# Patent

## Title
Natural killer cells from placenta

## Abstract

Provided herein are placental perfusate, placental perfusate cells, placenta-derived intermediate natural killer cells, combined natural killer cells from placenta and umbilical cord blood, and combinations thereof. Also provided herein are compositions comprising the same, and methods of using placental perfusate, placental perfusate cells, placenta-derived intermediate natural killer cells, and combined natural killer cells and combinations thereof, to suppress the growth or proliferation of tumor cells, cancer cells, and the like, and to treat individuals having tumor cells. Also provided herein are methods of treating an individual having a tumor or graft-versus-host disease with placental perfusate, placental perfusate-derived cells, natural killer cells from placenta, e.g., from placental perfusate, and/or combined natural killer cells comprising natural killer cells from placenta, e.g., from placental perfusate, and umbilical cord blood.

## Background
This application claims benefit of U.S. Provisional Patent Application No. 61/761,176, filed Feb. 5, 2013 and U.S. Provisional Patent Application No. 61/780,115, filed Mar. 13, 2013, the disclosures of which are incorporated by reference herein in their entirety.


1. FIELD
Presented herein are methods of suppressing the growth or proliferation of tumor cells by contacting the tumor cells with placental perfusate, placental perfusate-derived cells, natural killer cells from placenta, e.g., from placental perfusate, and/or combined natural killer cells comprising natural killer cells from placenta, e.g., from placental perfusate, and umbilical cord blood. Also provided herein are methods of producing a unique population of natural killer cells from placenta, e.g., from placental perfusate, e.g., human placental perfusate. Further provided herein are methods of using the placental perfusate, and the natural killer cells therefrom, to suppress the proliferation of tumor cells. Also provided herein are methods of treating an individual having a tumor or graft-versus-host disease with placental perfusate, placental perfusate-derived cells, natural killer cells from placenta, e.g., from placental perfusate, and/or combined natural killer cells comprising natural killer cells from placenta, e.g., from placental perfusate, and umbilical cord blood.
2. BACKGROUND
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. Pat. No. 7,045,146 and U.S. Pat. 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. No one to date has described the use of placental perfusate, or populations of placental cells from perfusate, in the suppression of tumor cell proliferation.
Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells do not express T-cell antigen receptors (TCR), CD3 or surface immunoglobulins (Ig) B cell receptor, but usually express the surface markers CD16 (FcγRIII) and CD56 in humans. NK cells are cytotoxic; small granules in their cytoplasm contain special proteins such as perforin and proteases known as granzymes. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell through which the granzymes and associated molecules can enter, inducing apoptosis. One granzyme, granzyme B (also known as granzyme 2 and cytotoxic T-lymphocyte-associated serine esterase 1), is a serine protease crucial for rapid induction of target cell apoptosis in the cell-mediated immune response.
NK cells are activated in response to interferons or macrophage-derived cytokines. Activated NK cells are referred to as lymphokine activated killer (LAK) cells. NK cells possess two types of surface receptors, labeled “activating receptors” and “inhibitory receptors,” that control the cells' cytotoxic activity.
Among other activities, 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. Activated and expanded 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.
In spite of the advantageous properties of NK cells in killing tumor cells and virus-infected cells, they remain difficult to work with and to apply in immunotherapy, primarily due to the difficulty in maintaining their tumor-targeting and tumoricidal capabilities during culture and expansion. Thus, there is a need in the art for a ready supply of natural killer cells.
3. SUMMARY
In one aspect, provided herein is the use of placental perfusate; cells from placental perfusate, e.g., total nucleated cells from placental perfusate; combinations of placental perfusate cells and cord blood cells; and/or natural killer cells from placenta, e.g., natural killer cells from placental perfusate or natural killer cells obtained by digestion of placental tissue, to suppress tumor cell proliferation.
In one aspect, provided herein is a method of suppressing the proliferation of a tumor cell, or population of tumor cells, comprising contacting the tumor cell or population of tumor cells with human placental perfusate. In a specific embodiment of this method, the tumor cell is a blood cancer cell. In another specific embodiment, the tumor cells are blood cancer cells. In another specific embodiment, the tumor cell is a solid tumor cell. In another specific embodiment, the tumor cells are solid tumor cells. In another embodiment, 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. In another specific embodiment, said contacting takes place in vitro. In another specific embodiment, said contacting takes place in vivo. In a more specific embodiment, said in vivo contacting takes place in a human.
In another specific embodiment, said placental perfusate is perfusate that has been passed through placental vasculature, e.g., only through placental vasculature. In another specific embodiment, said placental perfusate has been passed through the placental vasculature and collected from the maternal face of the placenta. In another specific embodiment, all, or substantially all (e.g., greater than 90%, 95%, 98% or 99%) of cells in said placental perfusate are fetal cells. In another specific embodiment, the placental perfusate comprises fetal and maternal cells. In a more specific embodiment, the fetal cells in said placental perfusate comprise less than about 90%, 80%, 70%, 60% or 50% of the cells in said perfusate. In another specific embodiment, said perfusate is obtained by passage of a 0.9% NaCl solution through the placental vasculature. In another specific embodiment, said perfusate comprises a culture medium. In another specific embodiment, said perfusate has been treated to remove a plurality of erythrocytes.
As used herein, the phrase “natural killer cell(s) from placenta” does not include natural killer cells from umbilical cord blood or placental blood.
In another aspect, provided herein is 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. In another specific embodiment, said plurality of placental perfusate cells are, or comprise, total nucleated cells from placental perfusate. In another specific embodiment, 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. In another specific embodiment, said contacting takes place in vitro. In another specific embodiment, said contacting takes place in vivo. In a more specific embodiment, said in vivo contacting takes place in a mammal, e.g., a human. In another specific embodiment, said placental perfusate cells have been treated to enrich for at least one type of cell, e.g., CD56+ cells. In another specific embodiment, said placental perfusate cells are CD56+ placental cells. In a more specific embodiment, 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. In another specific embodiment, said CD56+ cells are selected by CD56-conjugated microbeads. In another specific embodiment, said CD56+ cells comprise cells that exhibit detectably lower expression of NKG2D, NKp46 or CD94 than an equivalent number of CD56+CD16+ natural killer cells. In another specific embodiment, the PINK cells are CD3−. In a more specific embodiment, at least 50% of the cells in said placental perfusate cells are said CD56+ cells. In a more specific embodiment, wherein the CD56+ cells are at least 50% of said placental perfusate 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. In specific embodiments, said contacting is contacting in vitro. In another embodiment, said contacting is contacting in vivo, e.g., in a mammal, e.g., a human.
In another aspect, provided herein is 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. In a specific embodiment, the natural killer cells from placenta are natural killer cells obtained from placental perfusate. In another specific embodiment, the natural killer cells are natural killer cells obtained by physical disruption and/or enzymatic digestion of placental tissue. In another specific embodiment, the natural killer cells are CD56+CD16− natural killer cells, e.g., PINK cells. In another specific embodiment, 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. In another specific embodiment, the natural killer cells are CD3−. In a specific embodiment, the plurality of natural killer cells is at least 80% of the cells in a population of cells that comprises the natural killer cells. In another specific embodiment, said contacting takes place in vitro. In another specific embodiment, said contacting takes place in vivo. In a more specific embodiment, said in vivo contacting takes place in a mammal, e.g., a human.
In another specific embodiment of the method, 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. In another specific embodiment, said plurality of natural killer cells, e.g., PINK cells, expresses one or more of the microRNAs hsa-miR-100, hsa-miR-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, hsa-miR-520g, hsa-miR-520h, hsa-miR-564, hsa-miR-566, hsa-miR-618, and/or hsa-miR-99a at a detectably higher level than peripheral blood natural killer cells.
In another specific embodiment, said plurality of natural killer cells, e.g., PINK 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. In a more specific embodiment, said immunomodulatory compound is lenalidomide or pomalidomide. In another specific embodiment, said plurality of natural killer cells, e.g., PINK 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. In another specific embodiment, 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. In another specific embodiment, said plurality of natural killer cells, e.g., PINK cells, express one or more of ACTB, BAX, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, IL1A, 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.
In another embodiment, 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.
In another embodiment, the combined natural killer cells are natural killer cells from a mixed source, e.g., placenta (e.g., placental perfusate) and umbilical cord blood. For example, 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. In one embodiment, natural killer cells are isolated from combined umbilical cord blood and human placental perfusate. Combined natural killer cells may also be referred to herein as “pNK” cells. In more specific embodiments, 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, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, or the like. In one embodiment, 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. In one embodiment, the cells from placenta and another source are combined prior to isolation of combined natural killer cells. In yet another embodiment, 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.
In specific embodiments, the combined natural killer cells are not cultured. In other embodiments, the combined natural killer cells are cultured.
In certain embodiments, 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.
In certain embodiments, 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. In other specific embodiments, 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.
In certain embodiments, 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. In certain embodiments, the combined natural killer cells express one or more of 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 at a detectably lower amount than an equivalent number of natural killer cells from peripheral blood.
In a specific embodiment of any of the above methods, the tumor cell is a solid tumor cell. In another specific embodiment, the tumor cell is a liquid tumor cell, e.g., a blood tumor cell. In more specific embodiments, 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.
In another aspect, provided herein is a composition comprising isolated placental CD56+, CD16− natural killer cells, e.g., PINK cells. In a specific embodiment, said placental natural killer cells are isolated from placental perfusate. In another specific embodiment, said placental natural killer cells are isolated from placenta by physical disruption and/or enzymatic digestion of placental tissue. In another specific embodiment, said natural killer cells comprise at least 50% of cells in the composition. In a specific embodiment, said natural killer cells comprise at least 80% of cells in the composition. In another specific embodiment, said composition comprises isolated CD56+, CD16− natural killer cells. In a more specific embodiment, said CD56+, CD16+ natural killer cells are from a different individual than said CD56+, CD16− natural killer cells. In another specific embodiment, said isolated CD56+, CD16− natural killer cells are from a single individual. In a more specific embodiment, said isolated CD56+, CD16− natural killer cells comprise natural killer cells from at least two different individuals. In another specific embodiment, said placental natural killer cells, e.g., said PINK cells, are expanded.
In a more specific embodiment, the composition comprises placental natural killer cells and natural killer cells from another source. In a specific embodiment, said other source is cord blood and/or umbilical cord blood. In another specific embodiment, said other source is peripheral blood. In more specific embodiments, 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, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, or the like. In specific embodiments, the composition comprises natural killer cells obtained from a combination of placental cells (e.g., placental perfusate) and cells from another source (e.g., umbilical cord blood).
In another specific embodiment, the composition comprises isolated placental perfusate. In a more specific embodiment, said placental perfusate is from the same individual as said natural killer cells. In another more specific embodiment, said placental perfusate comprises placental perfusate from a different individual than said natural killer cells. In another specific embodiment, all, or substantially all (e.g., greater than 90%, 95%, 98% or 99%) of cells in said placental perfusate are fetal cells. In another specific embodiment, the placental perfusate comprises fetal and maternal cells. In a more specific embodiment, the fetal cells in said placental perfusate comprise less than about 90%, 80%, 70%, 60% or 50% of the cells in said perfusate. In another specific embodiment, said perfusate is obtained by passage of a 0.9% NaCl solution through the placental vasculature. In another specific embodiment, said perfusate comprises a culture medium. In another specific embodiment, said perfusate has been treated to remove a plurality of erythrocytes.
In another specific embodiment, the composition comprises placental perfusate cells. In a more specific embodiment, said placental perfusate cells are from the same individual as said natural killer cells. In another more specific embodiment, said placental perfusate cells are from a different individual than said natural killer cells. In another specific embodiment, 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. In another more specific embodiment of any of the above embodiments comprising placental perfusate, said placental perfusate comprises placental perfusate from at least two individuals. In another more specific embodiment of any of the above embodiments comprising placental perfusate cells, 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.
In another aspect, provided herein is 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. In one embodiment, provided herein is 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. In a specific embodiment, the placental cells are, or comprise, placental perfusate cells, e.g., total nucleated cells from placental perfusate. In another specific embodiment, said plurality of placental cells are, or comprise, placental cells obtained by mechanical and/or enzymatic digestion of placental tissue. In another embodiment, said isolating is performed using one or more antibodies. In a more specific embodiment, said one or more antibodies comprises one or more of antibodies to CD3, CD16 or CD56. In a more specific embodiment, said isolating comprises isolating CD56+ cells from CD56− cells in said plurality of placental cells. In a more specific embodiment, said isolating comprises isolating CD56+, CD16− placental cells from placental cells that are CD56− or CD16+. In a more specific embodiment, said isolating comprises isolating CD56+, CD16−, CD3− placental cells from placental cells that are CD56−, CD16+, or CD3+. In another embodiment, 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+, CD16− natural killer cells.
In certain embodiments of the above methods, the placental perfusate cells have been expanded in culture. In certain embodiments, the placental perfusate cells and cells from another source (e.g., umbilical cord blood) have been expanded in culture. In certain embodiments, natural killer cells obtained from placenta (e.g., placental perfusate) or obtained from a mixed source (e.g., placenta and umbilical cord blood) have been expanded in culture. In various embodiments, 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. In a specific embodiment, said cells have been expanded in the presence of a feeder layer and/or in the presence of at least one cytokine. In a more specific embodiment, said feeder layer comprises K562 cells or peripheral blood mononuclear cells (PBMCs), or a combination of both. In another more specific embodiment, said at least one cytokine is interleukin-2.
In another embodiment, provided herein is 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. In certain embodiments, said individual has a solid tumor. In certain other embodiments, the individual has a blood cancer. In specific embodiments, 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.
In another embodiment, provided herein is a method of treating an individual having graft-versus-host disease. In a more specific embodiment, said graft-versus-host disease develops after an allogeneic bone marrow transplant. In another more specific embodiment, said graft-versus-host disease develops after a solid organ transplant. In another more specific embodiment, said graft-versus-host disease develops after a composite tissue allograft. In another more specific embodiment, said graft-versus-host disease is reduced in grade by at least one step by said administering. In another more specific embodiment, said graft-versus-host disease does not progress beyond grade II within 100 days after transplantation as a result of said administering. In another more specific embodiment, said graft-versus-host disease does not progress beyond grade I within 100 days after transplantation as a result of said administering.
3.1. Definitions
As used herein, “combined 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.
As used herein, “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 CD16. PINK cells are not obtained solely from cord blood or peripheral blood.
As used herein, “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.
As used herein, “placental perfusate cells” means nucleated cells, e.g., total nucleated cells, isolated from, or isolatable from, placental perfusate.
As used herein, “tumor cell suppression,” “suppression of tumor cell proliferation,” and the like, 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.



4. BRIEF DESCRIPTION OF THE FIGURES
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 (IFNγ) by placental perfusate-derived intermediate natural killer cells (PINK) cells alone or in the presence of KG-1a tumor cells. PINK cells and KG-1a cells were cultured alone or in combination at a ratio of 1:1. Y axis: picograms of IFNγ 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-1a tumor cells. PINK cells and KG-1a cells were cultured alone or in combination at a ratio of 1:1. Y axis: picograms of GM-CSF produced by the cultures.
FIG. 3 depicts cytotoxicity of PINK cells to KG-1a 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-1a 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-1a 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-1a 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 IFNγ production by HPP cells co-cultured with KG-1a 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: IFNγ levels per milliliter after 24 hours co-culture.
FIG. 10 Production of IFNγ 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). IFNγ levels were determined by Luminex assay (HCYTO-60K-03, Millipore). X axis: tumor cell line tested. Y axis: picograms of IFNγ produced by HPP or UCB cells, compared to picograms of IFNγ produced in the absence of tumor cells.
FIG. 11 depicts the reduction in tumor size upon administration of 2×107 human placental perfusate (HPP) cells to mice having KG-1 cell tumors approximately 332 mm3 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 mm3.
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. E=NK cells; T=tumor 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 CD16, 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. (B) Cell cycle analysis at different phases from ex vivo expanded pNK at different time points as indicated (n=5).
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.



