotoxic to acute myelogenous leukemia cells, as demonstrated in a cytotoxicity assay and by Luminex analysis of NK cell cytokine secretion.
• CD56 microbead-enriched NK cells from HPP were mixed with KG- la acute myelogenous leukemia cells at a 1 : 1 ratio. After incubation for 24 hours, supernatant was collected and subjected to Luminex analysis of IFN- ⁇ and GM-CSF secretion. Increased levels of IFN- ⁇ and GM-CSF was observed after 24h incubation of CD56- enriched HPP cells with KG- la cells as shown in FIG. 2.
• CFSE carboxyfluoroscein succinimidyl ester
• Lactate Dehydrogenase (LDH)-Release Assay The LDH-release assay was performed using the CYTOTOX 96® colorimetric cytotoxicity assay kit (Promega, Cat# G1780). In this assay, cultured NK cells, comprising a combination of CD56 + CD16 ⁇ cells and
• CD56 CD16 cells derived from matched HPP/UCB were effector cells, and tumor cells were target cells. Effector cells and target cells were placed in 96-well U-bottom tissue culture plates and incubated at various effector-target (E:T) ratios in 100 ⁇ RPMI 1640 without phenol red (Invitrogen, Cat# 11835-030) supplemented with 2% human AB serum (Gemini, Cat# 100-512). Cultures were incubated for 4h at 37°C in 5% C0 2 .
• E:T effector-target
• %Cytotoxicity (Sample - Effector Spontaneous - Target Spontaneous)/(Target maximum - Target Spontaneous)* 100.
• Certain tumor types may be more responsive to NK cells than others.
• PINK cells To analyze susceptibility of tumor cells to cultured PINK cells, twelve different tumor cell lines, cocultured with PINK cells, were analyzed in an LDH release assay. The 12 tumor cell lines included human chronic myelogenous leukemia (CML), lymphoma, retinoblastoma (RB), and multiple myeloma (MM) (Table 8).
• CML chronic myelogenous leukemia
• RB retinoblastoma
• MM multiple myeloma
• E:T effector to target ratio of 10: 1 significant cytotoxicity of cultured PINK cells was seen towards K562 cells (CML) at 88.6% ⁇ 5.6%, U937 cells (lymphoma) at 89.2% ⁇ 9.8%, WERI-RB-1 cells (RB) at 73.3% ⁇ 11.8%, RPMI8226 cells (MM) at 61.3% ⁇ 1.3%, and U266 cells (MM) at 57.4% ⁇ 4.7% (Table 9).
• RNA isolation and purification Isolated or expanded NK cells were subjected to
• RNA preparation using RNAQUEOUS®-4PCR Kit (Ambion, Cat #AM1914).
• NK cells 0.5 to 1.5 x 10 6 cells
• the sample lysate was then mixed with an ethanol solution, and applied to a silica-based filter which selectively and quantitatively binds mRNA and the larger ribosomal RNAs; very small RNAs such as tRNA and 5S ribosomal RNA were not quantitatively bound.
• the filter was then washed to remove residual DNA, protein, and other contaminants, and the RNA was eluted in nuclease-free water containing a trace amount of EDTA to chelate heavy metals.
• the silica filter was housed in a small cartridge which fits into the RNase-free microfuge tubes supplied with the kit.
• the sample lysate, wash solutions, and elution solution were moved through the filter by centrifugation or vacuum pressure.
• the RNA was treated with the ultra-pure DNase 1 provided with the kit to remove trace amounts of DNA.
• the DNase and divalent cations were removed by a reagent also provided with the kit.
• the concentration and purity of the recovered RNA was determined by measuring its absorbance at 260 and 280 nm.
• qRT-PCR Quantitative real-time analysis. Isolated RNA was then used for cDNA synthesis using TAQMAN® Reverse Transcription Reagents (Applied Biosystems, Cat #N8080234) followed by real-time PCR analysis by the 7900HT Fast Real-Time PCR System using Human Immune Array (Applied Biosystems, Cat# 4370573) and Human MicroRNA Array (Applied Biosystems, Cat# 4384792).