5. DETAILED DESCRIPTION
Provided herein is the use of 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. In particular, provided herein are natural killer (NK) cells, and 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. Provided herein are also 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. Methods of obtaining cells from placental tissue are described in Section 5.2, below. Placental perfusate-derived natural killer cells, e.g., placental intermediate natural killer (PINK) cells, and methods of obtaining the cells, are described in Section 5.3, below. 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.
5.1. Placental Perfusate
5.1.1. Cell Collection Composition
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. Application Publication No. 2007/0190042, which are incorporated herein by reference in their entireties.
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.
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-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor; and/or an oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide, perfluorodecyl bromide, etc.).
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 RNase, or a DNase, or the like. Such 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, LIBERASE® enzyme, hyaluronidase, and the like.
The cell collection composition can comprise a bacteriocidally or bacteriostatically effective amount of an antibiotic. In certain non-limiting embodiments, the antibiotic is a macrolide (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. In a particular embodiment, 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/l, or about 40 g/1 to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present at about 25 μM to about 100 μM); 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 μM to about 25 μM); 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 μM to about 5 μM).
5.1.2. Collection and Handling of Placenta
Generally, a human placenta is recovered shortly after its expulsion after birth. In a preferred embodiment, 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. Preferably, 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. For example, 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.
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. Pat. No. 5,372,581; Hessel et al., U.S. Pat. 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. Such cord blood recovery may be performed commercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell. Preferably, the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.
Typically, 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. In another embodiment, the placenta is transported in a cord blood collection kit substantially as described in U.S. Pat. No. 7,147,626. Preferably, the placenta is delivered to the laboratory four to twenty-four hours following delivery. In certain embodiments, the proximal umbilical cord is clamped, preferably within 4-5 cm (centimeter) of the insertion into the placental disc prior to cord blood recovery. In other embodiments, 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. (centigrade). Suitable anticoagulant solutions are well known in the art. For example, a solution of heparin or warfarin sodium can be used. In a preferred embodiment, 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.
5.1.3. Placental Perfusion
Methods of perfusing mammalian placentae are disclosed, e.g., in Hariri, U.S. Pat. Nos. 7,045,148 and 7,255,879, and in U.S. Application Publication No. 2007/0190042, entitled “Improved Composition for Collecting and Preserving Organs”, the disclosures of which are hereby incorporated by reference herein in their entireties.
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. In one embodiment, 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. Preferably, 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. The sterile connection apparatus is connected to a perfusion manifold.
In preparation for perfusion, 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. In one embodiment, 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. In another embodiment, 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).
In one embodiment, for example, 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.
In another embodiment, 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. 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. 2004/0048796. After cleaning the placenta of all visible blood clots and residual blood, e.g., using sterile gauze, 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. Alternatively, 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.
In one embodiment, 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.
The first collection of perfusion fluid from a mammalian placenta during the 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. 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. In various embodiments, 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. Typically, 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., β-mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 0.5 μg/ml). In one embodiment, 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. In a preferred embodiment, 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.
5.1.4. Placental Perfusate and Placental Perfusate Cells
Placental perfusate comprises a heterogeneous collection of cells. Typically, placental perfusate is depleted of erythrocytes prior to use. Such depletion can be carried out by known methods of separating red blood cells from nucleated blood cells. In certain embodiment, the perfusate or perfusate cells are cryopreserved. In certain embodiments, the cells from placental perfusate are neither expanded nor cultured prior to cryopreservation. In certain embodiments, the placental perfusate comprises, or the perfusate cells comprise, only fetal cells, or a combination of fetal cells and maternal cells.
Typically, placental perfusate from a single placental perfusion comprises about 100 million to about 500 million nucleated cells. In certain embodiments, the placental perfusate or perfusate cells comprise stem cells. In certain embodiments, 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. In other embodiments, placental perfusate and placental perfusate cells comprise adherent placental stem cells, e.g., CD34− stem cells. In other embodiment, the placental perfusate and placental perfusate cells comprise, e.g., endothelial progenitor cells, osteoprogenitor cells, and natural killer cells. In certain embodiments, 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.
5.2. Disruption and Digestion of Placental Tissue to Obtain PINK Cells
Placental natural killer cells, e.g., PINK cells, can be obtained from placental tissue that has been mechanically and/or enzymatically disrupted.
Placental tissue can be disrupted using one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, an RNase, or a DNase, or the like. Such 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, LIBERASE® enzyme, hyaluronidase, and the like. Typically after digestion, the digested tissue is passed through a strainer or filter to remove partially-digested cell clumps, leaving a substantially single-celled suspension.
After a suspension of placental cells is obtained, natural killer cells can be isolated using, e.g., antibodies to CD3 and CD56. In a specific embodiment, placental natural killer cells are isolated by selecting for cells that are CD56+ to produce a first cell population; contacting said first cell population with antibodies specific for CD3 and/or CD16; and removing cells from said first cell population that are CD3+ or CD56+, thereby producing a second population of cells that is substantially CD56+ and CD3−, CD56+ and CD16−, or CD56+, CD3− and CD16−.
In one embodiment, magnetic beads are used to isolate placental natural killer cells from a suspension of placental cells. The cells may be isolated, e.g., using a magnetic activated cell sorting (MACS) technique, a method for separating particles based on their ability to bind magnetic beads (e.g., about 0.5-100 μm diameter) that comprise one or more specific antibodies, e.g., anti-CD56 antibodies. A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically recognizes a particular cell surface molecule or hapten. The beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker. In one embodiment, these cells can then isolated and re-mixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation.
5.3. Placental Natural Killer Cells
In one aspect, provided herein is the isolation, characterization, and use of natural killer cells obtainable from placenta, e.g., from placental perfusate and/or from mechanically and/or enzymatically-disrupted placental tissue, and of compositions comprising such natural killer cells. In a specific embodiment, the placental natural killer cells are “placental intermediate natural killer cells,” or “PINK” cells, are characterized as being CD56+CD16−, i.e., displaying the CD56 cellular marker and lacking the CD16 cellular marker, e.g., as determined by flow cytometry, e.g., fluorescence-activated cell sorting using antibodies against CD16 and CD56, as described above. As such, provided herein are isolated PINK cells and isolated pluralities of PINK cells. Also provided herein are isolated pluralities of cells comprising CD56+CD16− PINK cells in combination with CD56+CD16+ natural killer cells. In more specific embodiments, the CD56+CD16+ natural killer cells can be isolated from placenta, or from another source, e.g., peripheral blood, umbilical cord blood, bone marrow, or the like. Thus, in various other embodiments, PINK cells can be combined with CD56+CD16+ natural killer cells, e.g., in ratios of, for example, about 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3, 9:2, 1:10, 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 or about 9:1. As used in this context, “isolated” means that the cells have been removed from their normal environment, e.g., the placenta.
In certain embodiments, the PINK cells are CD3−.
In other embodiments, the PINK cells do not exhibit one or more cellular markers exhibited by fully mature natural killer cells (e.g., CD16), or exhibit such one or more markers at a detectably reduced level compared to fully mature natural killer cells, or exhibit one or more cellular markers associated with natural killer cell precursors but not fully mature natural killer cells. In a specific embodiment, a PINK cell provided herein expresses NKG2D, CD94 and/or NKp46 at a detectably lower level than a fully mature NK cell. In another specific embodiment, a plurality of PINK cells provided herein expresses, in total, NKG2D, CD94 and/or NKp46 at a detectably lower level than an equivalent number of fully mature NK cells.
In certain embodiments, PINK cells express one or more of the microRNAs hsa-miR-100, hsa-miR-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, hsa-miR-520g, hsa-miR-520h, hsa-miR-564, hsa-miR-566, hsa-miR-618, and/or hsa-miR-99a at a detectably higher level than peripheral blood natural killer cells.
In certain embodiments, the placental natural killer cells, e.g., PINK cells, have been expanded in culture. In certain other embodiments, the placental perfusate cells have been expanded in culture. In a specific embodiment, said placental perfusate cells have been expanded in the presence of a feeder layer and/or in the presence of at least one cytokine. In a more specific embodiment, said feeder layer comprises K562 cells or peripheral blood mononuclear cells. In another more specific embodiment, said at least one cytokine is interleukin-2.
In another embodiment, provided herein is an isolated plurality (e.g., population) of PINK cells. In another specific embodiment, the isolated population of cells is produced by CD56-microbead isolation of cells from placental perfusate. In various specific embodiments, the population comprises at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least about 99% PINK cells. In another embodiment, the plurality of PINK cells comprises, or consists of, PINK cells that have not been expanded; e.g., are as collected from placental perfusate. In another embodiment, the plurality of PINK cells comprise, or consist of, PINK cells that have been expanded. Methods of expanding natural killer cells have been described, e.g., in Ohno et al., U.S. Patent Application Publication No. 2003/0157713; see also Yssel et al., J. Immunol. Methods 72(1):219-227 (1984) and Litwin et al., J. Exp. Med. 178(4):1321-1326 (1993) and the description of natural killer cell expansion in Example 1, below.
In other embodiments, the isolated plurality of PINK cells does not exhibit one or more cellular markers exhibited by fully mature natural killer cells (e.g., CD16), or exhibits such one or more markers at a detectably reduced level compared to fully mature natural killer cells, or exhibits one or more cellular markers associated with natural killer cell precursors but not associated with fully mature natural killer cells. In a specific embodiment, a PINK cell provided herein expresses NKG2D, CD94 and/or NKp46 at a detectably lower level than a fully mature NK cell. In another specific embodiment, a plurality of PINK cells provided herein expresses, in total, NKG2D, CD94 and/or NKp46 at a detectably lower level than an equivalent number of fully mature NK cells.
In certain specific embodiments, the population of PINK cells expresses one or more of the microRNAs hsa-miR-100, hsa-miR-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, hsa-miR-520g, hsa-miR-520h, hsa-miR-564, hsa-miR-566, hsa-miR-618, and/or hsa-miR-99a at a detectably higher level than peripheral blood natural killer cells. In another specific embodiment, the population of PINK cells expresses a detectably higher amount of granzyme B than an equivalent number of peripheral blood natural killer cells.
In other embodiments, the PINK cells provided herein have been expanded in culture. In specific embodiments, the PINK cells have been cultured, e.g., expanded in culture, for at least, about, or at most 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 or 28 days. In a specific embodiment, the PINK cells are cultured for about 21 days.
In another embodiment, provided herein is an isolated population of cells, e.g., placental cells, comprising PINK cells. In a specific embodiment, the isolated population of cells is total nucleated cells from placental perfusate, e.g., placental perfusate cells, comprising autologous, isolated PINK cells. In another specific embodiment, the population of cells is an isolated population of cells produced by CD56-microbead isolation of cells from placental perfusate. In various specific embodiments, the population comprises at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least about 99% PINK cells.
Because 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). In one embodiment, the PINK cells are derived only from fetal placental cells, e.g., cells obtained from closed-circuit perfusion of the placenta (see above) wherein the perfusion produces perfusate comprising a substantial majority, or only, fetal placental cells. In another embodiment, the PINK cells are derived from fetal and maternal cells, e.g., cells obtained by perfusion by the pan method (see above), wherein the perfusion produced perfusate comprising a mix of fetal and maternal placental cells. Thus, in one embodiment, provided herein is a population of placenta-derived intermediate natural killer cells, the substantial majority of which have the fetal genotype. In another embodiment, provided herein is a population of placenta-derived intermediate natural killer cells that comprise natural killer cells having the fetal genotype and natural killer cells having the maternal phenotype.
Also provided herein are populations of placenta-derived intermediate natural killer cells that comprise natural killer cells from a non-placental source. For example, in one embodiment, provided herein is population of PINK cells that also comprises natural killer cells from umbilical cord blood, peripheral blood, bone marrow, or a combination of two or more of the foregoing. In one embodiment, provided herein is a population of PINK cells that also comprises natural killer cells from umbilical cord blood. The populations of natural killer cells comprising PINK cells and natural killer cells from a non-placental source can comprise the cells 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, 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, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, or about 1:100, or the like.
Further provided herein are combinations of umbilical cord blood and isolated PINK cells. In various embodiments, cord blood is combined with PINK cells at about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, or 5×108, or more, PINK cells per milliliter of cord blood.
Also provided herein are methods of isolating PINK cells. In one embodiment, PINK cells are collected by obtaining placental perfusate, then contacting the placental perfusate with a composition that specifically binds to CD56+ cells, e.g., an antibody against CD56, followed by isolating of CD56+ cells on the basis of said binding to form a population of CD56+ cells. The population of CD56+ cells comprises an isolated population of natural killer cells. In a specific embodiment, CD56+ cells are contacted with a composition that specifically binds to CD16+ cells, e.g., an antibody against CD16, and the CD16+ cells from the population of CD56+ cells. In another specific embodiment, CD3+ cells are also excluded from the population of CD56+ cells.
In one embodiment, PINK cells are obtained from placental perfusate as follows. Post-partum human placenta is exsanguinated and perfused, e.g., with about 200-800 mL of perfusion solution, through the placental vasculature only. In a specific embodiment, the placenta is drained of cord blood and flushed, e.g., with perfusion solution, through the placental vasculature to remove residual blood prior to said perfusing. The perfusate is collected and processed to remove any residual erythrocytes. Natural killer cells in the total nucleated cells in the perfusate can be isolated on the basis of expression of CD56 and CD16. In certain embodiments, the isolation of PINK cells comprises isolation using an antibody to CD56, wherein the isolated cells are CD56+. In another embodiment, the isolation of PINK cells comprises isolation using an antibody to CD16, wherein the isolated cells are CD16−. In another embodiment, the isolation of PINK cells comprises isolation using an antibody to CD56, and exclusion of a plurality of non-PINK cells using an antibody to CD16, wherein the isolated cells comprise CD56+, CD16− cells.
Cell separation can be accomplished by any method known in the art, e.g., fluorescence-activated cell sorting (FACS), or, preferably, magnetic cell sorting using microbeads conjugated with specific antibodies. Magnetic cell separation can be performed and automated using, e.g., an AUTOMACSTM Separator (Miltenyi).
In another aspect, provided herein is 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. In a specific embodiment, the placental cells are, or comprise, placental perfusate cells, e.g., total nucleated cells from placental perfusate. In another specific embodiment, said plurality of placental cells are, or comprise, placental cells obtained by mechanical and/or enzymatic digestion of placental tissue. In another embodiment, said isolating is performed using one or more antibodies. In a more specific embodiment, said one or more antibodies comprises one or more of antibodies to CD3, CD16 or CD56. In a more specific embodiment, said isolating comprises isolating CD56+ cells from CD56− cells in said plurality of placental cells. In a more specific embodiment, said isolating comprises isolating CD56+, CD16− placental cells, e.g., placental natural killer cells, e.g., PINK cells, from placental cells that are CD56− or CD16+. In a more specific embodiment, said isolating comprises isolating CD56+, CD16−, CD3− placental cells from placental cells that are CD56−, CD16+, or CD3+. In another embodiment, 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+, CD16− natural killer cells.
5.4. Placental Natural Killer Cells from Matched Perfusate and Cord Blood
Further provided herein are natural killer cells obtained, and obtainable from, combinations of matched units of placental perfusate and umbilical cord blood, referred to herein as combined natural killer cells. In certain embodiments, “matched units,” as used herein, 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. In certain embodiments, such matched units are referred to as “donor matched” units. In certain embodiments, “matched units,” as used herein, indicates that the NK cells are obtained from placental perfusate cells, and umbilical cord blood cells, wherein the umbilical cord blood cells are not obtained from umbilical cord blood from the placenta from which the placental perfusate is obtained, but are obtained from umbilical cord blood of an immunologically matched individual, i.e., the placental perfusate cells and umbilical cord blood cells, and thus the natural killer cells from each, are from different but immunologically matched individuals. In certain embodiments, such matched units are referred to as “HLA matched” units.
In certain embodiments, 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, 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, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, or about 1:100, or the like.
In certain embodiments, the combined natural killer cells comprise only, or substantially only, natural killer cells that are CD56+ and CD16−. In certain specific embodiments, the combined natural killer cells comprise at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% CD56+CD16− natural killer cells (e.g., PINK cells). In certain other embodiments, the combined natural killer cells comprise NK cells that are CD56+ and CD16−, and NK cells that are CD56+ and CD16+. In particular embodiments, the combined natural killer cells comprise a detectably lower number of CD56+CD16+ natural killer cells than an equivalent number of natural killer cells from peripheral blood.
In certain embodiments, the combined natural killer cells comprise natural killer cells that are CD56+ and CD3−. In certain specific embodiments, the combined natural killer cells comprise at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% CD56+CD3− natural killer cells. In certain specific embodiments, the combined natural killer cells comprise a detectably higher number of CD56+CD3− 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+CD16− 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 lower number of CD3−CD56+CD16− 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+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 lower number of CD3−CD56+ NKp46+ 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 lower number of CD3−CD56+ NKp30+ 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 lower number of CD3−CD56+2B4+ 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 lower number of CD3−CD56+CD94+ 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 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.
In one embodiment, the combined natural killer cells comprise a detectably greater amount of KIR2DL2/3+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In certain specific embodiments, the combined natural killer cells comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or more KIR2DL2/3+ natural killer cells.
In one embodiment, the combined natural killer cells comprise a detectably greater amount of NKp30+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In certain specific embodiments, the combined natural killer cells comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or more NKp30+ natural killer cells.
In one embodiment, the combined natural killer cells comprise a detectably greater amount of 2B4+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In certain specific embodiments, the combined natural killer cells comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or more 2B4+ natural killer cells.
In one embodiment, the combined natural killer cells comprise a detectably greater amount of CD94+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In certain specific embodiments, the combined natural killer cells comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99.5% or more CD94+ natural killer cells.
In one embodiment, 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.
In one embodiment, combined natural killer cells have a different microRNA (miRNA) expression profile compared to natural killer cells from peripheral blood. In a specific embodiment, the combined natural killer cells detectably express one or more of miRNAs hsa-miR-337, hsa-miR-422a, hsa-miR-549 and hsa-miR-618. In one embodiment, the combined natural killer cells detectably express hsa-miR-337, hsa-miR-422a, hsa-miR-549 and hsa-miR-618. In one embodiment, the combined natural killer cells express one or more of 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. In one embodiment, the combined natural killer cells express 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. In a specific embodiment, combined natural killer cells have increased expression of hsa-miR-155 compared to an equivalent number of peripheral blood natural killer cells.
In another embodiment, the combined natural killer cells do not detectably express one or more of 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. In another embodiment, 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. In another embodiment, the combined natural killer cells express one or more of 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 at a detectably lower amount than an equivalent number of natural killer cells from peripheral blood. In another embodiment, the combined natural killer cells express 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 at a detectably lower amount than an equivalent number of natural killer cells from peripheral blood.
In certain embodiments, the combined natural killer cells express one or more of the following miRNAs at a detectably higher level (e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 90-fold, 100-fold, 250-fold, 500-fold, or 1000-fold or more) than an equivalent number of natural killer cells from peripheral blood: hsa-miR-211, hsa-miR-520c, hsa-miR-125b, hsa-miR-100, hsa-miR-326, hsa-miR-519c, hsa-miR-515-5p, hsa-miR-450, hsa-miR-198, hsa-miR-522, hsa-miR-518e, hsa-miR-497, hsa-miR-566, hsa-miR-519d, hsa-miR-627, hsa-miR-524, hsa-miR-520g, hsa-miR-302c, hsa-miR-512-3p, and hsa-miR-520h.
In certain embodiments, the combined natural killer cells express one or more of the following miRNAs at a detectably lower level (e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, 90-fold, 100-fold, 250-fold, 500-fold, or 1000-fold or more) than an equivalent number of natural killer cells from peripheral blood: hsa-miR-331, hsa-miR-186, hsa-miR-17-5p, hsa-miR-26a, hsa-miR-133b, hsa-miR-181b, hsa-miR-222, hsa-miR-197, hsa-miR-146b, hsa-miR-342, hsa-miR-181d, hsa-miR-155, hsa-miR-484, hsa-let-7g, hsa-miR-200c, hsa-miR-181c, hsa-miR-191, hsa-miR-596, hsa-miR-142-5p, hsa-miR-95, hsa-let-7a, hsa-miR-21, hsa-miR-152, hsa-miR-642, hsa-miR-24, hsa-miR-10a, hsa-miR-429, hsa-let-7b, and hsa-miR-199b.
In another embodiment, the combined natural killer cells express a detectably higher amount of granzyme B than an equivalent number of peripheral blood natural killer cells.
In certain embodiments, combined natural killer cells induce interferon (IFN)-γ at an increased level compared to an equivalent number of peripheral blood natural killer cells.
In certain embodiments, combined natural killer cells have greater cytotoxicity, e.g., against tumor cells or tumor cell lines, compared to an equivalent number of peripheral blood natural killer cells. Cytotoxicity may be measured using any method known in the art, for example, a lactate dehydrogenase assay or a fluorescence-activated cell sorting (FACS)-based PKH26/TP-PRO-3 assay and, for example, a tumor cell line such as, e.g., K562, U937, WERI-RB-1, RPMI8226, HCT-116, or U266.
In one embodiment, the combined natural killer cells have not been cultured or expanded. In another embodiment, 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. In a particular embodiment, the combined natural killer cells are expanded for more than 14 days. In certain embodiments, the combined natural killer cells are expanded for less than 28 days. In a particular embodiment, the combined natural killer cells are expanded for 21 days. In certain embodiments, expansion is ex vivo. In certain embodiments, expansion of combined natural killer cells in the absence of feeder cells. In certain embodiments, expansion of combined natural killer cells is in the presence of feeder cells. In certain embodiments, feeder cells are added to combined natural killer cells during expansion, for example, after 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more after initiating the culture (with or without feeder cells). Any feeder cells known in the art used in the culture or expansion of natural killer cells, e.g., PBMCs or K562 cells, may be used as feeder cells. In one embodiment, a combination of PBMCs and K562 cells are used as feeder cells, for example in a ratio of 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1 PBMC:K562. In one embodiment, the feeder cells comprise PBMCs and K562 at a ratio of 1:1.
In certain embodiments, combined natural killer cells are expanded, e.g., ex vivo, for about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more days. In one embodiment, ex vivo expansion of combined natural killer cells is for about 21 days. In certain embodiments, feeder cells are added during expansion. In one embodiment, addition of feeder cells to the culture during expansion enhances the extent of NK cell expansion compared to expansion in the absence of additional feeder cells. In one embodiment, fresh feeder cells are added when a majority of cultured NK cells are in S-phase, as determined using techniques known in the art (e.g., using BrdU and 7-aminoactinomycin-D (7-AAS) staining) In one embodiment, additional feeder cells are added after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days of culturing. In one embodiment, additional feeder cells are added at day 7 of culturing.
In certain embodiments, expansion of combined natural killer cells results in an increase in the proportion of cells that are CD56+CD3− compared to combined natural killer cells that are not expanded or which have been expanded for less than 21 days. In certain embodiments, expansion of combined natural killer cells results in increased expression of NKG2D, NKp46, NKp44 and/or NKp30 compared to combined natural killer cells that are not expanded or which have been expanded for less than 21 days. In certain embodiments, expansion of combined natural killer cells results in decreased expression of the bidirectional receptor 2B4 compared to combined natural killer cells that are not expanded or which have been expanded for less than 21 days.
In certain embodiments, expanded combined natural killer cells have increased or decreased expression of certain miRNAs—see for example Table 23 below—compared to an equivalent number of combined natural killer cells that have not been expanded or that have been expanded for less than 21 days. In one embodiment, miRNA hsa-miR-155 is upregulated in 21-day expanded combined natural killer cells compared to combined natural killer cells that have not been expanded, have been expanded for less than 21 days, and/or have been expanded for more than 21 days (e.g., 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or more).
In certain embodiments, expanded (e.g., 21-day expanded) combined natural killer cells have greater cytotoxicity, e.g., against tumor cells or tumor cell lines, compared to an equivalent number of combined natural killer cells that have not been expanded, have been expanded for less than 21 days, and/or have been expanded for more than 21 days (e.g., 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or more).
In certain embodiments, expanded combined natural killer cells induce interferon (IFN)-γ at an increased level compared to an equivalent number of combined natural killer cells that have not been expanded, have been expanded for less than 21 days, and/or have been expanded for more than 21 days (e.g., 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or more).