• Lenalidomide and pomalidomide are chemical analogs of thalidomide with enhanced anti-cancer and anti-inflammatory activities. To study if lenalidomide and pomalidomide are chemical analogs of thalidomide with enhanced anti-cancer and anti-inflammatory activities. To study if lenalidomide and pomalidomide are chemical analogs of thalidomide with enhanced anti-cancer and anti-inflammatory activities. To study if lenalidomide and
• pomalidomide could enhance PINK cell cytotoxicity, ex vivo cultured (day 19) PINK cells were pre-treated with lenalidomide or pomalidomide for 24 hours followed by co-culturing with target colorectal carcinoma cell line HCT-116. Lenalidomide-treated NK cells demonstrated 42.1% cytotoxicity and pomalidomide -treated NK cells showed 47.4% cytotoxicity, while control untreated PINK cells showed only 24.3% cytotoxicity.
• Table 1 OA, 10B qRT-PCT analysis of lenalidomide- and pomalidomide -treated cultured PINK cells compared to untreated cells.
• 10A Fold change of gene expression between lenalidomide-treated and lenalidomide-untreated samples for genes listed. The paired t-test is used to determine if fold changes are equal in lenalidomide-treated and -untreated samples.
• 10B Fold change of gene expression between pomalidomide-treated and pomalidomide-untreated samples for 25 genes listed. The paired t-test is used to determine if fold changes are equal in treated and untreated samples.
• CSF2 - colony stimulating factor 2 (granulocyte -macrophage)
• TNFRSF18 tumor necrosis factor receptor superfamily, member 18
• CSF2 - colony stimulating factor 2 (granulocyte -macrophage)
• GZMB - granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1)
• ILIA interleukin 1
• LTA - lymphotoxin a (TNF superfamily, member 1)
• PRF1 perforin 1 (pore forming protein)
• PTGS2 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)
• NK cells derived from donor matched umbilical cord blood and placental perfusate were effector cells, while tumor cells were target cells. Tumor cells were labeled with PKH26 (Sigma-Aldrich Catalog # PKH26-GL) (see, e.g., Lee-MacAry et al., J. Immunol. Meth.
• CML K562 cells human chronic myeloid lymphoma (CML) K562 cells were labeled with PKH26, which inserts into cell plasma membrane, and placed in 96-well U- bottomed tissue culture plates. Placental (combo) or peripheral blood NK cells cultured for 21 days were mixed with K562 cells at effector to target (E:T) ratios of 10: 1, 5: 1, 2.5: 1 and 1.25: 1 in RPMI 1640 supplemented with 10% v/v FBS. After a 4 hour incubation time, cells were harvested and TO-PRO-3 was added to cell cultures followed by flow cytometry for the presence of PKH26 and TO-PRO-3.
• CML chronic myeloid lymphoma
• Cytotoxicity was expressed as percentage of PKH26 TO-PRO-3 + dead cells within the total PKH26 + target tumor cells. Both placental NK cells and peripheral blood NK cells showed substantial toxicity towards K562 cells at all E:T ratios tested (FIG. 4). Significantly higher toxicity of placental NK cells than peripheral blood NK cells towards K562 cells was observed at two E:T ratios, 10: 1 and 5: 1 (FIG. 4).
• This Example demonstrates that human placental perfusate cells are cytotoxic to tumor cells, and that the cytotoxicity of total nucleated cells from HPP (TNC-HPP) on KG- la was higher than that of TNC from matched UCB.
• Total nucleated cells from HPP or umbilical cord blood (UCB) were mixed with KG- 1 a cells at ratios of 1 : 1 , 5: 1, 10: 1, 20: 1 or 100: 1. After 24h or 48h incubation, cells were harvested and examined for the presence of CFSE by FACS analysis (BD FACSCanto, BD Bioscience). Tumor cells cultured alone were used as controls. Cytotoxicity was defined as: (1-CFSEsampie/CFSEcontroi)* 100%. Significant cytotoxicity was shown at the 100: 1 ratio. See FIG. 5.
• TNC-HPP was stimulated with lOOU/mL, or lOOOU/mL of IL-2, while HPP cultured with RPMI medium was used as control.
• IL-2 appears to increase the cytotoxicity of the TNC-HPP. See FIG. 7.
• Table 1 1 Tumor Cell Types Tested for Cytotoxic Effect of Placental Perfusate
• AML Human acute myelogenous leukemia
• CML Human leukemia
• the IFN- ⁇ , TNF-a, and GM-CSF cytokine release profile of HPP cells cocultured with tumor cell lines was analyzed, and compared to that of UCB cells co-cultured with the tumor cell lines, at different time points by multiplexed Luminex assay.
• flow cytometry-based intracellular characterization of co-cultured cells was performed, with results demonstrating that these cytokines were not produced in tumor cells.
• Quantitative RT-PCR was also performed to examine the expression of IFN- ⁇ , TNF- ⁇ , and GM-CSF using Applied Biosystems FAST 7900HT instrument and primers.
• Concentrations of cytokines were determined using a Luminex assay.
• HPP cells The secretion level of IFN- ⁇ in HPP cells was higher than that of the matched UCB cells upon IL-2 treatment. Higher expression of IFN- ⁇ was confirmed by RT-PCR analysis of cells from matched HPP and UCB. These results show that HPP cells exhibit higher anti-leukemic activity as compared to UCB cells and this higher activity is associated with a significant increase in IFN- ⁇ production.
• IFN- ⁇ production in HPP cells, and in umbilical cord blood cells, during co- culture with a panel of tumor cell lines was analyzed using the tumor cells lines listed in Table 1 , above.
• HPP cells and tumor cells were co-cultured for 24 hours or 48 hours at a ratio of 50: 1, using 10 4 tumor cells and 5 x 10 5 HPP cells.
• CCRF-CEM, J.RT3-T3.5, K562, KG1, KG- la, KU812, NC1-H1417, U-937 and WERl-RB-1 cell lines the increase in IFN- ⁇ production in HPP cells at 24 hours co-culture exceeded that of umbilical cord blood cells co-cultured with these cell lines for the same time. See FIG. 10A.
• this Example demonstrates the effectiveness of human placental perfusate in vivo against tumor cells using a NOD/SCID mouse xenograft tumor model.
• Dulbecco's medium supplemented with 20% of fetal bovine serum (growth medium) at 37°C in 95% air/5%) C0 2 and 100% humidity.
• Medium in the culture was changed every other day and cells were passaged weekly. KG-1 cells grow as suspensions. Therefore, for changing medium or passaging cells, the cell suspensions were collected in centrifuge tubes and centrifuged at 2,000 rpm for 10 min in a SORVALL ® HERAEUS ® rotor (part no. 75006434). The supernatant was discarded and an appropriate amount of the cell pellet was resuspended in the growth medium for continuation of culturing.
• KG-1 Cell Preparation for Implantation For cell implantation to mice, cells were harvested by centrifugation as described above. Cell pellets were collected and re-suspended in phosphate buffered saline. To determine the number of cells to be implanted to the mice, an aliquot of the cell suspension was counted using a hemacytometer. Trypan Blue dye was used to exclude the non- viable cells in the suspension.
• HPP Cell Preparation for Implantation For HPP storage and thawing, samples were received frozen in a dry shipper container in good condition. On the day of thawing, HPP units were removed from the cryofreezer (one at a time) and placed into ziptop plastic bags. The bags were then placed into a 37°C water bath with gentle agitation until mostly thawed (a small frozen piece remaining in the bag). The bags were then removed from the water bath, the units were removed from the zip-top bags, and the units were gently inverted until completely thawed. The units were then placed into a laminar flow hood and the outer surface of the blood bag was sterilized by spraying with 70%> ethanol.