Further provided herein are combinations of umbilical cord blood and combined natural killer cells. In various embodiments, cord blood is combined with combined natural killer cells at about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 combined natural killer cells per milliliter of cord blood.
5.5. Perfusate/Cell Compositions
In addition to placental perfusate, placental perfusate cells, combined natural killer cells, and placental natural killer cells, e.g., placental intermediate natural killer cells, provided herein are compositions comprising the perfusate or cells, for use in suppressing the proliferation of a tumor cell or plurality of tumor cells.
5.5.1. Combinations of Placental Perfusate, Perfusate Cells and Placenta-Derived Intermediate Natural Killer Cells
Further provided herein are compositions comprising combinations of the placental perfusate, placental perfusate cells, placental intermediate natural killer cells, and/or combined natural killer cells described in Sections 5.1, 5.3, or 5.4 above. In one embodiment, for example, provided herein is a volume of placental perfusate supplemented with a plurality of placental perfusate cells and/or a plurality of placental natural killer cells, e.g., placental intermediate natural killer cells, for example, obtained from placental perfusate cells or placental tissue mechanically or enzymatically disrupted. In specific embodiments, for example, each milliliter of placental perfusate is supplemented with about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more placental perfusate cells, placental intermediate natural killer cells, and/or combined natural killer cells. In another embodiment, a plurality of placental perfusate cells is supplemented with placental perfusate, placental intermediate natural killer cells, and/or combined natural killer cells. In another embodiment, a plurality of placental intermediate natural killer cells is supplemented with placental perfusate, placental perfusate cells, and/or combined natural killer cells. In certain embodiments, when perfusate is used for supplementation, the volume of perfusate is about, greater than about, or less than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total volume of cells (in solution) plus perfusate. In certain other embodiments, when placental perfusate cells are combined with a plurality of PINK cells and/or combined natural killer cells, the placental perfusate 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. In certain other embodiments, 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. In certain other embodiments, 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. In certain other embodiments, when PINK cells, combined natural killer cells or placental perfusate cells are used to supplement placental perfusate, the volume of solution (e.g., saline solution, culture medium or the like) in which the cells are suspended comprises about, greater than about, or less than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total volume of perfusate plus cells, where the PINK cells are suspended to about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more cells per milliliter prior to supplementation.
In other embodiments, any of the above combinations is, in turn, combined with umbilical cord blood or nucleated cells from umbilical cord blood.
Further provided herein is pooled placental perfusate that is obtained from two or more sources, e.g., two or more placentas, and combined, e.g., pooled. Such pooled perfusate can comprise approximately equal volumes of perfusate from each source, or can comprise different volumes from each source. The relative volumes from each source can be randomly selected, or can be based upon, e.g., a concentration or amount of one or more cellular factors, e.g., cytokines, growth factors, hormones, or the like; the number of placental cells in perfusate from each source; or other characteristics of the perfusate from each source. Perfusate from multiple perfusions of the same placenta can similarly be pooled.
Similarly, provided herein are 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.
Further provided herein are placental perfusate, placental perfusate cells, and placental intermediate natural killer cells, and pools of the same or combinations of the same, that have been assayed to determine the degree or amount of tumor suppression (that is, the potency) to be expected from, e.g., a given number of placental perfusate or PINK cells, or a given volume of perfusate. For example, 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.
In certain embodiments, 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. Such units can be provided so as to contain a specified number of, e.g., placental perfusate cells, placental intermediate natural killer cells, or both, e.g., 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more cells per milliliter, or 1×104 1×1010, 5×1010, 1×1011 or more cells per unit. Such units can be provided to contain specified numbers of any two, or all three, of placental perfusate, placental perfusate cells, and/or PINK cells.
In the above combinations of placental perfusate, placental perfusate cells and/or PINK cells, any one, any two, or all three of the placental perfusate, placental perfusate cells and/or PINK cells can be autologous to a recipient (that is, obtained from the recipient), or homologous to a recipient (that is, obtained from at last one other individual from said recipient).
Any of the above combinations or pools of PINK cells, placental perfusate cells and/or placental perfusate can comprise CD56+CD16+ natural killer cells from, e.g., placental perfusate, peripheral blood, umbilical cord blood, bone marrow, or the like. In specific embodiments, the combinations comprise about, at least about, or at most about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106 or more such natural killer cells per milliliter, or 1×104, 5×104, 1×105 1010, 1×1011 or more cells per unit. The CD56+CD16+ 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).
Preferably, 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.
Also provided herein are compositions comprising placental intermediate natural killer cells, alone or in combination with placental perfusate cells and/or placental perfusate. Thus, in another aspect, provided herein is a composition comprising isolated CD56+, CD16− 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. In a specific embodiment, said natural killer cells comprise at least 80% of cells in the composition. In another specific embodiment, said composition comprises isolated CD56+, CD16+ natural killer cells. In a more specific embodiment, said CD56+, CD16+ natural killer cells are from a different individual than said CD56+, CD16− natural killer cells. In another specific embodiment, said natural killer cells are from a single individual. In a more specific embodiment, said isolated natural killer cells comprise natural killer cells from at least two different individuals. In another specific embodiment, the composition comprises isolated placental perfusate. In a more specific embodiment, said placental perfusate is from the same individual as said natural killer cells. In another more specific embodiment, said placental perfusate comprises placental perfusate from a different individual than said natural killer cells. In another specific embodiment, the composition comprises placental perfusate cells. In a more specific embodiment, said placental perfusate cells are from the same individual as said natural killer cells. In another more specific embodiment, said placental perfusate cells are from a different individual than said natural killer cells. In another specific embodiment, the composition additionally comprises isolated placental perfusate and isolated placental perfusate cells, wherein said isolated perfusate and said isolated placental perfusate cells are from different individuals. In another more specific embodiment of any of the above embodiments comprising placental perfusate, said placental perfusate comprises placental perfusate from at least two individuals. In another more specific embodiment of any of the above embodiments comprising placental perfusate cells, said isolated placental perfusate cells are from at least two individuals.
5.5.2. Compositions Comprising Adherent Placental Stem Cells
In other embodiments, 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, is supplemented with adherent placental stem cells. Such stem cells are described, e.g., in Hariri U.S. Pat. Nos. 7,045,148 and 7,255,879. Adherent placental stem cells are not trophoblasts.
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., 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 or more adherent placental cells. The adherent placental stem cells in the combinations can be, e.g., adherent placental stem cells that have been cultured for, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 population doublings, or more.
Adherent placental stem cells, when cultured in primary cultures or in cell culture, adhere to the tissue culture substrate, e.g., tissue culture container surface (e.g., tissue culture plastic). Adherent placental stem cells in culture assume a generally fibroblastoid, stellate appearance, with a number of cytoplasmic processes extending from the central cell body. 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, and populations of placental stem cells, useful in the compositions and methods provided herein, express a plurality of markers that can be used to identify and/or isolate the stem cells, or populations of cells that comprise the stem cells. The adherent placental stem cells, and adherent stem cell populations useful in the compositions and methods provided herein include stem cells and stem cell-containing cell populations obtained directly from the placenta, or any part thereof (e.g., amnion, chorion, amnion-chorion plate, placental cotyledons, umbilical cord, and the like). The adherent placental stem cell population, in one embodiment, is a population (that is, two or more) of adherent placental stem cells in culture, e.g., a population in a container, e.g., a bag.
Adherent placental stem cells generally express the markers CD73, CD105, CD200, HLA-G, and/or OCT-4, and do not express CD34, CD38, or CD45. Adherent placental stem cells can also express HLA-ABC (MHC-1) and HLA-DR. These markers can be used to identify adherent placental stem cells, and to distinguish placental stem cells from other stem cell types. Because the placental stem cells can express CD73 and CD105, they can have mesenchymal stem cell-like characteristics. However, because 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. In the same manner, the lack of expression of CD34, CD38 and/or CD45 identifies the adherent placental stem cells as non-hematopoietic stem cells.
In one embodiment, the adherent placental stem cells are CD200+, HLA-G+, wherein the stem cells detectably suppress cancer cell proliferation or tumor growth. In a specific embodiment, said adherent stem cells are also CD73+ and CD105+. In another specific embodiment, said adherent stem cells are also CD34−, CD38− or CD45−. In a more specific embodiment, said adherent stem cells are also CD34−, CD38−, CD45−, CD73+ and CD105+. In another embodiment, said adherent stem cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
In another embodiment, the adherent placental stem cells are CD73+, CD105+, CD200+, wherein said stem cells detectably suppress cancer cell proliferation or tumor growth. In a specific embodiment of said populations, said adherent stem cells are HLA-G+. In another specific embodiment, said adherent stem cells are CD34−, CD38− or CD45−. In another specific embodiment, said adherent stem cells are CD34−, CD38− and CD45−. In a more specific embodiment, said adherent stem cells are CD34−, CD38−, CD45−, and HLA-G+. In another specific embodiment, said adherent placental stem cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
In another embodiment, the adherent placental stem cells are CD200+, OCT-4+, wherein said stem cells detectably suppress cancer cell proliferation or tumor growth. In a specific embodiment, said adherent stem cells are CD73+ and CD105+. In another specific embodiment, said adherent stem cells are HLA-G+. In another specific embodiment, said adherent stem cells are CD34−, CD38− and CD45−. In a more specific embodiment, said adherent stem cells are CD34−, CD38−, CD45−, CD73+, CD105+ and HLA-G+. In another specific embodiment, the adherent placental stem cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.
In another embodiment, the adherent placental stem cells are CD73+, CD105+ and HLA-G+, wherein said adherent stem cells detectably suppress cancer cell proliferation or tumor growth. In a specific embodiment of the above plurality, said adherent stem cells are also CD34−, CD38− or CD45−. In another specific embodiment, said adherent stem cells are also CD34−, CD38− and CD45−. In another specific embodiment, said adherent stem cells are also OCT-4+. In another specific embodiment, said adherent stem cells are also CD200+. In a more specific embodiment, said adherent stem cells are also CD34−, CD38−, CD45−, OCT-4+ and CD200+.
In another embodiment, 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. In a specific embodiment, said adherent stem cells are also CD34−, CD38− or CD45−. In another specific embodiment, said adherent stem cells are also CD34−, CD38− and CD45−. In another specific embodiment, said adherent stem cells are also OCT-4+. In a more specific embodiment, said adherent stem cells are also OCT-4+, CD34−, CD38− and CD45−.
In another embodiment, the adherent placental stem cells are OCT-4+ stem cells, wherein said adherent placental stem cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies, and wherein said stem cells have been identified as detectably suppressing cancer cell proliferation or tumor growth.
In various embodiments, 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. In a specific embodiment of the above populations, said stem cells are CD73+ and CD105+. In another specific embodiment, said stem cells are CD34−, CD38−, or CD45−. In another specific embodiment, said stem cells are CD200+. In a more specific embodiment, said stem cells are CD73+, CD105+, CD200+, CD34−, CD38−, and CD45−. In another specific embodiment, 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.
In a more specific embodiment of any of the above embodiments, the adherent placental cells express ABC-p (a placenta-specific ABC transporter protein; see, e.g., Allikmets et al., Cancer Res. 58(23):5337-9 (1998)).
In another embodiment, the adherent placental stem cells are CD29+, CD44+, CD73+, CD90+, CD105+, CD200+, CD34− and CD133−. In another embodiment, the adherent placental stem cells, the placental stem cells constitutively secrete IL-6, IL-8 and monocyte chemoattractant protein (MCP-1).
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, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 or more adherent placental stem cells.
5.5.3. Compositions Comprising Placental Stem Cell Conditioned Media
Also provided herein is the use of 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. In various embodiments, 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. In other embodiments, 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. In another embodiment, 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. In certain embodiments, 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.
Thus, in one embodiment, provided herein is 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, CD105, and CD200, or express CD200 and OCT-4, or express CD73, CD105, and HLA-G, or express CD73 and CD105 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. In a specific embodiment, the composition further comprises a plurality of said placental stem cells. In another specific embodiment, the composition comprises a plurality of non-placental cells. In a more specific embodiment, 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. In another specific embodiment, the composition comprises an anti-proliferative agent, e.g., an anti-MIP-1α or anti-MIP-1β antibody.
In a specific embodiment, 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. For example, the conditioned culture medium or supernatant can be obtained from a culture comprising about 1×105 placental stem cells, about 1×106 placental stem cells, about 1×107 placental stem cells, or about 1×108 placental stem cells, or more. In another specific embodiment, the conditioned culture medium or supernatant is obtained from a co-culture comprising about 1×105 to about 5×105 placental stem cells and about 1×105 tumor cells; about 1×106 to about 5×106 placental stem cells and about 1×106 tumor cells; about 1×107 to about 5×107 placental stem cells and about 1×107 tumor cells; or about 1×108 to about 5×108 placental stem cells and about 1×108 tumor cells.
In a specific embodiment, the conditioned medium suitable for administration to a 70 kg individual comprises supernatant conditioned by about 70 million placental stem cells in about 200 mL culture medium.
Conditioned medium can be condensed to prepare an administrable pharmaceutical-grade product. For example, 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. In a specific embodiment, for example, 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.
5.6. Preservation of Perfusate and Placental Cells
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. Such 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 Aug. 16, 2007. In one embodiment, 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.
In one embodiment, 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 perfluorocarbon, wherein said 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. For example, the placenta can be perfused with the placental cell collection composition, and placental cells, e.g., total nucleated placental cells, are isolated therefrom. In a specific embodiment, the inhibitor of apoptosis is a caspase inhibitor. In another specific embodiment, said inhibitor of apoptosis is a JNK inhibitor. In a more specific embodiment, said JNK inhibitor does not modulate differentiation or proliferation of said stem cells. In another embodiment, the placental cell collection composition comprises said inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in separate phases. In another embodiment, the placental cell collection composition comprises said inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in an emulsion. In another embodiment, the placental cell collection composition additionally comprises an emulsifier, e.g., lecithin. In another embodiment, said apoptosis inhibitor and said perfluorocarbon are between about 0° C. and about 25° C. at the time of contacting the placental cells. In another more specific embodiment, 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. In another more specific embodiment, said contacting is performed during transport of said population of stem cells. In another more specific embodiment, said contacting is performed during freezing and thawing of said population of stem cells.
In another embodiment, 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.
In a specific embodiment, the organ-preserving compound is UW solution (described in U.S. Pat. No. 4,798,824; also known as VIASPANTM; see also Southard et al., Transplantation 49(2):251-257 (1990) or a solution described in Stern et al., U.S. Pat. No. 5,552,267. In another embodiment, said organ-preserving composition is hydroxyethyl starch, lactobionic acid, raffinose, or a combination thereof. In another embodiment, the placental cell collection composition additionally comprises an oxygen-carrying perfluorocarbon, either in two phases or as an emulsion.
In another embodiment of the method, 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. In another embodiment, placental cells are contacted with said stem cell collection compound after collection by perfusion.
Typically, during placental cell collection, enrichment and isolation, it is preferable to minimize or eliminate cell stress due to hypoxia and mechanical stress. In another embodiment of the method, therefore, placental perfusate, a placental cell, or population of placental cells, is exposed to a hypoxic condition during collection, enrichment or isolation for less than six hours during said preservation, wherein a hypoxic condition is a concentration of oxygen that is less than normal blood oxygen concentration. In a more specific embodiment, said population of placental cells is exposed to said hypoxic condition for less than two hours during said preservation. In another more specific embodiment, 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.
The placental cells, natural killer cells from placenta, or combined natural killer cells provided herein can be cryopreserved, e.g., in cryopreservation medium in small containers, e.g., ampoules. Suitable 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 1° 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.
5.7. Use of Placental Perfusate, PINK Cells, and Combined Natural Killer Cells to Suppress Tumor Cell Growth
Also provided herein are methods of suppressing the growth, e.g., proliferation, of tumor cells using placental perfusate, cells isolated from placental perfusate, isolated combined natural killer cells, or isolated placental natural killer cells, e.g., placenta-derived intermediate natural killer cells.
In one embodiment, provided herein is 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.
As used herein, “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. In another embodiment, “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. In another embodiment, “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.
In certain embodiments, 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. In certain other embodiments, 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. Thus, in another embodiment, provided herein is 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.
As used herein, “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. Contacting of placental perfusate, placental perfusate cells and/or PINK cells that accomplishes any one or more of such therapeutic benefits is said to be therapeutically beneficial.
In certain embodiments, 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, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, or 5×1010 placental perfusate cells, combined natural killer cells and/or placenta-derived intermediate natural killer cells. In other embodiments, 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, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, or 5×1010 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 intermediate natural killer cells, and tumor cells in said individual. For example, 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.
Further provided herein is 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. In various embodiments, provided herein is 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.
In one specific embodiment, for example, 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. In specific embodiments, for example, each milliliter of placental perfusate is supplemented with about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106 or more placental perfusate cells, combined natural killer cells or placental intermediate natural killer cells. In other specific embodiments, 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 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more PINK cells, combined natural killer cells, and/or placental perfusate cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 or more PINK cells, combined natural killer cells, and/or placental perfusate cells.
In another specific embodiment, 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. In more specific embodiments, about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more placental perfusate cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 or more placental perfusate cells, are supplemented with about, or at least about, 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more PINK cells and/or combined natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 or more PINK cells or combined natural killer cells. In other more specific embodiments, about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more placental perfusate cells, PINK cells, and/or combined natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106 more placental perfusate 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, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mL of perfusate, or about 1 unit of perfusate.
In another specific embodiment, 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. In more specific embodiments, about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more placental intermediate natural killer cells, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 or more PINK cells, are supplemented with about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more placental perfusate cells and/or combined natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 or more placental perfusate cells and/or combined natural killer cells. In other more specific embodiments, about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more placental perfusate cells and/or combined natural killer cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 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, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mL of perfusate, or about 1 unit of perfusate.
In another embodiment, 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. In specific embodiments, the placental perfusate, perfusate cells, combined natural killer cells or PINK cells are supplemented with about 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108 or more adherent placental stem cells per milliliter, or 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, 1×107, 5×107, 1×108, 5×108, 1×109, 5×109, 1×1010, 5×1010, 1×1011 or more adherent placental stem cells.
In another embodiment, 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 104, 105, 106, 107, 108, 109 or 1010 cells.
In other embodiments, 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. In other embodiments, the placental perfusate, placental perfusate cells, PINK cells, combined natural killer cells and combinations and pools of the same are processed prior to use. For example, 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; lyophilization and rehydration, etc. Similarly, 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.
In another embodiment, provided herein is 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. In certain embodiments, 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. Thus, 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.
In a specific embodiment, the tumor cells are blood cancer cells. In various specific embodiments, 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.
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. Such 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; 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; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; 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 hydrochloride.
Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; 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; 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; 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); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; 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; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; 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; 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; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen (GENASENSE®); O6-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; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; 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 demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; 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; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; 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.
In another embodiment, provided herein is a method of treating an individual, e.g., a transplant recipient or individual who will receive a transplant, that has, or is experiencing a symptom of, or is at risk for developing, graft-versus-host disease (GVHD), 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. In certain embodiments, 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.
5.8. Treatment of Natural Killer Cells with Immunomodulatory Compounds
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. Thus, provided herein is 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. In another embodiment, provided herein is 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. In another embodiment, provided herein is 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. In a specific embodiment, said population of natural killer cells is contained within placental perfusate cells, e.g., total nucleated cells from placental perfusate.
In specific embodiments of the above embodiments, the natural killer cells are CD56+, CD16− placental intermediate natural killer cells (PINK cells). In another specific embodiment of the above embodiments, the natural killer cells are combined natural killer cells, i.e., natural killer cells from matched placental perfusate and umbilical cord blood.
In another specific embodiment, 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. In another specific embodiment, said plurality of natural killer cells, e.g., PINK cells, contacted with said immunomodulatory compound express one or more of ACTB, BAX, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, IL1A, 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 immunomodulatory compound for a time and in a concentration sufficient for the placental perfusate cells to demonstrate detectably increased cytotoxicity towards said plurality of tumor cells compared to an equivalent number of placental perfusate cells not contacted with the immunomodulatory compound. In another embodiment, provided herein is 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 pharmaceutically acceptable salts, solvates, and prodrugs thereof.
As used herein and unless otherwise indicated, the terms “immunomodulatory compounds” encompass small organic molecules that markedly inhibit TNF-α, LPS induced monocyte IL-1β and IL-12, and partially inhibit IL-6 production. In specific examples, the immunomodulatory compounds are lenalidomide, pomalidomide or thalidomide.
Specific examples of immunomodulatory compounds, include, but are not limited to, cyano and carboxy derivatives of substituted styrenes such as those disclosed in U.S. Pat. No. 5,929,117; 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such as those described in U.S. Pat. Nos. 5,874,448 and 5,955,476; the tetra substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines described in U.S. Pat. No. 5,798,368; 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines (e.g., 4-methyl derivatives of thalidomide), including, but not limited to, those disclosed in U.S. Pat. Nos. 5,635,517, 6,476,052, 6,555,554, and 6,403,613; 1-oxo and 1,3-dioxoisoindolines substituted in the 4- or 5-position of the indoline ring (e.g., 4-(4-amino-1,3-dioxoisoindoline-2-yl)-4-carbamoylbutanoic acid) described in U.S. Pat. No. 6,380,239; isoindoline-1-one and isoindoline-1,3-dione substituted in the 2-position with 2,6-dioxo-3-hydroxypiperidin-5-yl (e.g., 2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-one) described in U.S. Pat. No. 6,458,810; a class of non-polypeptide cyclic amides disclosed in U.S. Pat. Nos. 5,698,579 and 5,877,200; aminothalidomide, as well as analogs, hydrolysis products, metabolites, derivatives and precursors of aminothalidomide, and substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles such as those described in U.S. Pat. Nos. 6,281,230 and 6,316,471; and isoindole-imide compounds such as those described in U.S. patent publication no. 2003/0045552 A1, U.S. Pat. No. 7,091,353, and WO 02/059106. The entireties of each of the patents and patent applications identified herein are incorporated herein by reference. Immunomodulatory compounds do not include thalidomide.
In certain embodiments, 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. Pat. No. 5,635,517, which is incorporated herein by reference in its entirety. These compounds have the structure I:




in which one of X and Y is C═O, the other of X and Y is C═O or CH2, and R2 is hydrogen or lower alkyl, in particular methyl. Specific immunomodulatory compounds include, but are not limited to:

    1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;
    1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline;
    1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline;
    1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline;
    1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; and
    1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline.


Other specific immunomodulatory compounds belong to a class of substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as those described in U.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and 6,476,052, and WO 98/03502, each of which is incorporated herein by reference. Representative compounds are of formula:




in which:
one of X and Y is C═O and the other of X and Y is C═O or CH2;
(i) each of R1, R2, R3, and R4, 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 R1, R2, R3, and R4 is —NHR5 and the remaining of R1, R2, R3, and R4 are hydrogen;
R5 is hydrogen or alkyl of 1 to 8 carbon atoms;
R6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, or halo;
provided that R6 is other than hydrogen if X and Y are C═O and (i) each of R1, R2, R3, and R4 is fluoro or (ii) one of R1, R2, R3, or R4 is amino.
Compounds representative of this class are of the formulas:




wherein R1 is hydrogen or methyl. In a separate embodiment, encompassed is the use of enantiomerically pure forms (e.g. optically pure (R) or (S) enantiomers) of these compounds.
Still other specific immunomodulatory compounds belong to a class of isoindole-imides disclosed in U.S. Patent Application Publication Nos. US 2003/0096841 and US 2003/0045552, and WO 02/059106, each of which are incorporated herein by reference. Representative compounds are of formula II:




and pharmaceutically acceptable salts, hydrates, solvates, clathrates, enantiomers, diastereomers, racemates, and mixtures of stereoisomers thereof, wherein:

one of X and Y is C═O and the other is CH2 or C═O;
R1 is H, (C1-C8)alkyl, (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heterocycloalkyl, (C0-C4)alkyl-(C2-C5)heteroaryl, C(O)R3, C(S)R3, C(O)OR4, (C1-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5, C(O)NHR3, C(S)NHR3, C(O)NR3R3′, C(S)NR3R3′ or (C1-C8)alkyl-O(CO)R5;
R2 is H, F, benzyl, (C1-C8)alkyl, (C2-C8)alkenyl, or (C2-C8)alkynyl;
R3 and R3′ are independently (C1-C8)alkyl, (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heterocycloalkyl, (C0-C4)alkyl-(C2-C5)heteroaryl, (C0-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5, (C1-C8)alkyl-O(CO)R5, or C(O)OR5;
R4 is (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C1-C4)alkyl-OR5, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heterocycloalkyl, or (C0-C4)alkyl-(C2-C5)heteroaryl;
R5 is (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl, or (C2-C5)heteroaryl;
each occurrence of R6 is independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl, (C2-C5)heteroaryl, or (C0-C8)alkyl-C(O)O—R5 or the R6 groups can join to form a heterocycloalkyl group;
n is 0 or 1; and
* represents a chiral-carbon center.
In specific compounds of formula II, when n is 0 then R1 is (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heterocycloalkyl, (C0-C4)alkyl-(C2-05)heteroaryl, C(O)R3, C(O)OR4, (C1-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5, C(S)NHR3, or (C1-C8)alkyl-O(CO)R5;
R2 is H or (C1-C8)alkyl; and R3 is (C1-C8)alkyl, (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl, (C0-C4)alkyl-(C1-C6)heterocycloalkyl, (C0-C4)alkyl-(C2-C5)heteroaryl, (C5-C8)alkyl-N(R6)2; (C0-C8)alkyl-NH—C(O)O—R5; (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5, (C1-C8)alkyl-O(CO)R5, or C(O)OR5; and the other variables have the same definitions.
In other specific compounds of formula II, R2 is H or (C1-C4)alkyl.
In other specific compounds of formula II, R1 is (C1-C8)alkyl or benzyl.
In other specific compounds of formula II, R1 is H, (C1-C8)alkyl, benzyl, CH2OCH3, CH2CH2OCH3, or




In another embodiment of the compounds of formula II, R1 is




wherein Q is O or S, and each occurrence of R7 is independently H, (C1-C8)alkyl, (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl, halogen, (C0-C4)alkyl-(C1-C6)heterocycloalkyl, (C0-C4)alkyl-(C2-C5)heteroaryl, (C0-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5, (C1-C8)alkyl-C(O)OR5, (C1-C8)alkyl-O(CO)R5, or C(O)OR5, or adjacent occurrences of R7 can be taken together to form a bicyclic alkyl or aryl ring.

In other specific compounds of formula II, R1 is C(O)R3.
In other specific compounds of formula II, R3 is (C0-C4)alkyl-(C2-C5)heteroaryl, (C1-C8)alkyl, aryl, or (C0-C4)alkyl-OR5.
In other specific compounds of formula II, heteroaryl is pyridyl, furyl, or thienyl.
In other specific compounds of formula II, R1 is C(O)OR4.
In other specific compounds of formula II, the H of C(O)NHC(O) can be replaced with (C1-C4)alkyl, aryl, or benzyl.
Further examples of the compounds in this class include, but are not limited to: [2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl]-amide; (2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-carbamic acid tert-butyl ester; 4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-acetamide; N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropyl-carboxamide; 2-chloro-N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}acetamide; N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-3-pyridylcarboxamide; 3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidine-2,6-dione; 2-(2,6-dioxo(3-piperidyl))-4-(benzylamino)isoindoline-1,3-dione; N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamide; N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyridylcarboxamide; N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}heptanamide; N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furylcarboxamide; {N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methyl acetate; N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)pentanamide; N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarboxamide; N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(butylamino)carboxamide; N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamino)carboxamide; and N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylamino)carboxamide.
Still other specific immunomodulatory compounds belong to a class of isoindole-imides disclosed in U.S. Patent Application Publication No. 2002/0045643, International Publication No. WO 98/54170, and U.S. Pat. No. 6,395,754, each of which is incorporated herein by reference. Representative compounds are of formula III:




and pharmaceutically acceptable salts, hydrates, solvates, clathrates, enantiomers, diastereomers, racemates, and mixtures of stereoisomers thereof, wherein:
one of X and Y is C═O and the other is CH2 or C═O;
R is H or CH2OCOR′;
(i) each of R1, R2, R3, or R4, 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 R1, R2, R3, or R4 is nitro or —NHR5 and the remaining of R1, R2, R3, or R4 are hydrogen;
R5 is hydrogen or alkyl of 1 to 8 carbons
R6 hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;
R′ is R7—CHR10—N(R8R9);
R7 is m-phenylene or p-phenylene or —(CnH2n)— in which n has a value of 0 to 4;
each of R8 and R9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R8 and R9 taken together are tetramethylene, pentamethylene, hexamethylene, or —CH2CH2X1CH2CH2— in which X1 is —O—, —S—, or —NH—;
R10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and
* represents a chiral-carbon center.
Other representative compounds are of formula:




wherein:
one of X and Y is C═O and the other of X and Y is C═O or CH2;
(i) each of R1, R2, R3, or R4, 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 R1, R2, R3, and R4 is —NHR5 and the remaining of R1, R2, R3, and R4 are hydrogen;
R5 is hydrogen or alkyl of 1 to 8 carbon atoms;
R6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;
R7 is m-phenylene or p-phenylene or —(CnH2n)— in which n has a value of 0 to 4;
each of R8 and R9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R8 and R9 taken together are tetramethylene, pentamethylene, hexamethylene, or —CH2CH2X1CH2CH2— in which X1 is —O—, —S—, or —NH—;
R10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl.
Other representative compounds are of formula:




in which
one of X and Y is C═O and the other of X and Y is C═O or CH2;
each of R1, R2, R3, and R4, 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 R1, R2, R3, and R4 is nitro or protected amino and the remaining of R1, R2, R3, and R4 are hydrogen; and
R6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.
Other representative compounds are of formula:




in which:
one of X and Y is C═O and the other of X and Y is C═O or CH2;
(i) each of R1, R2, R3, and R4, 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 R1, R2, R3, and R4 is —NHR5 and the remaining of R1, R2, R3, and R4 are hydrogen;
R5 is hydrogen, alkyl of 1 to 8 carbon atoms, or CO—R7—CH(R10)NR8R9 in which each of R7, R8, R9, and R10 is as herein defined; and
R6 is alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.
Specific examples of the compounds are of formula:




in which:
one of X and Y is C═O and the other of X and Y is C═O or CH2;
R6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, chloro, or fluoro;
R7 is m-phenylene, p-phenylene or —(CnH2n)— in which n has a value of 0 to 4;
each of R8 and R9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R8 and R9 taken together are tetramethylene, pentamethylene, hexamethylene, or —CH2CH2X1CH2CH2— in which X1 is —O—, —S— or —NH—; and
R10 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-1,3-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., U.S. Pat. No. 5,635,517, incorporated herein by reference). The compounds are available from Celgene Corporation, Warren, N.J. 4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione has the following chemical structure:




The compound 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione has the following chemical structure:




In another embodiment, specific immunomodulatory compounds encompass polymorphic forms of 3-(4-amino-1-oxo-1,3 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 A1, which is incorporated herein by reference. For example, Form A 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 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 2θ, 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-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione discovered thus far.
Form B of 3-(4-amino-1-oxo-1,3 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 2θ, 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-1-oxo-1,3 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 2θ, 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-1-oxo-1,3 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 2θ, 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-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione in water and by a slow evaporation of 3-(4-amino-1-oxo-1,3 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 2θ, 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 2θ, and has a differential scanning calorimetry melting temperature maximum of about 269° C.
Form G 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 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 2θ, and has a differential scanning calorimetry melting temperature maximum of about 267° C.
Form H of 3-(4-amino-1-oxo-1,3 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 2θ, and has a differential scanning calorimetry melting temperature maximum of about 269° C.
Other specific immunomodulatory compounds usable in the methods provided herein include, but are not limited to, 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such as those described in U.S. Pat. Nos. 5,874,448 and 5,955,476, each of which is incorporated herein by reference. Representative compounds are of formula:




wherein Y is oxygen or H2 and
each of R1, R2, R3, and R4, independently of the others, is hydrogen, halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino.
Other specific immunomodulatory compounds usable in the methods provided herein include, but are not limited to, the tetra substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines described in U.S. Pat. No. 5,798,368, which is incorporated herein by reference. Representative compounds are of formula:




wherein each of R1, R2, R3, and R4, independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms.

Other specific immunomodulatory compounds that can be used in the methods provided herein include, but are not limited to, 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines disclosed in U.S. Pat. No. 6,403,613, which is incorporated herein by reference. Representative compounds are of formula:




in which
Y is oxygen or H2,
a first of R1 and R2 is halo, alkyl, alkoxy, alkylamino, dialkylamino, cyano, or carbamoyl, the second of R1 and R2, independently of the first, is hydrogen, halo, alkyl, alkoxy, alkylamino, dialkylamino, cyano, or carbamoyl, and
R3 is hydrogen, alkyl, or benzyl.
Specific examples of the compounds are of formula:




wherein a first of R1 and R2 is 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 R1 and R2, 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
R3 is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl. Specific examples include, but are not limited to, 1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.
Other compounds that can be used in the methods provided herein are of formula:




wherein a first of R1 and R2 is 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 R1 and R2, 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
R3 is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl.
Other specific immunomodulatory compounds that can be used in the methods provided herein include, but are not limited to, 1-oxo and 1,3-dioxoisoindolines substituted in the 4- or 5-position of the indoline ring described in U.S. Pat. No. 6,380,239 and U.S. Application Publication No. 2006/0084815, which are incorporated herein by reference. Representative compounds are of formula:




in which the carbon atom designated C* constitutes a center of chirality (when n is not zero and R1 is not the same as R2); one of X1 and X2 is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X1 or X2 is hydrogen; each of R1 and R2 independent of the other, is hydroxy or NH-Z; R3 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 X1 is amino, and n is 1 or 2, then R1 and R2 are not both hydroxy; and the salts thereof.
Further compounds that can be used in the methods provided herein are of formula:




in which the carbon atom designated C* constitutes a center of chirality when n is not zero and R1 is not R2; one of X1 and X2 is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X1 or X2 is hydrogen; each of R1 and R2 independent of the other, is hydroxy or NH-Z; R3 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.

Specific examples of compounds that can be used in the methods provided herein include, but are not limited to, 2-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid and 4-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid, which have the following structures, respectively, and pharmaceutically acceptable salts, solvates, prodrugs, and stereoisomers thereof:




Other representative compounds are of formula:




in which the carbon atom designated C* constitutes a center of chirality when n is not zero and R1 is not R2; one of X1 and X2 is amino, nitro, alkyl of one to six carbons, or NH-Z, and the other of X1 or X2 is hydrogen; each of R1 and R2 independent of the other, is hydroxy or NH-Z; R3 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]-1,3-dioxo-1,3-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]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-4-phenylcarbamoyl-butyric acid, and 2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-pentanedioic acid, which have the following structures, respectively, and pharmaceutically acceptable salts, solvate, prodrugs, and stereoisomers thereof:




Other specific examples of the compounds are of formula:




wherein one of X1 and X2 is nitro, or NH-Z, and the other of X1 or X2 is hydrogen;
each of R1 and R2, independent of the other, is hydroxy or NH-Z;
R3 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; and
n has a value of 0, 1, or 2;
provided that if one of X1 and X2 is nitro, and n is 1 or 2, then R1 and R2 are other than hydroxy; and
if —COR2 and —(CH2)nCOR1 are different, the carbon atom designated C* constitutes a center of chirality. Other representative compounds are of formula:




wherein one of X1 and X2 is alkyl of one to six carbons;
each of R1 and R2, independent of the other, is hydroxy or NH-Z;
R3 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; and
n has a value of 0, 1, or 2; and
if —COR2 and —(CH2)nCOR1 are different, the carbon atom designated C* constitutes a center of chirality.
Still other specific immunomodulatory compounds include, but are not limited to, isoindoline-1-one and isoindoline-1,3-dione substituted in the 2-position with 2,6-dioxo-3-hydroxypiperidin-5-yl described in U.S. Pat. No. 6,458,810, which is incorporated herein by reference. Representative compounds are of formula:




wherein:
the carbon atoms designated * constitute centers of chirality;
X is —C(O)— or —CH2—;
R1 is alkyl of 1 to 8 carbon atoms or —NHR3;
R2 is hydrogen, alkyl of 1 to 8 carbon atoms, or halogen; and
R3 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,
cycloalkyl of 3 to 18 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 —COR4 in which
R4 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,
cycloalkyl of 3 to 18 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.
Compounds provided herein can either be commercially purchased or prepared according to the methods described in the patents or patent publications disclosed herein. Further, 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 al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind., 1972).
It should be noted that if there is a discrepancy between a depicted structure and a name given that structure, the depicted structure is to be accorded more weight. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it.
5.9. Administration of PINK Cells, Human Placental Perfusate, or Combined Natural Killer Cells
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. In various embodiments, 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. In one embodiment, the cells are administered to an individual intravenously. In another embodiment, the cells are administered to the individual at the site of a tumor, e.g., a solid tumor. In a specific embodiment in which the individual has a tumor at more than one site, the cells are administered to at least two, or all, tumor sites. In certain other embodiments, 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. In certain specific embodiments, 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.
In one embodiment, the cells provided herein are seeded onto a natural matrix, e.g., a placental biomaterial such as an amniotic membrane material. Such 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% H2O) 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. 2004/0048796, the disclosure of which is hereby incorporated by reference in its entirety.
In another embodiment, 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. Suitable hydrogels for such compositions include self-assembling peptides, such as RAD16. In one embodiment, 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. In some embodiments, the hydrogel or matrix of the invention is biodegradable.
In some embodiments of the invention, 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).
In some embodiments, 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. Examples of 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. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.
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. Such a framework 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.
Examples of scaffolds that can be used in the present invention 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(ε-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. 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 HEALOSTM (DePuy, Inc., Raynham, Mass.) and VITOSS®, RHAKOSSTM, and CORTOSS® (Orthovita, Malvern, Pa.). The framework can be a mixture, blend or composite of natural and/or synthetic materials.
In another embodiment, 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.
The placental stem cells of the invention can, in another embodiment, be seeded onto foam scaffolds that may be composite structures. Such 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. In some embodiments, 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.
In some embodiments, 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 PURSPANTM (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.
6. EXAMPLES
6.1. Example 1
The present example demonstrates the isolation, culture and characterization of natural killer cells from human placental perfusate and umbilical cord blood.
Isolation of Natural Killer Cells.
Natural killer cells were isolated from 8 units of human placental perfusate (HPP), and from 4 units of umbilical cord blood (UCB, also referred to herein as “CB”), using CD56-conjugated microbeads. 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 μL of buffer per 107 total cells with 20 μL 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 107 total cells was added, and the mixture was then centrifuged at 300g for 10 minutes. The supernatant was pipetted off completely. The cell pellet was resuspended up to 108 cells in 500 μL 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 2×3 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. A 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. Among the whole CD56+, CD3− natural killer cell population, the percentage range of CD56+, CD16− natural killer cells (that is, PINK cells) was 56.6 to 87.2 (median 74.2, average 65.5) from HPP, and the percentage range of CD56+, CD16− 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).
In other experiments, natural killer cells were isolated using a magnetic negative selection kit that targets cell surface antigens on human blood cells (CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA-DR, glycophorin A). 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 approximately 30 mL of ice cold FACS buffer (RPMI 1640, without phenol red, plus 5% FBS), and then 60 mL ice cold ammonium chloride (Catalog #07850, Stem Cell) was added, the solution was vortexed and then incubated on ice for 5 minutes. The mononuclear cells were then washed with FACS buffer 3 times and then centrifuged at 1500 rpm for 8 minutes. The cell number was counted and the cell pellet was resuspended in 5×107 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 μm mesh nylon strainer (Catalog #352340, BD Falcon) before proceeding to isolation. Isolation is automated by the device RoboSep (Catalog #20000, Stem Cell) and the program “Human NK Negative Selection 19055 and high recovery” (50 μL/mL cocktail addition, 100 μL/mL Microparticle addition, 10 and 5 minute incubations, 1×2.5 minute separations) with Human NK Cell Enrichment Kit (Catalog #19055, Stem Cell) including EasySep Negative Selection Human NK Cell Enrichment Cocktail and EasySep Magnetic Microparticles. A CD56+CD3− population was thus collected and ready for further analysis.
Expansion of Natural Killer Cells.
In general, natural killer cells were expanded as follows. 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(1):219-227 (1984) and Litwin et al., J. Exp. Med. 178(4):1321-1326 (1993). Briefly, Start Medium includes IMDM (Invitrogen) with 10% FCS (Hyclone), containing the following reagents with final concentration of 35 μg/mL transferrin (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 2×10−5M ethanolamine (Sigma-Aldrich), 1 μg/mL oleic acid (Sigma-Aldrich), 1 μg/mL linoleic acid (Sigma-Aldrich), 0.2 μg/mL palmitic acid (Sigma-Aldrich), 2.5 μg/mL BSA (Sigma-Aldrich) and 0.1 μg/mL Phytohemagglutinin (PHA-P, Sigma-Aldrich). CD56+CD3− NK cells were resuspended at 2.5×105 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 1×106 per mL. NK cells were cultured for 5-6 days at 37° C. in 5% CO2. After 5-6 days and then every 3-4 days an equal volume of Maintenance Medium (IMDM with 10% FCS, 2% Human AB serum, antibiotics, L-glutamine and 400 units of IL-2 per mL) was added to the culture. NK cells were harvested at day 21.
Characterization of Natural Killer Cells from Placenta and Umbilical Cord Blood.
Donor matched HPP and CB was thawed, and the cells were washed with FACS buffer (RPMI-1640 with 5% FBS). Natural killer cells were then enriched with CD56 microbeads using the ROBOSEP® magnetic separation system (StemCell Technologies) as instructed by the manufacturer. The 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-CD16 FITC, anti-NKG2D APC, anti-NKp46 APC, anti-CD94 PE(R&D), anti-NKB1 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 In Vivo,” J. Exp. Med. 203(4):1033-1043 (2006); Eagle & Trowsdale, “Promiscuity and the Single Receptor: NKG2D,” Nature Reviews Immunology 7, 737-744 (2007); Walzer et al., “Natural Killer Cells: From CD3−NKp46+ to Post-Genomics Meta-Analyses,” Curr. Opinion Immunol. 19:365-372 (2007). As shown in Table 1, expression of KIR3DL1, KIR2DL2/L3, NKG2D, and CD94 (and NKp46; data not shown) was not significantly different between an enriched CD56+ cell population from HPP and an HLA-matched CD56+ cell population from umbilical cord blood (CB).







TABLE 1







Percentage of NK cells bearing certain


marker combinations. Mean of 3 samples.










Mean (%)












CB
HPP
p value
















CD3−CD56+
0.6
0.7
0.799



CD3−CD56+CD16−
53.9
58.7
0.544



CD3−CD56+CD16+
46.1
41.3
0.544



CD3−CD56+KIR3DL1+
5.8
7.3
0.762



CD3−CD56+KIR2DL2/L3+
10.7
9.9
0.89



CD3−CD56+NKG2D+
60.3
58.5
0.865



CD3−CD56+CD94+
74.6
76.8
0.839










6.2. Example 2
Donor matched mononucleated cells of umbilical cord blood and placental perfusate (combo) were mixed and washed with FACS buffer (RPMI-1640 with 5% FBS) once and immunophenotypically characterized using the antibodies listed in Table 2 on a BD FACSCANTOTM system (BD Biosciences). The data were analyzed by FLOWJO® software (Tree Star).







TABLE 2







List of antibodies used in immunophenotypic characterization.









Item
vendor
Cat No.





FITC anti-hu CD3
BD Bioscience
555332


FITC anti-hu CD3
Miltenyi
130-080-401


APC-Cy7 anti-hu CD3
BD Bioscience
557832


FITC anti-hu CD16
BD Bioscience
555406


PE-Cy5 anti-hu CD16
BD Bioscience
555408


PE anti-hu CD56
BD Bioscience
555516


PE anti-hu CD56
Miltenyi
130-090-755


PE-CY5 anti-hu CD56
BD Bioscience
555517


PE-Cy7 anti-hu CD56
BD Bioscience
557747


PE anti-hu CD94
R&D
FAB-1058P


PE anti-hu KIR-NKAT2 (2DL2/L3)
BD Bioscience
556071


PE anit-hu NKB1(3DL1)
BD Bioscience
555967


APC anit-hu NKG2D
BD Bioscience
558071


APC anit-hu NKp46
BD Bioscience
558051


PE anti-hu CD226
BD Bioscience
559789


PE anit-hu NKp44
BD Bioscience
558563


PE anti-hu NKp30
BD Bioscience
558407


PE anti-hu 2B4
BD Bioscience
550816


Isotype FITC mouse IgG1
BD Bioscience
340755


Isotype FITC mouse IgG2b
BD Bioscience
556577


Isotype PE mouse IgG1
BD Bioscience
340761


Isotype PE mouse IgG2b
BD Bioscience
555743


Isotype PerCP mouse IgG1
BD Bioscience
340762


Isotype PE-Cy5 mouse IgG2b
BD Bioscience
555744


Isotype APC mouse IgG1
BD Bioscience
340754


Isotype APC mouse IgG2a
BD Bioscience
555576


Isotype APC-Cy7 mouse IgG1
BD Bioscience
348802


Isotype PE-Cy7 mouse IgG1
BD Bioscience
348798









Immunophenotypic Characterization of Placental NK Cells and Peripheral Blood (PB) NK Cells.
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 CD16, 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 CD16, but are capable of producing cytokines and chemokines upon activation. Individual 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.
FACS analysis was performed on placental (combo) NK and peripheral blood NK cells using fluorescence-conjugated mAbs against specific NK receptors. Among 11 NK subsets characterized, the numbers of cells in seven out of 11 NK subsets (CD3−CD56+CD16−, CD3−CD56+CD16+, CD3−CD56+KIR2DL2/3+, CD3−CD56+NKp46+, CD3−CD56+NKp30+, CD3−CD56+2B4+ and CD3−CD56+CD94+) showed significant difference (p<0.05) between placental NK and peripheral blood NK cells (accounted for 64% difference) (Table 3A; see also Tables 3B and 3C).







TABLE 3A







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.











Combo (16 units)
PB (13 units)



Surface markers
Mean %
Mean %
P Value













CD3−CD56+
2.2
2.4
0.728


CD3−CD56+CD16−
60.9
21.4
0.000


CD3−CD56+CD16+
39.1
78.6
0.000


CD3−CD56+KIR3DL1
12.3
7.1
0.099


CD3−CD56+KIR2DL2/L3
21.9
9.5
0.004


CD3−CD56+NKG2D
42.1
29.9
0.126


CD3−CD56+NKp46
7.0
18.9
0.011


CD3−CD56+CD226
16.0
26.7
0.135


CD3−CD56+NKp44
9.5
4.9
0.073


CD3−CD56+NKp30
39.1
19.0
0.006


CD3−CD56+2B4
11.1
4.5
0.019


CD3−CD56+CD94
71.3
26.2
0.000









Tables 3B and 3C show the phenotypic characterization of CD3−CD56+CD16− 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.











TABLE 3B






Combo
PB



Surface markers
Mean %
Mean %
P Value


















CD3−CD56+CD16−
62.3
14.1
0.000


CD3−CD56+CD16−KIR3DL1
7.8
1.5
0.004


CD3−CD56+CD16−NKG2D
43.5
42.7
0.941


CD3−CD56+CD16−KIR2DL2/L3
13.6
2.4
0.000


CD3−CD56+CD16−NKp46
6.7
43.6
0.001


CD3−CD56+CD16−CD94
69.8
48.5
0.057


CD3−CD56+CD16−CD226
7.6
4.9
0.068


CD3−CD56+CD16−NKp44
3.4
0.6
0.076


CD3−CD56+CD16−NKp30
46.7
22.0
0.000


CD3−CD56+CD16−2B4
3.7
0.5
0.078



















TABLE 3C






Combo
PB



Surface markers
Mean %
Mean %
P Value


















CD3−CD56+CD16+
37.7
85.9
0.000


CD3−CD56+CD16+KIR3DL1
21.5
8.9
0.014


CD3−CD56+CD16+NKG2D
42.1
28.5
0.066


CD3−CD56+CD16+KIR2DL2/L3
34.5
12.1
0.000


CD3−CD56+CD16+NKp46
10.4
14.5
0.242


CD3−CD56+CD16+CD94
72.9
23.8
0.000


CD3−CD56+CD16+CD226
35.5
32.6
0.347


CD3−CD56+CD16+NKp44
22.6
6.4
0.016


CD3−CD56+CD16+NKp30
45.7
19.7
0.000


CD3−CD56+CD16+2B4
31.2
6.1
0.008









60.9% of placental NK cells are CD56+CD16− (placenta-derived intermediate natural killer (PINK) cells) while only 21.4% of peripheral blood NK cells are CD56+CD16−. After cultivation for 21 days, the percentage of four out 11 NK subsets (CD3−CD56+KIR2DL2/3+, CD3−CD56+NKp46+, CD3−CD56+NKp44+ and CD3−CD56+NKp30+) showed significant difference (p<0.05) between placental and peripheral blood NK cells.
In addition, in a separate experiment, it was determined that, after cultivation for 21 days, placental and peripheral blood NK cells demonstrated unique cytokine profiles, particularly for IL-8, as determined by LUMINEX® assay (Table 4).











TABLE 4








PB
Combo



Cytokine
(pg/mL)
(pg/mL)




















IL-13
1.26
1.89



IL-8
6.61
15.77



IL-10
1.26
2.23



TNFa
0.28
0.34



MCP-1
10.49
11.32










MicroRNA Profiling of Placental NK Cells And Peripheral Blood 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 1.5×106 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. 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 RNAs became immobilized. This RNA 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. One miRNA, designated hsa-miR-199b, was found to be unique for peripheral blood NK cells.







TABLE 5







miRNA profiling for piNK cells and PB NK cells 


via qRT-PCR.










Sanger



miRNA ID
Accession No.
Sequence





hsa-miR-100
MIMAT0000098
aacccguagauccgaacuugug


 


hsa-miR-127
MIMAT0000446
ucggauccgucugagcuuggcu


 


hsa-miR-211
MIMAT0000268
uucccuuugucauccuucgccu


 


hsa-miR-302c 
MIMAT0000717
uaagugcuuccauguuucagugg


 


hsa-miR-326
MIMAT0000756
ccucugggcccuuccuccag


 


hsa-miR-337
MIMAT0000754
uccagcuccuauaugaugccuuu


 


hsa-miR-497
MIMAT0002820
cagcagcacacugugguuugu


 


hsa-miR-512-
MIMAT0002823
aagugcugucauagcugagguc


3p




 


hsa-miR-515-
MIMAT0002826
uucuccaaaagaaagcacuuucug


5p




 


hsa-miR-517b
MIMAT0002857
ucgugcaucccuuuagaguguu


 


hsa-miR-517c
MIMAT0002866
aucgugcauccuuuuagagugu


 


hsa-miR-518a
MIMAT0002863
aaagcgcuucccuuugcugga


 


hsa-miR-518e
MIMAT0002861
aaagcgcuucccuucagagug


 


hsa-miR-519d
MIMAT0002853
caaagugccucccuuuagagug


 


hsa-miR-520g
MIMAT0002858
acaaagugcuucccuuuagagugu


 


hsa-miR-520h
MIMAT0002867
acaaagugcuucccuuuagagu


 


hsa-miR-564
MIMAT0003228
aggcacggugucagcaggc


 


hsa-miR-566
MIMAT0003230
gggcgccugugaucccaac


 


hsa-miR-618
MIMAT0003287
aaacucuacuuguccuucugagu


 


hsa-miR-99a
MIMAT0000097
aacccguagauccgaucuugug









Immunophenotypic Characterization of Cultured Placental NK Cells and Uncultured NK Cells. The overall properties of cultured PINK cells were evaluated by extensive immunophenotypic studies and cytotoxicity assays. To determine the phenotype of expanded NK cells, expression of NK receptors (NKRs) 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. From day 0 to day 21 the expression of NKG2D was increased from 60.9%±4.8% to 86%±17.4% (p value of 0.024); NKp46 was increased from 10.5%±5.4% to 82.8%±9.0% (p value of 0.00002); NKp44 was increased from 9.6%±6.5% to 51.6%±27.5% (p value of 0.022); and 2B4 was decreased from 13.0%±7.1% to 0.65%±0.5% (p value of 0.009%)(Table 6). Under these culture conditions the inhibitory KIRs including KIR3DL1 (killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail 1, an inhibitory receptor) and KIR2DL2/L3 (killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail 2 and long cytoplasmic tail 3; inhibitory receptors) remained not affected during 21-day expansion. The changes in the expression NKRs were further correlated with a marked increase in cytolytic activity at day 21 versus day 14 against K562 cells (63%±15% versus 45%±4%, p value of 0.0004). These findings have led to identification of the putative markers of NK cells which correlate well with the NK cell cytotoxicity activity.







TABLE 6







Phenotypic characterization of piNK cells before and


after 21-day cultivation. Standard deviation (Stdev)


was calculated for population means for 5 donors.










Day 0
Day 21












Mean %
Stdev
Mean %
Stdev















CD3−CD56+
2.9
1.1
85.5
8.6


CD3−CD56+CD16−
62.6
20.2
27.8
8.3


CD3−CD56+CD16+
37.4
20.2
72.2
8.3


CD3−CD56+KIR3DL1+
22.7
4.2
20.0
16.7


CD3−CD56+KIR2DL2/L3+
28.4
4.2
29.6
6.4


CD3−CD56+NKG2D+
60.9
4.8
86.0
17.4


CD3−CD56+NKp46+
10.5
5.4
82.8
8.9


CD3−CD56+CD226+
19.5
7.4
14.1
13.3


CD3−CD56+NKp44+
9.6
6.5
51.6
27.5


CD3−CD56+NKp30+
58.9
7.0
76.5
19.4


CD3−CD56+2B4+
13.0
7.1
0.6
0.5


CD3−CD56+CD94+
79.7
4.9
63.9
19.4









Membrane Proteomic Profiling of Cultured Placental NK Cells and Cultured Peripheral Blood NK Cells via Lipid-Based Protein Immobilization Technology and Linear Ion Trap LC/MS. Membrane Protein Purification: Placental natural killer cells from combined placental perfusate and cord blood cells, and PB NK cells, cultured for 21 days, were incubated for 15 min with a protease inhibitor cocktail solution (P8340, Sigma Aldrich, St. Louis, Mo.; contains 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A, E-64, bestatin, leupeptin, and aprotinin, without metal chelators) prior to cell lysis. The cells were then lysed by the addition of a 10 mM HCl solution, without detergents, and centrifuged for 10 min at 400g to pellet and remove the nuclei. The post-nuclear supernatant was transferred to an ultracentriguation tube and centrifuged on a WX80 ultracentrifuge with T-1270 rotor (Thermo Fisher Scientific, Asheville, N.C.) at 100,000g for 150 minutes generating a membrane protein pellet.
Generation, Immobilization and Digestion of Proteoliposomes:
The membrane protein pellet was washed several times using NANOXIS® buffer (10 mM Tris, 300 mM NaCl, pH 8). The membrane protein pellet was suspended in 1.5 mL of NANOXIS® buffer and then tip-sonicated using a VIBRA-CELLTM VC505 ultrasonic processor (Sonics & Materials, Inc., Newtown, Conn.) for 20 minutes on ice. The size of the proteoliposomes was determined by staining with FM1-43 dye (Invitrogen, Carlsbad, Calif.) and visualization with fluorescence microscopy. The protein concentration of the proteoliposome suspension was determined by a BCA assay (Thermo Scientific). The proteoliposomes were then injected onto an LPITM Flow 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 μg/mL (Princeton Separations, Adelphi, N.J.) 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 EP-PAK® cartridge (Waters Corporation, Milford, Mass.).
Strong Cation-Exchange (SCX) Fractionation:
Tryptic peptides were reconstituted in a 0.1% formic acid/water solution and loaded onto a strong-cation exchange (SCX) TOP-TIPTM column (PolyLC, Columbia, Md.), a pipette tip packed with 30 μm 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×150 mm 3 μm 200 Å MAGIC C18 column (Michrom Bioresources, Inc., Auburn, Calif.) 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 μL/min. Eluted peptides were analyzed on an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, Calif.) that employed ten data-dependent MS/MS scans following each full scan mass spectrum.
Bioinformatics:
Six RAW files corresponding to the 6 salt fractions that were collected for each tumor cell line (AML, CML) were searched as a single search against the IPI Human Database using an implementation of the SEQUEST algorithm on a SORCERERTM SOLOTM workstation (Sage-N Research, San Jose, Calif.). A peptide mass tolerance of 1.2 amu was specified, oxidation of methionine was specified as a differential modification, and carbamidomethylation was specified as a static modification. A Scaffold software implementation of the Trans-Proteomic Pipeline (TPP) was used to sort and parse the membrane proteomic data. Proteins were considered for analysis if they were identified with a peptide probability of 95%, protein probability of 95% and 1 unique peptide. Comparisons between membrane proteomic datasets were made using custom Perl scripts that were developed in-house.
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.