• Blood bags were cut open using sterile scissors, and cells were transferred to sterile 50 ml conical tubes (1 tube for each HPP unit; 2 tubes for each UCB unit) by sterile pipette.
• 10 mL thawing buffer (2.5% human albumin, 5% dextran 40) was slowly added to each tube with gentle mixing (over a period of 2.2 - 2.9 minutes).
• Each blood bag was then rinsed with 10 mL thawing buffer, which was then slowly added to the 50 ml conical tube (over a period of 0.7 - 1.3 min).
• each unit was stored on wet ice prior to centrifugation. All tubes were centrifuged for 10 min (440 x g at 10°C), supernatants were aspirated using sterile pipettes, and pellets were gently disrupted by shaking the tube. A 1-ml aliquot of vehicle (PBS + 1% fetal calf serum) was added to one of the tubes, and the tube was mixed by gentle swirling. Using a 2- ml pipette, the contents were transferred to a second tube and then to a third tube and then a fourth tube. The emptied tubes were washed with 0.2 ml dilution buffer.
• vehicle PBS + 1% fetal calf serum
• HPP dose preparation After counting, HPP cells were diluted to 1 x 10 8 cells/ml by adding vehicle. HPP cells were then stored on ice until syringes were loaded. The elapsed time between thawing the first unit and completion of dose preparation was less than 3 hours.
• mice were implanted with 5 million viable KG-1 cells S/C at the flank region.
• the mice were separated such that four to five mice were housed in a micro-isolation cage system with wood chip bedding. Sterilized rodent feed and water was provided ad libitum.
• the mice were closely monitored twice a week for tumor growth. The first measurable tumor was observed on Day 25. Body weights were then recorded once a week and tumor measurements recorded twice a week with a caliper.
• the animals were randomized into three separate groups, with tumor volumes averaging about 300-350 mm 3 . See Table 12, below. The first group consisted of four control mice with an average tumor volume of 312 mm 3 .
• mice Two of these mice were implanted intravenously (IV), and two intra-tumorally (IT), with 200 ⁇ 1 and 50 ⁇ 1 of a vehicle solution, respectively.
• the second group with an average tumor volume of 345 mm 3 consisted of four mice implanted intravenously with 200 ⁇ 1 of HPP cells per mouse (2 x 10 7 cells).
• the last group, implanted IT with 50 ⁇ 1 of HPP cells per mouse also consisted of four mice with an average tumor volume of 332 mm 3 .
• Table 12 Experimental groups for in vivo tumor suppression experiment.
• the tumor volumes (TV) were measured until Day 66 (day 14 post HPP-cell implantation) when the TV of the control group reached an average of 2921 mm 3 .
• the IV treatment group at the end of study had an average TV of 2076 mm 3
• the IT group had a TV of 2705 mm 3 .
• the IT group showed a modest 20% inhibition whereas the IV group showed more than 35% inhibition of tumor growth compared to the control group. Inhibition in the IT group was demonstrable. See FIG. 11.
• This example describes generation and characterization of combined natural killer cells from donor matched full-term human placenta perfusate cells and umbilical cord blood (UCB) cells. 6.7.1. MATERIALS AND METHODS
• UCB peripheral blood mononuclear cells
• PBMCs peripheral blood mononuclear cells obtained from buffy coat (Blood Center, NJ) were prepared as another source of NK cells. After washing, cell pellets were resuspended at 5 x 10 7 cells/ml in RoboSep buffer (StemCell Technologies).
• 0.1 mg/ml DNase I (StemCell Technologies) solution was added to the cell suspension to a final concentration of 100 ⁇ /ml, mixed gently by pipette and incubated for 15 minutes at room temperature prior to isolation by EasySep® NK Cell Enrichment Kit (StemCell Technologies).
• Human NK Cell Enrichment Cocktail containing monoclonal antibodies to the following human cell surface antigens: CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA-DR, and glycophorin A, was added to the cell suspension at a final concentration of 50 ⁇ /ml and incubated for 10 minutes.
• Start Medium was composed of IMDM (ATCC) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 35 mg/ml transferrin (Sigma-Aldrich), 5 ⁇ g/ml insulin (Sigma-Aldrich), 20 ⁇ ethanolamine (Sigma-Aldrich), 1 ⁇ g/ml oleic acid (Sigma-Aldrich), 1 ⁇ g/ml linoleic acid (Sigma-Aldrich), 0.2 ⁇ g/ml palmitic acid (Sigma-Aldrich), 2.5 ⁇ g/ml BSA (Sigma-Aldrich) and 0.1 ⁇ g/ml Phytohemagglutinin (PHA-P, Sigma- Aldrich).
• FBS fetal bovine serum
• FBS fetal bovine serum
• transferrin Sigma-Aldrich
• 5 ⁇ g/ml insulin Sigma-Aldrich
• 20 ⁇ ethanolamine S
• NK cells were resuspended at approximately 2.5 x 10 5 /ml in Start Medium plus Penicillin-Streptomycin (Invitrogen) and 200 IU/ml IL-2 (R&D Systems). Mitomycin C (Sigma- Aldrich)-treated PBMC and K562 (ATCC) cells were added to Start Medium as feeder cells at a final concentration of 1 x 10 6 /ml for PBMC cells and 1 x 10 6 /ml for K562 cells. To initiate NK cell expansion, the feeder - NK cell suspension was transferred into a gas permeable culture bag (American Fluoroseal) and was cultured in an incubator at 37°C in 5% C0 2 .
• a gas permeable culture bag American Fluoroseal
• BrdU/7-AAD Cell Cycle Analysis Expanded cells were labeled with BrdU (BD Bioscience) and cultured at 37°C in 5% C0 2 for 24 hours. The cells were harvested, fixed and stained with anti-BrdU and 7-AAD following the protocol provided by the manufacturer. The cell cycle data was collected via FACSCalibur (BD Biosciences) and analysis was accomplished with Flow Jo (Tree Star, Inc.).
• MNCs enriched NK cells from the donor-matched human placenta-derived stem cells and UCB, or expanded cells from day 7, 14, 21 cultures
• CD56-PerCP/-PE/-PE-Cy7 CD3-FITC/-APC-Cy7, CD16-FITC/-PerCP
• CD158b-PE killer-immunoglobulin-like receptor [KIR] 2DL2/2DL3
• CD158el-PE KIR3DL1
• NKG2D-APC NKp46-APC
• NKp44-PE NKp30-PE
• CD226-PE 2B4-PE
• CD94-PE R&D Systems
• PKH26/TO-PRO-3 Cytotoxicity Assay NK cell in vitro cytotoxicity was examined using NK cells as effector cells and various tumor cell lines as target cells.
• Target cells were labeled with PKH26 (Sigma-Aldrich) (Ferlazzo et al, 2004; Lee-MacAryet al, 2001), placed in 96-well U-bottom tissue culture plates and incubated with effector cells at various effector to target (E:T) ratios in 200 ⁇ RPMI 1640 supplemented with 10% FBS. After 4 hours incubation at 37°C in 5% C02, cells were harvested and TO-PRO-3 (Invitrogen) was added to cultures at 1 ⁇ final concentration followed by FACS analysis using BD FACSCanto I.
• Cytotoxicity was expressed as the percentage of dead cells (PKH26+TO-PRO-3+) within the total PKH26+ target tumor cells.
• RNA samples were subjected to cDNA synthesis using TAQMAN ® Reverse Transcription Reagents (Applied Biosystems) followed by real-time PCR analysis by the 7900HT Fast Real-Time PCR System. Human MicroRNA Arrays (Applied Biosystems) were used for gene expression profiling and miRNA profiling.