TABLE 7





PROTEINS SPECIFIC FOR
PROTEINS


PLACENTAL NK CELLS
SPECIFIC FOR PB NK CELLS







Aminopeptidase N
Fibroblast growth factor receptor 4



precursor


Apolipoprotein E
Immunity-associated nucleotide 4-like 1



protein


Atrophin-1 interacting
Integrin alpha-L precursor


protein 1


Innexin inx-3
Integrin beta-2 precursor


Integrin alpha-2 precursor
Integrin beta-4 precursor


Integrin beta-5 precursor
Membrane-bound lytic murein



transglycosylase D precursor


Mast cell surface glycoprotein
Oxysterol binding protein-related protein 8


GP49B precursor


Ryanodine receptor 1
Perforin 1 precursor









6.3. Example 3
This example demonstrates that placental intermediate natural killer cells are cytotoxic towards tumor 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.
In the cytokine secretion assay, CD56 microbead-enriched NK cells from HPP were mixed with KG-1a acute myelogenous leukemia cells at a 1:1 ratio. After incubation for 24 hours, supernatant was collected and subjected to Luminex analysis of IFN-γ and GM-CSF secretion. Increased levels of IFN-γ and GM-CSF was observed after 24h incubation of CD56-enriched HPP cells with KG-1a cells as shown in FIG. 2.
Cytotoxicity of PINK Cells
In a cytotoxicity assay utilizing PINK cells, target tumor cells were labeled with carboxyfluoroscein succinimidyl ester (CFSE). CFSE is a vital stain that is non-toxic to cells, and is partitioned between daughter cells during cell division. The cells were then placed in 96-well U-bottomed tissue culture plates and incubated with freshly isolated CD56+CD16− PINK cells at effector-target (E:T) ratios of 20:1, 10:1, 5:1 and 1:1 in RPMI 1640 supplemented with 10% FBS. After a 4 hour incubation time, cells were harvested and examined by flow cytometry for the presence of CFSE. The number of target cells recovered from culture without NK cells was used as a reference. Cytotoxicity is defined as: (1−CFSEsample/CFSEcontrol)*100%. Significant tumor cell cytotoxicity was observed at the 20:1 ratio. See FIG. 3.
Tumor Cell Susceptibility to Cultured PINK Cells
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 μl 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% CO2. After incubation, 50 μl supernatant was transferred to enzymatic assay plate, LDH activity was detected as provided by the manufacturer, and absorption was measured at 490 nm in an ELISA reader (Synergy HT, Biotek). The degree of cytotoxicity was calculated according to the following equation: % Cytotoxicity=(Sample−Effector Spontaneous−Target Spontaneous)/(Target maximum−Target Spontaneous)*100.
Certain tumor types may be more responsive to NK cells than others. 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). The NK cell cytotoxicity was measured by the LDH release assay after 4-hour co-culture.







TABLE 8







ATCC Tumor cell lines










Name
Description







CCRF-CEM
Human leukemia



KG-1
Human acute myeloid leukemia



KG-1A
Human acute myeloid leukemia



K562
Human chronic myeloid leukemia



KU812
Human chronic myeloid leukemia



U-937
Human histiocytic lymphoma



WERI-RB-1
Human retinoblastoma



HCC2218
Human breast cancer



RPMI 8226
Human multiple myeloma



HCT116
Human colorectal carcinoma



HT29
Human colorectal adenocarcinama



U266
Human multiple myeloma










At effector to target (E:T) 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).







TABLE 9







Differential susceptibility of tumor cells to cultured piNK cells. Standard


error of the mean (S.E.M.) was calculated for average cytotoxicity


from 3 donors.











Cell Line
% Cytotoxicity
S.E.M















CCRF-CEM
7.6
1.2



KG-1
20.5
1.5



KG-1a
6.0
3.2



K562
88.6
5.6



KU812
40.3
8.2



U937
89.2
9.8



WERI-RB-1
73.3
11.8



RPM18226
61.3
1.3



U266
57.4
4.7



HCT-116
61.0
5.1



HCC2218
14.8
3.7



HT-29
45.6
6.0










Enhancement of PINK Cell Cytotoxicity by Treatment with Lenalidomide and Pomalidomide

RNA Isolation and Purification.
Isolated or expanded NK cells were subjected to RNA preparation using RNAQUEOUS®-4PCR Kit (Ambion, Cat #AM1914). In brief, NK cells (0.5 to 1.5×106 cells) were lysed in a guanidinium lysis solution. 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. After elution from the filter the RNA was treated with the ultra-pure DNase 1 provided with the kit to remove trace amounts of DNA. Finally, 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.
Quantitative Real-Time (qRT-PCR) 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 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.
Quantitative real-time PCR (qRT-PCR) and flow cytometry analyses showed that the pomalidomide-elicited enhancement of NK cell cytotoxicity was correlated with increased granzyme B (GZMB) gene expression (60%±1.7% increase) (Table 10) and increased percentage of GZMB-positive NK cells (25% increase). In addition, expression of GM-CSF was increased in lenalidomide (232%±1.6% increase) and pomalidomide (396%±0.3% increase)-treated PINK cells (Table 10A, 10B).
Table 10A, 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.













TABLE 10A







Veh
Len.
Veh-stdev
Len.-stdev
P Value





















BAX
1
1.39
0.06
0.02
0.05


CCL5
1
1.24
0.11
0.07
0.04


CCR5
1
0.9
0.07
0.08
0.02


CD68
1
4.04
0.05
0.13
0.01


CD8A
1
1.3
0.01
0.02
0.02


CSF2
1
2.32
0.14
0.02
0.02


FAS
1
1.11
0.02
0.04
0.04


GUSB
1
1.13
0.04
0.07
0.05


IL2RA
1
1.26
0.03
0.01
0.03


TNFRSF18
1
0.7
0.1
0.16
0.04





BAX—BCL2-associated X protein


CCL5—chemokine (C-C motif) ligand 5


CCR5—chemokine (C-C motif) receptor 5


CSF2—colony stimulating factor 2 (granulocyte-macrophage)


FAS—TNF receptor superfamily, member 6


GUSB—β glucuronidaseβ


IL2RA—α interleukin 2 receptor


TNFRSF18—tumor necrosis factor receptor superfamily, member 18



















TABLE 10B







Veh
Pom.
Veh-stdev
Pom.-stdev
P Value





















ACTB
1
0.77
0.01
0
0.01


BAX
1
2.23
0.06
0
0.01


CCL2
1
5.46
0.01
0.37
0.02


CCL3
1
2.2
0.04
0.16
0.02


CCL5
1
1.78
0.11
0.04
0.02


CCR5
1
0.68
0.07
0
0.05


CD68
1
8.74
0.05
0.19
0


CD80
1
1.59
0.13
0.19
0.02


CD8A
1
2.39
0.01
0.08
0.01


CSF1
1
1.41
0.07
0.05
0.01


CSF2
1
3.96
0.14
0
0.01


ECE1
1
1.56
0.06
0.12
0.02


FAS
1
1.34
0.02
0.03
0.01


GNLY
1.01
1.96
0.18
0.02
0.05


GUSB
1
1.76
0.04
0.01
0.01


GZMB
1
1.59
0.06
0.02
0.03


IL10
1.02
1.52
0.31
0.22
0.04


IL1A
1.01
2.61
0.19
0.12
0.01


IL2RA
1
1.58
0.03
0.06
0.01


IL8
1
1.62
0.04
0.06
0.04


LTA
1
2.88
0.02
0.21
0.02


PRF1
1
1.17
0.07
0.1
0.05


PTGS2
1
1.68
0.01
0.05
0.02


SKI
1
1.96
0.04
0.02
0.01


TBX21
1.01
2.05
0.14
0.2
0.01





ACTB—β-actin


BAX—BCL2-associated X protein


CCL2—chemokine (C-C motif) ligand 2


CCL3—chemokine (C-C motif) ligand 3


CCL5—chemokine (C-C motif) ligand 5


CCR5—chemokine (C-C motif) receptor 5


CSF1—colony stimulating factor 1 (macrophage)


CSF2—colony stimulating factor 2 (granulocyte-macrophage)


ECE1—endothelin converting enzyme 1


FAS—TNF receptor superfamily, member 6


GNLY—granulysin


GUSB—glucuronidase-β


GZMB—granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1)


IL1A—α interleukin 1


IL2RA—interleukin 2 receptor-α


IL8—interleukin 8


IL10—interleukin 10


LTA—lymphotoxin α (TNF superfamily, member 1)


PRF1—perforin 1 (pore forming protein)


PTGS2—prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)


SKI—v-ski sarcoma viral oncogene homolog (avian)


TBX21—T-box 21






Cytotoxicity of Combined Natural Killer Cells

In a separate cytotoxicity assay, cultured 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. 252(1-2):83-92 (2001)), which inserts into cell plasma membrane due to its lipophilic aliphatic residue, then placed in 96-well U-bottom tissue culture plates and incubated with cultured NK cells at various effector-target (E:T) ratios in 200 μl RPMI 1640 supplemented with 10% FBS. Cultures were incubated for 4h at 37° C. in 5% CO2. After incubation, cells were harvested and TO-PRO-3 (Invitrogen Catalog # T3605), a membrane-impermeable DNA stain, was added to cultures to 1 μM final concentration followed by FACS analysis using a FACSCANTOTM system. Cytotoxicity was expressed as percentage of dead cell (PKH26+TO-PRO-3+) within the total PKH26+ target tumor cells.
In this cytotoxicity assay, 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. 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).
6.4. Example 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-1a was higher than that of TNC from matched UCB. Total nucleated cells from HPP or umbilical cord blood (UCB) were mixed with KG-1a 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 FACSCANTOTM, BD Bioscience). Tumor cells cultured alone were used as controls. Cytotoxicity was defined as: (1-CFSEsample/CFSEControl)*100%. Significant cytotoxicity was shown at the 100:1 ratio. See FIG. 5.
In a separate experiment, the cytotoxicity of total nucleated cells from HPP was compared to that of total nucleated cells from umbilical cord blood. Matched TNC-HPP or UCB was mixed with KG-1a cells at ratios of 0.78:1, 1.56:1, 3.12:1, 6.25:1, 12.5:1, 25:1, 50:1 or 100:1. TNC-HPP showed consistently higher cytotoxicity at all ratios compared to that of UCB. See FIG. 6.
In another experiment, 24 hours prior to incubation with KG-1a cells, TNC-HPP was stimulated with 100 U/mL, or 1000 U/mL of IL-2, while HPP cultured with RPMI medium was used as control. At a ratio of 6.25 NK cells per KG-1a cells and above, IL-2 appears to increase the cytotoxicity of the TNC-HPP. See FIG. 7.
The experiments were repeated using a broader array of tumor cell types, as shown in Table 11, using 5×105 HPP cells and 1×104 tumor cells.







TABLE 11





Tumor Cell Types Tested for Cytotoxic Effect of Placental Perfusate


















HCC2218
Human primary ductal carcinoma



CCRF-CEM
Human leukemia



J.RT3-T3.5
Human acute T cell leukemia



K562
Human chronic myeloid lymphoma (CML)



KG-1
Human acute myelogenous leukemia



KG-1a
Human acute myelogenous leukemia (AML)



KU812
Human leukemia (CML)



NCI-H1417
Human lung carcinoma



SNU-CI
Human colon adenocarcinoma



U-937
Human histiocytic lymphoma



WERI-RB-1
Human retinoblastoma



HCT-116
Human colorectal carcinoma



HT-29
Human colorectal adenocarcinoma



U266
Human myeloma










When HPP cells and tumor cells were co-cultured for 24 hours or 48 hours at a 50:1 ratio, the HPP cells showed substantial toxicity towards the tumor cells. For both times, co-culture resulted in the death of over 50% of the tumor cells. See FIGS. 8A and 8B.
6.5. Example 5
Without wishing to be bound by any particular mechanism of action responsible for mediating the potent anti-tumor effects of HPP cells, the IFN-γ, TNF-α, 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. To address the source of IFN-γ, TNF-α, and GM-CSF in the supernatant, flow cytometry-based intracellular characterization of co-cultured cells was performed, with results demonstrating that these cytokines were not produced in tumor cells.
Supernatants collected post-incubation were subjected to Luminex assay to determine the concentrations of IFN-γ, TNF-α, and GM-CSF (Cat# HCYTO-60K-03, Millipore). These three cytokines are related with NK cytotoxicity. (See, e.g., Imai et al., Blood 2005. 106(1):376-83.). Quantitative RT-PCR was also performed to examine the expression of IFN-γ, TNF-α, and GM-CSF using Applied Biosystems FAST 7900HT instrument and primers. Culture conditions were the same as for the co-culture cytotoxicity assays described above. Concentrations of cytokines were determined using a Luminex assay.
Secretion of IFN-γ, TNF-α, and GM-CSF from HPP cells cocultured with tumor cells was determined to be significantly higher than that of UCB cells. In one experiment, HPP cells were mixed with KG-1a cells at ratios of 0.78:1, 1.56:1, 3.12:1, 6.25:1, 12.5:1, 25:1, 50:1 or 100:1, in the presence or absence of 100 U/ml IL-2. TNC-HPP showed consistently increased production of IFN-γ in the presence of IL-2 compared to the absence of IL-2. IFN-γ levels at 24h were determined to increase about 5-26 fold (median: 16 fold); at 48h around 3 ̃65 fold (median: 27 fold), which was consistent with the results from cytotoxicity study. See FIG. 9.
In another experiment, 24 hours prior to incubation with KG-1a cells, TNC-HPP was stimulated with 100 U/mL, or 1000 U/mL of IL-2, while HPP cultured with RPMI media was used as control. HPP or matched UCB cells were incubated 24h with or without IL-2, before cocultured with KG-1a cells. The secretion of IFN-γ was most increased in HPP cells cocultured with K562 and KG-1a at 48h. When HPP cells were treated with 100 U/mL IL-2, the cytotoxicity of HPP cells on KG-1a at 24h and 48h was increased. The secretion level of IFN-γ in HPP cells was higher than that of the matched UCB cells upon IL-2 treatment. Higher expression of IFN-γ 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-γ production.
IFN-γ 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 104 tumor cells and 5×105 HPP cells. For CCRF-CEM, J.RT3-T3.5, K562, KG1, KG-1a, KU812, NC1-H1417, U-937 and WER1-RB-1 cell lines, the increase in IFN-γ 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. At 48 hours co-culture, the increase in IFN-γ production in HPP cells exceeded that of umbilical cord blood for all tumor cell lines. See FIG. 10B. Of the tumor cell lines, K562 cells induced the greatest increase in IFN-γ production in HPP cells at both 24 hours and 48 hours. Similar results were observed for TNF-α and GM-CSF.
Cell cycle analysis demonstrated that the percentage of KG-1a in S phase decreased 30% when cocultured with HPP as compared to KG-1a cells cultured alone. Further coculture experiments performed using different enriched fractions of HPP demonstrated that the anti-leukemic activity of HPP was largely attributed to the high concentration of unique immature natural killer cells characterized by high expression of CD56+, lack of expression of CD16.
6.6. Example 6: Suppression of Tumor Cell Proliferation In Vivo by Human Placental Perfusate Cells
6.6.1. Materials & Methods
The Example presented herein demonstrates suppression of tumor cell proliferation in vivo by human placental perfusate cells. Specifically, this Example demonstrates the effectiveness of human placental perfusate in vivo against tumor cells using a NOD/SCID mouse xenograft tumor model.
Culturing of KG-1 Cells.
KG-1 cells were maintained in Iscove's modified Dulbecco's medium supplemented with 20% of fetal bovine serum (growth medium) at 37° C. in 95% air/5% CO2 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. Next, 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).
After thawing, each unit was stored on wet ice prior to centrifugation. All tubes were centrifuged for 10 min (440×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.
For cell counting, a 25 μl aliquot of was transferred to a 15 ml conical tube containing 975 μl vehicle on ice. Erythrocytes were then lysed by adding 4 ml cold ammonium chloride lysis reagent and incubating on ice for 10 min. After incubation, 5 ml cold PBS was added to each tube and the tubes were centrifuged (10 min, 400×g, 10° C.). After RBC lysis, cells were counted by hemacytometer, using trypan blue to assess viability. Counting results were corrected for dilution and then divided by a lysis factor (0.46) to estimate the number of cells present before RBC lysis.
For HPP dose preparation, after counting, HPP cells were diluted to 1×108 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.
Before filling syringes, a 50 μl aliquot of the dosing material was set aside for post-dose verification by counting as described above. After dosing, remaining dose material was assessed for dose verification.
Study Design.
On Day 1, twenty-four NOD/SCID male mice (Jackson Laboratories) 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. On Day 52 post-implantation, the animals were randomized into three separate groups, with tumor volumes averaging about 300-350 mm3. See Table 12, below. The first group consisted of four control mice with an average tumor volume of 312 mm3. Two of these mice were implanted intravenously (IV), and two intra-tumorally (IT), with 200 μl and 50 μl of a vehicle solution, respectively. The second group with an average tumor volume of 345 mm3 consisted of four mice implanted intravenously with 200 μl of HPP cells per mouse (2×107 cells). The last group, implanted IT with 50 μl of HPP cells per mouse also consisted of four mice with an average tumor volume of 332 mm3.







TABLE 12







Experimental groups for in vivo tumor suppression experiment.











Tumor Volume On Day of


Animal #
HPP Treatment Group
HPP Implantation










Group 1 (Control)









1
IV 1
457


2
IT 2
429


3
IT 3
214


4
IV 4
147



Mean:
312







Group 2 (IV Cell Implantation)









5
1
466


6
2
209


7
3
217


8
4
487



Mean:
345







Group 3 (IT Cell Implantation)









9
1
491


10
2
256


11
3
296


12
4
285



Mean:
332





IV—200 μL implantation;


IT—50 μL implantation.