• the mean ACt from real-time PCR was calculated as ACt msan :::: raean(Ct samp ie) - mean(Ct s sanction do ), where Ct sampie is the Ct value of a miRNA and Ct ejado is the Ct value of the endogenous control.
• miRNAs were unique to pNK or PB NK if they met the following criteria: 1 ) ACt msaêt ⁇ 0; 2) 2*Ct en do so, where jACt, nealinger!
• ACt2 mean is the average ACt of PB NK. or expanded pNK. RNU44, one of the most highly abundant miRNAs, was used as the endogenous control. There were 8 control wells per sample, the average Ct of the control s was used to cal culate the ACt. The statistical method utilized herein eliminates miRNAs that are not abundant compared to the control.
• miRNA Target Prediction And Pathway Analysis A search for miRNAs that were unique to either PB NK and pNK, as well as those that were highly expressed in expanded pNK, was conducted within seven miRNA target gene prediction databases (Diana-microT, miRDB, miRTar, microRNA.org, MicroCosmTargets, picTar, and TargetScan) and three experimentally validated target gene databases (TarBase, miRecords, and miRTarBase) by using medium to high stringency search criteria. Genes that were predicted by five or more databases were considered as high confidence targets. Such targeted genes were then examined in pathway analysis (Ingenuity Systems) in order to determine the associated signaling pathways and cellular functions. Pathways were scored and ranked based on their p-values.
• Human placenta-derived stem cells were harvested from three placentas and analyzed for cell surface markers by flow cytometry in comparison to the donor-matched UCB.
• the CD56+CD3- NK cells were then examined using fluorescence-conjugated monoclonal antibodies against specific killer cell immunoglobulin (Ig)-like receptors (KIRs) and C-type lectin receptors (KLRs) (CD94, NKG2D).
• NK cells from 3 pairs of donor-matched human placenta-derived stem cells and UCB units exhibited phenotypic similarities, with no significant differences in expression of sub-populations such as CD56+CD16-, CD56+CD16+, NKG2D, CD94, KIR3DL1 and KIR2DL2/L3.
• NK cells from human placenta-derived stem cells and UCB NK cells showed comparable cytotoxicity against K562 cells at various E:T ratios, indicating functional similarity between human placenta- derived stem cell-derived and UCB NK cells after expansion (Figure 12B).
• pNK cells from 16 Combo units and NK cells from 13 units of buffy coat peripheral blood (PB) were subjected to immunophenotypic characterization.
• KIRs KIR3DL1, KIR2DL2/3
• KLRs CD94
• NKG2D nuclear factor receptors
• NKp46, NKp44 and NKp30 natural cytotoxicity receptors
• CD56+CD16-, CD56+CD16+, KIR2DL2/3+, NKp46+, NKp30+, 2B4+ and CD94+ ( Figure 13A and Table 13).
• the greater proportion of CD 16- cells observed in pNK cells compared to PB NK cells indicates that pNK cells comprise a more immature NK cell population than PB NK cells.
• miRNA target gene prediction analysis was performed as described in Materials and Methods. Fourteen highly expressed miRNAs in pNK and 24 highly expressed miRNA in PB NK were returned with more than one target gene (Table 16). In detail, several highly expressed miRNAs in PB NK including hsa-let-7a, hsa-let-7g, hsa-mir-133b, hsa-mir-181b, and hsa-mir-181d, that target the anti-apoptotic genes Bcl-2 and Bcl-2L were identified. Additionally, miRNA hsa-mir-146b is highly expressed in PB NK cells and its target TRAF6 has been reported to down-regulate NF-kB activity, suppress cell proliferation and enhance
• TRAF6 plays a critical role in innate and adaptive immunity (Chiffoleau, et al., 2003, J. Immunol. 171, 5751-5759) in conjunction with genes such as MAPK14, IL6, and FOS, which are targeted by hsa-mir-24, hsa- mir-7a, and hsa-mir-222 respectively, all found in this study to be highly expressed in PB NK. Furthermore, a hsa-mir-181 group (hsa-mir-181b, hsa-mir-181c, hsa-mir-181d) that was 3-fold higher in PB NK was identified.