On Day 66, 14 days after the implantation of HPP cells, the study was terminated due to high tumor volumes.
6.6.2. Results
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 mm3. The IV treatment group at the end of study had an average TV of 2076 mm3, and the IT group had a TV of 2705 mm3. With respect to % increase in the TV post-treatment, 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.
6.7. Example 7
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
Processing of Human Placenta Derived Stem Cells and UCB.
Donated full-term placentas were perfused with normal saline to recover human placenta derived stem cells. Human placenta-derived stem cells were subsequently processed to remove red blood cells, non-viable cells and tissue debris followed by cryopreservation. Human placenta-derived stem cells were neither expanded nor cultured during processing.
Isolation of Natural Kills Cells from Human Placenta-Derived Stem Cells and UCB.
The donor-matched cryopreserved human placenta-derived stem cells and UCB were initially thawed, combined, and washed with RPMI 1640 (without phenol red) (Gibco) containing 5% v/v fetal bovine serum (FBS; Hyclone laboratories). In some NK cell expansion experiments, peripheral blood mononuclear cells (PBMCs) obtained from buffy coat (Blood Center, NJ) were prepared as another source of NK cells. After washing, cell pellets were resuspended at 5×107 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 μl/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 μl/ml and incubated for 10 minutes. EasySep® Magnetic Microparticles were then added to a final concentration of 100 μl/ml and incubated for 5 minutes. Enrichment of NK cells were then performed using a fully automated cell separator, RoboSep, according to the protocol provided by the manufacturer (StemCell Technologies). A CD56+CD3− population was thus collected and ready for further analysis or cultivation.
Ex Vivo Expansion of pNK Cells.
Enriched placental CD56+CD3−NK cells were cultured in Start Medium based on a modification of previously described protocols (Yssel et al., 1984, J. Immunol. Methods. 72, 219−227). Briefly, Start Medium was composed of IMDM (ATCC) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 35 mg/ml transferrin (Sigma-Aldrich), 5 μg/ml insulin (Sigma-Aldrich), 20 μM ethanolamine (Sigma-Aldrich), 1 μg/ml oleic acid (Sigma-Aldrich), 1 μg/ml linoleic acid (Sigma-Aldrich), 0.2 μg/ml palmitic acid (Sigma-Aldrich), 2.5 μg/ml BSA (Sigma-Aldrich) and 0.1 μg/ml Phytohemagglutinin (PHA-P, Sigma-Aldrich). NK cells were resuspended at approximately 2.5×105/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 1×106/ml for PBMC cells and 1×106/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% CO2. After culturing for 5 to 7 days, expanded cell populations were fed with Maintenance Medium for up to 21 days. Maintenance Medium was composed of IMDM supplemented with 10% FBS, 2% Human AB serum (Gemini), Penicillin-Streptomycin and 200 IU/ml IL-2. Total cell number and cell viability were examined using Easycount (Immunicon) and Easycount Viasure Kit (Immunicon). Fold expansion was calculated using the formula as: fold=absolute number of CD56+CD3−NK cells on day 21/absolute number of NK cells on day 0.
BrdU/7-AAD Cell Cycle Analysis.
Expanded cells were labeled with BrdU (BD Bioscience) and cultured at 37° C. in 5% CO2 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 a FACSCALIBURTM system (BD Biosciences) and analysis was accomplished with FLOWJO® software (Tree Star, Inc.).
Immunophenotypic Characterization.
The phenotype of mononuclear cells (MNCs) or enriched NK cells from the donor-matched human placenta-derived stem cells and UCB, or expanded cells from day 7, 14, 21 cultures, was analyzed by multi-color flow cytometry. Cells were stained with fluorochrome-conjugated monoclonal antibodies against human blood surface antigens: CD56-PerCP/-PE/-PE-Cy7, CD3-FITC/-APC-Cy7, CD16-FITC/-PerCP, CD158b-PE (killer-immunoglobulin-like receptor [KIR] 2DL2/2DL3), CD158e1-PE (KIR3DL1), NKG2D-APC, NKp46-APC, NKp44-PE, NKp30-PE, CD226-PE, 2B4-PE (all purchased from BD Biosciences Pharmingen) and CD94-PE (R&D Systems). All analyses were performed using a FACSCANTO ITM system (BD Biosciences) and FLOWJO® analysis software.
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-MacAry et 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 μl RPMI 1640 supplemented with 10% FBS. After 4 hours incubation at 37° C. in 5% CO2, cells were harvested and TO-PRO-3 (Invitrogen) was added to cultures at 1 μM final concentration followed by FACS analysis using BD a FACSCANTO ITM system. Cytotoxicity was expressed as the percentage of dead cells (PKH26+TO-PRO-3+) within the total PKH26+ target tumor cells.
Lactate Dehydrogenase (LDH) Release Assay.
Alternatively, NK cell in vitro cytotoxicity was examined by LDH release assay using CYTOTOX 96® colorimetric cytotoxicity assay kit (Promega). In this assay, effector cells and target cells were placed in 96-well U-bottom tissue culture plates and incubated at various E:T ratios in 100 μl RPMI 1640 without phenol red (Invitrogen) supplemented with 2% human AB serum and incubated for 4 hours at 37° C. in 5% CO2. After incubation, 50 μl supernatant was transferred to the enzymatic assay plate for detection of LDH activity as instructed by the manufacturer. Cytotoxicity was calculated using the following equation: % Cytotoxicity=(experimental release−effector spontaneous release−target spontaneous release)/(target maximum release−target spontaneous release)*100.
miRNA Preparation And Quantitative PCR (QPCR).
miRNA was isolated from 0.5 to 1.5×106 cells using a MIRVANATM miRNA Isolation Kit (Ambion) following the protocol provided by manufacturer. The concentration and purity of the recovered small RNA was determined by measuring its absorbance at 260 and 280 nm. Purified 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. For each miRNA, the mean ΔCt from real-time PCR was calculated as ΔCtmean=mean(Ctsample)−mean(Ctendo), where Ctsample is the Ct value of a miRNA and Ctendo is the Ct value of the endogenous control. miRNAs were unique to pNK or PB NK if they met the following criteria: 1) ΔCtmean<0; 2)|ΔCtmean|≧2*Ctendo_SD, where |ΔCtmean| is the absolute value of ΔCtmean and Ctendo_SD is the standard deviation of the Ctendo; and 3) the miRNAs that satisfied the previous two criteria were exclusive to the cell type. The rationale for using the aforementioned criteria was to confirm that a miRNA is abundant in at least one donor sample, in comparison to the endogenous control. Donor samples without detectable levels of a miRNA were numerically ignored when averaging the Ct values. A negative ΔCtmean along with two standard deviations from the control gene ensures that the particular gene is relatively abundant. Additionally, all of the standard deviations of the reference genes were less than 0.25, thereby confirming the quality of the control. Significantly expressed miRNAs between pNK and PB NK, as well those between Day 0 pNK and Day 21 pNK were determined by a two-sample t-test (p value of 0.01 significance level) on the ΔCt values. Expression fold changes were calculated according to R=2(ΔCt1_mean−ΔCt2_mean) where R is the fold change (Livak, K. J. and Schmittgen, T. D, 2001, Methods. 4, 402-408); ΔCt1_mean is the average ΔCt of pNK on day 0, and ΔCt2_mean is the average ΔCt 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 controls was used to calculate the ΔCt. 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.
6.7.2. Results and Discussion
6.7.2.1. Phenotypic Profile of NK Cells from Human Placenta-Derived Stem Cells
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. NK cell composition analyzed by cell surface marker expression (CD56+CD3−) revealed no substantial difference between human placenta-derived stem cells (0.70%±0.24%) and the donor-matched UCB (0.63%±0.36%) (n=3). 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). The two-sample t-test was used to determine if population means were equal in human placenta-derived stem cells and UCB. As shown in FIG. 12A, 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. After NK expansion for 21 days separately, 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 (FIG. 12B).
6.7.2.2. Isolation and Characterization of pNK from Cryopreserved Combo Units
Human placenta-derived stem cells and donor matched UCB units were combined into one “Combo unit” to be used as the starting material for cell expansion.
Analyses demonstrate that nearly 90% of pNK cells were routinely recovered from the cryopreserved Combo units with greater than 70% CD56+CD3− cell purity.
pNK cells from 16 Combo units and NK cells from 13 units of buffy coat peripheral blood (PB) were subjected to immunophenotypic characterization. The expression of cell surface markers, including KIRs (KIR3DL1, KIR2DL2/3), KLRs (CD94), NKG2D, natural cytotoxicity receptors NCRs (NKp46, NKp44 and NKp30), 2B4 and CD226, was evaluated. Significant differences between pNK cells and PB NK cells were observed, including CD56+CD16−, CD56+CD16+, KIR2DL2/3+, NKp46+, NKp30+, 2B4+ and CD94+(FIG. 13A and Table 13). Most (79%) of the PB NK cells displayed the mature CD56+CD16+ phenotype, while a majority of the pNK population (over 60%) was immature CD56+CD16− cells. The greater proportion of CD16− 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.







TABLE 13







Comparison of subpopulations within pNK and PB NK cell phenotype










NK Characterization
Combo (16 Units)
PB (13 Units)













NK Sub-populations
Mean %
STDEV
Mean %
STDEV
P Value















CD16−
60.94
16.58
21.38
14.00
***


CD16+
39.05
16.58
78.63
14.01
***


KIR3DL1+
12.31
8.11
7.07
8.28
NS


KIR2DL2/L3+
21.89
8.65
9.46
11.31
**


NKG2D+
42.11
17.79
29.88
22.64
NS


NKp46+
6.98
4.33
18.86
13.97
*


CD226+
15.97
6.66
26.75
23.31
NS


NKp44+
9.48
5.27
4.89
6.40
NS


NKp30+
39.08
19.06
18.99
20.86
**


2B4+
11.07
5.90
4.46
6.45
*


CD94+
71.31
13.94
26.17
30.49
***





*p < 0.05;


**p < 0.01;


***p < 0.001;


NS = non-significant






The phenotype of pNK cells and PB NK cells was further explored by miRNA analysis using TAQMAN® Array Human MicroRNA Cards to compare expression of 365 miRNAs in pNK and PB NK cells. These analyses identified 4 miRNAs unique to pNK cells (hsa-miR-337, hsa-miR-422a, hsa-miR-549 and hsa-miR-618) and 8 miRNAs uniquely expressed by PB NK cells (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). Additionally, 20 miRNAs were expressed at a significantly higher level and 29 miRNAs were expressed at a significantly lower level in pNK cells compared to PB NK cells (Tables 14 and 15). These results demonstrate that pNK cells have a unique miRNA expression pattern compared to PB NK cells.







TABLE 14







Highly expressed miRNAs in pNK cells.











miRNA
Fold Increase in pNK
P Value















hsa-miR-211
5.26
6.73E−03



hsa-miR-520c
5.58
7.70E−03



hsa-miR-125b
7.46
9.26E−04



hsa-miR-100
11.19
4.29E−04



hsa-miR-326
14.50
5.05E−05



hsa-miR-519c
18.74
6.32E−03



hsa-miR-515-5p
20.99
6.88E−03



hsa-miR-450
21.31
3.00E−03



hsa-miR-198
27.97
3.41E−04



hsa-miR-522
33.63
2.56E−03



hsa-miR-518e
39.78
7.17E−03



hsa-miR-497
54.47
8.88E−03



hsa-miR-566
75.98
1.32E−04



hsa-miR-519d
96.65
3.47E−04



hsa-miR-627
98.54
3.42E−04



hsa-miR-524
106.36
7.97E−04



hsa-miR-520g
291.10
3.24E−04



hsa-miR-302c
396.53
3.55E−04



hsa-miR-512-3p
640.56
3.16E−05



hsa-miR-520h
1793.82
9.60E−05

















TABLE 15







Highly expressed miRNAs in PB NK cells.











miRNA
Fold Increase in PB NK
P Value















hsa-miR-331
1.43
5.33E−03



hsa-miR-186
1.90
4.54E−03



hsa-miR-17-5p
2.38
2.34E−03



hsa-miR-26a
2.66
2.36E−03



hsa-miR-133b
2.69
8.19E−04



hsa-miR-181b
2.77
4.42E−03



hsa-miR-222
2.83
5.76E−03



hsa-miR-197
3.00
5.48E−05



hsa-miR-146b
3.05
2.92E−03



hsa-miR-342
3.06
3.23E−04



hsa-miR-181d
3.08
3.41E−03



hsa-miR-155
3.12
8.24E−04



hsa-miR-484
3.18
1.23E−03



hsa-let-7g
3.18
2.08E−03



hsa-miR-200c
3.66
1.91E−03



hsa-miR-181c
3.83
2.72E−04



hsa-miR-191
4.06
3.16E−04



hsa-miR-596
4.14
7.06E−03



hsa-miR-142-5p
4.63
4.84E−04



hsa-miR-95
4.86
2.99E−03



hsa-let-7a
5.04
3.91E−04



hsa-miR-21
5.10
2.87E−04



hsa-miR-152
5.46
1.76E−03



hsa-miR-642
5.56
4.70E−04



hsa-miR-24
5.91
2.54E−05



hsa-miR-10a
14.56
5.71E−03



hsa-miR-429
31.74
5.70E−03



hsa-let-7b
108.34
4.66E−05



hsa-miR-199b
2819.55
3.05E−03










6.7.2.3. miRNA Target Gene Prediction
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 chemosensitivity (Paik, et al., 2011, Clin. Cancer Res. 17, 4761-4771). 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. These 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-γ production in primary CD56+ NK cells (Cichocki, et al., 2011, J. Immunol. 187, 6171-6175). Lastly, within the group of highly expressed miRNAs in pNK, miRNA:mRNA target pairs comprised of pluripotency markers in stem cells and cell cycle regulators, such as SOX2, BMPR1, SMO, AKT1, ATM, RAFT and MTOR, were identified most of which are not present in the targeted gene list (both experimental and validated) of miRNAs in PB NK.







TABLE 16







Differentially regulated miRs and their validated target genes in pNK compared to PB NK








miR
Genes










Highly Expressed miRNAs in PB NK



















hsa-let-7a
TRIM71
(7)
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)



TMEM2 
(5)
DHX57 
(3)
MRM1 
(2)
CCNA2 
(2)
CSNK1D 
(1)
UHRF1 
(1)



LIN28B 
(5)
FNDC3A 
(3)
POM121 
(2)
CCNF 
(2)
DOCK5 
(1)
C20ORF72 
(1)



CCNJ 
(5)
RDH10 
(3)
PXDN 
(2)
EDEM3 
(2)
FADS2 
(1)
SCAMP3 
(1)



CDC34 
(5)
SLC25A24 
(3)
SCYL1 
(2)
TRABD 
(2)
FAM96A 
(1)
C2ORF18 
(1)



IGF2BP2 
(5) 
SNAP23 
(3)
SLC25A32 
(2)
PLAGL2 
(2)
GPR56 
(1)
CIAO1 
(1)



DMD 
(5)
LIN28A 
(3)
SPRYD4 
(2)
FARP1 
(2)
IPO4 
(1)
BIRC6 
(1)



E2F6 
(5)
PRDM1 
(3)
TAF9B 
(2)
C7ORF58 
(2)
KIAA0409 
(1) 
AURKA 
(1)



HMGA1 
(4)
NRAS 
(3)
TTC9C 
(2)
LIN28 
(1)
NEDD4 
(1)
ALG3 
(1)



PGRMC1 
(4)
RRM2 
(3)
DLC1 
(2)
BCL7A 
(1)
OPRS1 
(1)
ARID3A 
(1)



THBS1 
(4)
CCND1 
(2)
CCND2 
(2)
ACTG1 
(1)
RHOB 
(1)
CCBL2 
(1)



PDE12 
(4)
ATP6V1F 
(2)
DICER1 
(2)
AARSD1 
(1)
RHOG 
(1)
RRP1B 
(1)



E2F5 
(4)
GEMIN7 
(2)
GTF2I 
(2)
ANAPC1 
(1)
SLC1A4 
(1)
TAB2 
(1)


















hsa-let-7g
HMGA2 
(6)
EIF4G2 
(5)
IGF2BP1 
(5)
COL1A2 
(3)
BCL2L1 
(1)



hsa-miR-10a
NCOR2 
(5)
MAP3K7 
(4)
HOXA1 
(1)
USF2 
(1)
BTRC 
(1)
















hsa-miR-133b
BCL2L2 
(3)
MCL1 
(2)





















hsa-miR-146b*
TRAF6 
(7)
IRAK1 
(5)
MMP16 
(2)
CARD10 
(1)




hsa-miR-152
DNMT1 
(5)


HLA-G
(2)























hsa-miR-155
SOCS1 
(5)
DET1 
(4)
SPI1 
(3)
SMAD1 
(2)
TM6SF1 
(1)
RAB34 
(1)



TSHZ3 
(5)
IKBKE 
(3)
MEIS1 
(2)
TRAM1 
(2)
FOXO3 
(1)
RAB6A 
(1)



HIVEP2 
(5)
PHF17 
(3)
SMAD2 
(2)
TRIP13 
(2)
ARL5B 
(1)
SYPL1 
(1)



TAB2 
(4)
BACH1 
(3)
CYR61 
(2)
FGF7 
(2)
ATG3 
(1)
VAMP3 
(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 
(4)
ZNF652 
(3)
CHAF1A 
(2)
C5ORF41 
(2) 
CBFB 
(1)
INPP5D 
(1)



DHX40 
(4)
MYB 
(3)
CLDN1 
(2)
IKBIP 
(2)
DNAJB1 
(1)
PAPOLA 
(1)



PICALM 
(4)
TLE4 
(3)
MYO10 
(2)
TWF1 
(2)
DSG2 
(1)
SERTAD2 
(1)



TRIM32 
(4)
CSF1R 
(3)
NARS 
(2)
CUX1 
(2)
FMNL2 
(1)
ERMP1 
(1)



ZIC3 
(4)
FAR1 
(3)
PHC2 
(2)
SLA 
(2)
PKN2 
(1)
C3ORF58 
(1)



KBTBD2 
(4)
ZNF236 
(3)
SDCBP 
(2)
AGTR1 
(1)
PRAF2 
(1)
HNRNPA3P1 
(1)
















MSH2 
(1)
PELI1 
(1)


















hsa-miR-17-5p
E2F1 
(1)
























hsa-miR-181b
GRIA2 
(4)
GATA6 
(4)
TIMP3 
(4)
MAP3K10 
(3)
NLK 
(2)
PLAG1 
(2)


















CYLD 
(2)
VSNL1 
(1)
KAT2B 
(1)
BCL2 
(1)




















hsa-miR-181c
GATA6 
(4)
KRAS 
(3)
NLK 
(2)
NOTCH4 
(2)
NOTCH2 
(1)

















hsa-miR-181d
GATA6 
(3)
NLK 
(2)
BCL2 
(1)

















hsa-miR-186
AKAP12 
(1)




















hsa-miR-191
TMC7 
(2)
SOX4 
(1)























hsa-miR-197
FBXW7 
(4)
DPH1 
(2)
UMPS 
(2)
CLIC1 
(1)
HNF4A 
(1)
FOXO3 
(1)



CHIC2 
(4)
ALMS1 
(2) 
CPNE6 
(2)
WDR6 
(1)
PEX13 
(1)
IL1R1 
(1)



ACVR1 
(4)
CES1 
(2)
RBM4 
(2)
NEK4 
(1)
C1ORF38 
(1)
CPSF1 
(1)



RAB28 
(3) 
ZNF302 
(2)
CYLD 
(2)
PIPOX 
(1)
MED16 
(1)
SNX1 
(1)



HNRNPD 
(2)
RAD51 
(2)
RFX1 
(2)
IGF2AS 
(1)
LRP4 
(1) 
KLF10 
(1)



GOLGB1 
(2)
RXRB 
(2)
IER3 
(1)
DCBLD2 
(1)
TSPYL1 
(1)
AGR2 
(1)
















TUSC2 
(1)
EHD2 
(1)






hsa-miR-199b
LAMC2 
(1)
HES1 
(1)























hsa-miR-200c
ZFPM2 
(6)
ZEB1 
(5)
ERRFI1 
(5)
ZEB2 
(5)
FN1 
(5)
UBE2I 
(3)



