• miRNAs target nemo-like kinase, a regulator of Notch signaling which plays an important role in the development of NK cells from CD34+ HSCs and IFN- ⁇ production in primary CD56+ NK cells (Cichocki, et al., 2011, J. Immunol. 187, 6171- 6175).
• miRNA:mRNA target pairs comprised of pluripotency markers in stem cells and cell cycle regulators, such as SOX2, BMPRl, SMO, AKTl, ATM, RAFl and MTOR, were identified most of which are not present in the targeted gene list (both experimental and validated) of miRNAs in PB NK.
• HMGA1 (4) LIN28A (3) ACP1 (2) E2F2 (2) SMOX (1 ) HMGA2 (6) THBS1 (4) CASP3 (3) RTCD1 (2) ITGB3 (2) MYC (1 ) UHRF2 (5) NRAS (3) PRDM1 (2) CCND2 (2) LIN28 (1 ) TUSC2 (1 ) MED28 (4) EIF2C4 (3) DICER1 (2) SLC20A1 (2) NF2 (1 ) BCL2 (1 ) ZFP36L1 (1 ) NKIRAS2 (1 ) EGR3 (1 ) IL6 (1 ) NEFM (1 ) hsa-let-7b* HMGA2 (6) CDC25A (3) GRPEL2 (2) NXT2 (2) CDIPT (1 ) SLC25A13 (1 )
• IGF2BP1 (5) AURKB (3) MARS2 (2) EIF2C3 (2) CDKAL1 (1 ) SLC25A1 (1 )
• IGF2BP2 (5) SNAP23 (3) (2) PLAGL2 (2) GPR56 (1) CIA01 (1)
• HMGA1 (4) NRAS (3) TTC9C (2) LIN28 (1) NEDD4 (1) ALG3 (1) PGRMC1
• HMGA2 (6) EIF4G2 (5] I IGF2BP1 (5) COL1A2 (3) BCL2L1 (1) hsa-miR-10a
• NCOR2 (5) MAP3K7(4) HOXA1 (1) USF2(1) BTRC (1) hsa-miR-133b BCL2L2 MCL1
• HIVEP2 (5) PHF17 (3) SMAD2 (2) TRIP13 (2) ARL5B (1) SYPL1 (1)
• JARID2 (4) RCN2 (3) TP53INP1 (2) KRAS (2) ATP6V1C1 (1) WDFY1 (1)
• CEBPB (4) RCOR1 (3) ANKFY1 (2) HIF1A(2) BET1 (1) ETS1 (1)
• ARID2 ZNF652 (3) CHAF1A (2) C50RF41 (2) CBFB (1) INPP5D (1)
• CHIC2 (4) ALMS1 (2) CPNE6(2) WDR6(1) PEX13 (1) IL1R1 (1)
• HNRNPD RAD51 (2) RFX1 (2) IGF2AS (1) LRP4(1) KLF10(1)
• FAM3C (3) TIAM1 (3) APAF1 (2) RHOB (2) SOCS5 (1) IL1B (1)
• CDKN1B (5) FOS (5) KIT (4) CDKN1C (3) PPP2R2A (2) MMP1 (1)
• ARID3B ENTPD4 (3) KLF13 (3) LIN28A (2) LIN28 (1) DDX19B (1)
• PRDM1 (4) KCNS3 (3) SLC7A6 (3) NKIRAS2 (2) (1) AKT1 (1)
• Expanded pNK cells were evaluated for cytolytic activity against a variety of tumor types.
• the cytolytic activity of expanded pNK was evaluated in a FACS-based

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