BAP1 
(2)
PTPN13 
(1)
BMI1 
(1)
JAG1 
(1)
TUBB3 
(1)




















hsa-miR-21
TGFBI 
(5)
GLCCI1 
(3)
RASGRP1 
(3)
SGK3 
(2)
ANKRD46 
(2)
SLC16A10 
(1)



NFIB 
(4)
SOX5 
(3)
MSH2 
(3)
RP2 
(2)
ACTA2 
(1)
TIMP3 
(1)



RECK 
(4)
JAG1 
(3)
PCBP1 
(3)
SERPINB5 
(2)
BTG2 
(1)
TGFBR2 
(1)



PDCD4 
(3)
BMPR2 
(3)
TOPORS 
(3)
SPRY2 
(2)
SESN1 
(1)
NCAPG 
(1)



FAM3C 
(3)
TIAM1 
(3)
APAF1 
(2)
RHOB 
(2)
SOCS5 
(1)
IL1B 
(1)

















RTN4 
(1)
PTX3 
(1)
CDK2AP1 
(1)






















hsa-miR-222
CDKN1B 
(5)
FOS 
(5)
KIT 
(4)
CDKN1C 
(3)
PPP2R2A 
(2)
MMP1 
(1)



















SOD2 
(1)
BBC3 
(1)
PTEN 
(1)
ICAM1
(1)
ESR1 
(1)




















hsa-miR-24*
CDKN1B 
(4)
TRIB3 
(4)
MAPK14 
(2)
FURIN 
(2)
BRCA1 
(1)
NOTCH1 
(1)



ACVR1B 
(4)
DND1 
(3)
NFAT5 
(2)
MLEC 
(2)
KIAA0152 
(1)
CDKN2A 
(1)

















KHSRP 
(1)
HNF4A 
(1)
TGFB1 
(1)






















hsa-miR-26a
SMAD1 
(5)
HMGA1 
(5)
CDK8 
(4)
HMGA2 
(3)
CCND2 
(2)
CCNE2 
(2)



STRADB 
(5)
GSK3B 
(5)
MTDH 
(4)
CPEB4 
(3)
CTGF 
(2)
LIF 
(2)



PTEN 
(5)
EZH2 
(4)
MAP3K2 
(4)
SERBP1 
(2)
CDC6 
(2)
CPEB2 
(1)
















CPEB3 
(1)
SMAD4 
(1)




















hsa-miR-331
ERBB2 
(2)
CDCA5 
(2)
KIF23 
(1)


















hsa-miR-342
BMP7 
(1)


GEMIN4 
(1)





















hsa-miR-429
ZFPM2 
(6)
ZEB1 
(6)
ERRFI1 
(4)
ZEB2 
(4)
WASF3 
(3)
BAP1 
(1)







Highly Expressed miRNAs in pNK



















hsa-miR-100
MTOR 
(3)
PLK1 
(1)
FGFR3 
(1)
IGF1R 
(1)
ATM
(1)




hsa-miR-125b
LACTB 
(6)
BMF 
(3)
SAMD10 
(3)
MKNK2 
(2)
BBC3 
(2)
DICER1 
(1)



BAK1 
(5)
BMPR1B 
(3)
EIF4EBP1 
(3)
CBFB 
(2)
QSOX2 
(2)
JUB 
(1)



ARID3B 
(5)
ENTPD4 
(3)
KLF13 
(3)
LIN28A 
(2)
LIN28 
(1)
DDX19B 
(1)



IRF4 
(5)
TOR2A 
(3)
ULK3 
(3)
IGF2 
(2)
C10ORF104 
(1)
PABPC1 
(1)



PRDM1 
(4)
KCNS3 
(3)
SLC7A6 
(3)
NKIRAS2 
(2)
B3GALT4 
(1)
AKT1 
(1)



SLC35A4 
(4)
LIN28B 
(3)
SLC7A1 
(3)
SEL1L 
(2)
UBE2I 
(1)
TP53 
(1)



CGN 
(4)
GRIN2A 
(3)
ERBB3 
(2)
ATXN1 
(2)
RBM8A 
(1)
CASC3 
(1)



PPAT 
(4)
STAT3 
(3)
CDKN2A 
(2)
RAF1 
(2)
IGFBP3 
(1)
E2F3 
(1)



CBX7 
(3)
LIF 
(3)
ABTB1 
(2)
CYP24A1 
(2)
MAN1A1 
(1)
RNF144A 
(1)



SGPL1 
(3)
SMARCD2 
(3)
ARID3A 
(2)
ABCC4 
(2)
SMO 
(1)
LYPLA2 
(1)

















PLEKHA8 
(1)
TP53INP1 
(1) 
VDR 
(1)

















hsa-miR-211
KCNMA1 
(3)







hsa-miR-302c
ESR1 
(2)






















hsa-miR-326
PKM2 
(2)

SMO 
(1)
GLI1 
(1)
NOTCH2 
(1)















hsa-miR-422a**
CYP8B1 
(2)







hsa-miR-519c
HIF1A 
(4)




















hsa-miR-519d
CDKN1A 
(4)
PPARA 
(3)






hsa-miR-520c
APP 
(3)
CD44 
(3)


















hsa-miR-520g
VEGFA 
(2)
























hsa-miR-520h
SMAD6 
(3)
ABCG2 
(2)
CDKN1A 
(2)
VEGFA 
(2)
ID1 
(1)
ID3 
(1)














hsa-miR-522
SOX2 
(1)




















hsa-miR-342
BMP7 
(1)


GEMIN4 
(1)





















hsa-miR-429
ZFPM2 
(6)
ZEB1 
(6)
ERRFI1 
(4)
ZEB2 
(4)
WASF3 
(3)
BAP1 
(1)





* and ** denote that the expression level is significantly higher (2 standard deviations higher) in at least one of the donors than the endogenous control in only the PB or pNK, respectively. Numbers in parentheses denote the number of databases which indicate that the gene is targeted by the miR, in addition to having been validated previously (e.g., by statistical analysis and by experiment).






6.7.2.4. Ex Vivo Expansion of pNK Cells
Starting from an average of 10 million pNK cells after the isolation procedure described above, a protocol to expand cytotoxic and helper T cells (Yssel et al., 1984) was used to optimize expansion of pNK cells. First, to optimize the feeder cell concentration, allogeneic PBMCs and K562 cells were tested at ratios of 1:10, 1:5 and 1:1, with the concentration of PBMC fixed at 1×106/ml. As shown in Table 17, the ratio of 1:1 (1×106/ml K562:1×106/ml PBMC) resulted in the greatest NK cell expansion of 98.4-fold (N=14), compared to 31.8-fold (N=3) for the 1:5 ratio and 53.1-fold (N=9) for the 1:10 ratio. Therefore, a 1:1 ratio was used for further pNK cultivation. Next, the ability of replenishment with fresh feeder cells to enhance NK expansion during the cultivation process was evaluated. To determine the best time window for replenishing with fresh feeder cells, cell growth kinetics of pNK cells at Day 7, 14, 21, and 28 were evaluated with BrdU and 7-aminoactinomycin-D (7-AAD) double-staining followed by flow cytometry. As seen in FIG. 14, at day 7, the majority of cultured NK cells were in S-phase, indicating that the cells were proliferating. The percentage of actively proliferating/dividing cells decreased substantially during subsequent culture, suggesting Day 7 was the optimal window for re-stimulation. As shown in Table 18, addition of fresh K562 and PBMC feeder cells at Day 7 resulted in a 3-fold increase in expansion of NK cells. This optimized 21-day NK culture method was repeated in 20 expansion experiments, yielding an average of 1.2×109 CD56+CD3−NK cells per donor with approximately 80% viability (FIG. 15A).







TABLE 17







Optimization of ratio of K562 to PBMC as feeder cells












Feeder

Fold Expansion*
Average Fold


Type of Feeders
Ratio
#Donor
Range
Expansion














K562:PBMC
 1:10
9
 6.2~130.6
53.13


K562:PBMC
1:5
3
10.64~53.76
31.81


K562:PBMC
1:1
14
 14.8~358.4
98.4





*Fold expansion = absolute number of CD56+ CD3− NK cells on Day 21/absolute number of NK cells on Day 0.













TABLE 18







Effect of feeder replenishing at different time points












Day of

Fold Expansion
Average Fold



Stimulation
#Donor
Range
Expansion
















Day 0
10
17.4~83.0 
40.9



Day 0 + Day 7
10
47.6~394.8
147.5










6.7.2.5. Characterization of Ex Vivo-Expanded pNK Cells
Immunophenotypic and miRNA changes were characterized in expanded pNK cells from 12 Combo units in comparison to unexpanded cells. Day 21 pNK cells showed a significant increase in the expression of activating receptors such as NKG2D, NKp46, NKp44 and NKp30, and a significant decrease in the expression of bidirectional receptor 2B4 compared to unexpanded cells, for example. The expression of inhibitory KIRs, including KIR3DL1 and KIR2DL2/L3 was similar for expanded and unexpanded cells (FIG. 15B and Table 19).







TABLE 19







Sub-population comparison of Day 21-expanded pNK versus unexpanded


pNK cells











Day 21 pNK
Day 0 pNK



NK Characterization
(12 units)
(16 Units)












NK Sub-populations
Mean %
STDEV
Mean %
STDEV
P Value















CD56+CD16−
35.98
18.18
60.94
16.58
**


CD56+CD16+
63.80
18.32
39.05
16.58
**


KIR3DL1+
17.92
13.44
12.31
8.11
NS


KIR2DL2/L3+
21.10
10.48
21.89
8.65
NS


NKG2D+
89.28
12.88
42.11
17.79
***


NKp46+
88.74
5.34
6.98
4.33
***


CD226+
18.79
12.14
15.97
6.66
NS


NKp44+
64.13
16.65
9.48
5.27
**


NKp30+
84.53
12.40
39.08
19.06
***


2B4+
0.89
0.99
11.07
5.90
***


CD94+
74.82
12.45
71.31
13.94
NS





*p < 0.05;


**p < 0.01;


***p < 0.001






Expansion and immunophenotype of PB NK using the optimized isolation and expansion procedure established for pNK (described above) in 9 donors was also characterized, and a comparison of Day 21-expanded pNK versus Day 21-expanded PB NK is shown in Table 20.







TABLE 20







Sub-populations comparison in Day 21-expanded pNK versus Day


21-expanded PB NK











Combo (12 Units)
PB (9 Units)













NK sub-populations
Mean %
STDEV
Mean %
STDEV
P Value















CD16−
35.98
18.18
28.39
7.78
NS


CD16+
63.80
18.32
71.50
7.78
NS


KIR3DL1+
17.92
13.44
8.83
6.44
NS


KIR2DL2/L3+
21.10
10.48
37.40
17.82
*


NKG2D+
89.28
12.88
73.27
20.06
NS


NKp46+
88.74
5.34
64.97
17.26
**


CD226+
18.79
12.14
4.80
1.72
NS


NKp44+
64.13
16.65
16.00
3.82
**


NKp30+
84.53
12.40
55.77
1.96
*


2B4+
0.89
0.99
2.16
1.76
NS


CD94+
74.82
12.45
71.76
20.35
NS





*p < 0.05;


**p < 0.01;


***p < 0.001






Moreover, expression of 23 miRNAs was increased while 31 other miRNAs were downregulated after ex vivo expansion (Table 21). One of the miRNAs found to be upregulated was hsa-miR-155, which when overexpressed has been shown to increase NK cell function via enhanced induction of IFN-γ (Trotta, et al., 2012, Blood 119, 3478-3485).







TABLE 21







Differentially regulated miRNAs during pNK ex vivo expansion












Fold Change in




miRNA
Day 21 vs Day 0
P Value















hsa-miR-520g
−2820.57
6.33E−05



hsa-miR-520h
−2803.02
4.35E−04



hsa-miR-518a
−2300.60
5.72E−05



hsa-miR-517b
−1669.82
8.11E−05



hsa-miR-451
−1626.65
2.40E−03



hsa-miR-518c
−609.88
2.93E−04



hsa-miR-127
−557.55
3.40E−04



hsa-miR-517a
−288.89
8.27E−05



hsa-miR-382
−273.34
1.77E−04



hsa-miR-519d
−245.09
7.13E−04



hsa-miR-486
−149.95
6.29E−03



hsa-miR-518b
−112.10
9.70E−05



hsa-miR-522
−85.30
2.58E−03



hsa-miR-376a
−72.14
2.95E−03



hsa-miR-198
−70.93
1.13E−03



hsa-miR-126
−51.46
1.97E−04



hsa-miR-487b
−47.68
3.32E−03



hsa-miR-519c
−47.52
4.88E−03



hsa-miR-518e
−34.89
5.06E−03



hsa-miR-433
−18.07
5.68E−04



hsa-miR-125b
−16.66
4.66E−04



hsa-miR-214
−16.38
5.74E−03



hsa-miR-130a
−12.98
4.13E−03



hsa-miR-518d
−10.75
8.73E−03



hsa-miR-99a
−7.91
2.03E−03



hsa-miR-515-3p
−4.93
2.50E−03



hsa-miR-95
−4.73
9.30E−04



hsa-miR-30a-3p
−3.06
1.20E−03



hsa-miR-30d
−2.59
4.32E−03



hsa-miR-26a
−1.75
7.81E−03



hsa-miR-191
−1.32
2.12E−03



hsa-miR-331
1.53
6.74E−03



hsa-miR-181c
1.71
1.24E−03



hsa-miR-142-3p
2.24
5.55E−03



hsa-miR-155
2.48
3.47E−03



hsa-miR-24
2.66
1.03E−03



hsa-miR-23a
3.07
8.50E−03



hsa-miR-142-5p
3.21
3.34E−04



hsa-let-7d
3.22
3.29E−03



hsa-miR-195
3.77
2.92E−04



hsa-miR-141
3.80
5.20E−03



hsa-miR-98
3.83
2.96E−03



hsa-miR-222
3.86
7.71E−03



hsa-miR-545
5.28
5.60E−03



hsa-miR-642
7.17
2.82E−04



hsa-miR-21
13.46
1.94E−04



hsa-miR-210
13.87
6.66E−03



hsa-miR-221
21.73
3.04E−03



hsa-miR-34c
45.37
5.26E−04



hsa-miR-135b
49.26
1.17E−03



hsa-miR-34a
66.38
8.08E−03



hsa-miR-10a
72.27
1.27E−03



hsa-miR-380-3p
921.50
1.18E−03



hsa-miR-520a
10892.33
2.38E−05










6.7.2.6. Antitumor Cytolytic Activity of Expanded pNK Cells
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 PKH26/TO-PRO-3 cytotoxicity assay against K562 cells. As shown in FIG. 15C, there was a significant enhancement of pNK cells at Day 21 versus Day 14 at an E:T ratio of 10:1 (63%±15% versus 45%±4%, p<0.001) against K562. By comparison, less cytotoxic/cytolytic activity was observed from PB NK cells (vs. pNK cells) at Day 21 (42%±7%) (FIG. 15C).
This increase in cytolytic activity after 21-day expansion of pNK cells is correlated with the increased expression in activating receptors and miRNAs. Extended cultivation to 28 days did not result in further increases in cytolytic activity against K562 cells.
Eleven additional tumor cells lines were co-cultured with Day 21 expanded pNK cells, and NK cell cytolytic activity at E:T ratios of 10:1, 5:1, 2:1 and 1:1 was measured in a 4-hour LDH release assay. At an E:T ratio of 10:1, expanded pNK cells exhibited greater than 50% cytotoxicity against multiple tumor cell lines, including U937 cells at 89.2%±9.8%; WERI-RB-1 cells at 73.3%±11.8%; RPMI8226 cells at 61.3%±1.3%; HCT-116 cells at 61%±5.1% and U266 cells at 57.4%±4.7%, as well as K562 cells at 88.6%±5.6% (FIG. 16). Cytolytic activity of expanded pNK cells against tumor lines was in a dose-dependent manner as shown through systemic titration of E:T ratios.
These results demonstrate that expanded pNKs have the ability to kill a variety of tumor cells, including both blood (e.g., leukemia) and solid tumors.
6.7.3. Conclusion
This example demonstrates that pNK cells, e.g., cells from human placenta-derived stem cells and UCB cells, which are distinct in phenotype and miRNA profile compared to PB NK cells, can be expanded to yield a clinically relevant quantity of a highly cytotoxic cells useful against a variety of tumor cell types.
6.8. Example 8
This example describes use of pNK cells for treating GVHD in patients receiving HLA-nonidentical (allogeneic) stem cell transplantation.
HLA-Nonidentical Donor Hematopoietic Stem Cell (HSC) Plus pNK Cell Transplantation in Patients with Hematologic Malignancies.
Individuals with hematological malignancies are given chemotherapy and total-body irradiation to stop the growth of cancer cells and to prevent the individual's immune system from rejecting the healthy donor's stem cells. Healthy HSCs from a donor that are infused into the individual help the individual's hematopoietic reconstitution. Individuals then undergo pNK cell transplantation to prevent GVHD or reduce the severity of GVHD in addition to preventing relapse and improving survival.
EQUIVALENTS
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

## Claims

1. A method of treating or preventing graft-versus-host disease, comprising administering natural killer cells isolated from human placental perfusate and umbilical cord blood to a patient with or at risk of developing graft-versus-host disease, wherein said patient has received a solid organ transplant or a composite tissue allograft.

2. The method of claim 1, wherein said human placental perfusate and umbilical cord blood has been treated to remove a plurality of erythrocytes.

3. The method of claim 1, wherein said natural killer cells comprise at least about 50% CD56+ cells.

4. The method of claim 1, wherein said natural killer cells comprise at least about 50% CD56+CD16− cells.

5. The method of claim 1, wherein said natural killer cells are contacted with an immunomodulatory compound in an amount and for a time sufficient for said cells to express detectably more granzyme B than an equivalent number of cells not contacted with said immunomodulatory compound.

6. The method of claim 5, wherein said immunomodulatory compound is lenalidomide or pomalidomide.

7. The method of claim 1, wherein said 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.

8. The method of claim 1, wherein said natural killer cells have not been cultured.

9. The method of claim 1, wherein said natural killer cells have been cultured.

10. The method of claim 9, wherein said natural killer cells have been cultured for about 21 days.

11. The method of claim 9, wherein said natural killer cells have been cultured in the presence of feeder cells.

12. The method of claim 11, wherein additional feeder cells are added to the culture at about day 7.

13. The method of claim 1, wherein said patient has received a solid organ transplant.

14. The method of claim 1, wherein said patient has received a composite tissue allograft.

15. The method of claim 1, wherein said graft-versus-host disease is reduced in grade by at least one step by said administering.

16. The method of claim 1, wherein said graft-versus-host disease does not progress beyond grade II within 100 days after transplantation as a result of said administering.

17. The method of claim 1, wherein said graft-versus-host disease does not progress beyond grade I within 100 days after transplantation as a result of said administering.

18. The method of claim 1, wherein said natural killer cells express one or both of miRNAs hsa-miR-422a and hsa-miR-549.

19. The method of claim 1, wherein the natural killer cells express one or more of miRNAs hsa-miR-125b, hsa-miR-198, hsa-miR-450, hsa-miR-519c, hsa-miR-520c, hsa-miR-522, hsa-miR-524, and hsa-miR-627 at a detectably greater amount than an equivalent number of natural killer cells from peripheral blood.

20. The method of claim 9, wherein the natural killer cells express one or more of miRNAs hsa-miR-450, hsa-miR-520c, hsa-miR-524, and hsa-miR-627 at a detectably greater amount than an equivalent number of natural killer cells from peripheral blood.