TWO-STEP IN VITRO METHOD FOR THE PRODUCTION OF A POPULATION OF ACTIVATED NATURAL EXTERMINATOR (NK) P

专利摘要:
METHOD AND METHOD OF TWO STEPS FOR THE PRODUCTION OF A POPULATION OF ACTIVATED NATURAL EXTERMINATING CELLS (NK), POPULATION OF ACTIVATED NK CELLS AND METHOD TO SUPPRESS THE PROLIFERATION OF TUMOR CELLS. The present invention provides methods for the production of natural killer cells (NK) using a two-step expansion and differentiation method. Also provided herein are methods for suppressing tumor cell poliferation, or for treating individuals with cancer or a viral infection, comprising administering the NK cells produced by the method to an individual with cancer or viral infection.
公开号:BR112013000822B1
申请号:R112013000822-9
申请日:2011-07-13
公开日:2021-02-23
发明作者:Robert J. Hariri；Mohammad A. Heidaran；Stephen Jasko；Lin Kan；Ajai Pal；Bhavani Stout；Vanessa Voskinarian-Berse；Andrew Zeitlin；Eric Law；Xiaokui Zhang
申请人:Anthrogenesis Corporation；
IPC主号:

专利说明:

1. RELATED REQUESTS
[0001] This application claims the benefit of Provisional US Patent Application number 61 / 363,981, filed on July 13, 2010, and Provisional US Patent Application number 61 / 497,897, filed on June 16 2011, the descriptions of which are incorporated herein in full as a reference. 2. FIELD OF THE INVENTION
[0002] Here are provided methods for producing a population of natural killer cells, for example, natural killer cells derived from placenta, for example, placental perfusate (for example, human placental perfusate), such as intermediate natural killer cells placenta, or other tissues, for example, umbilical cord blood or peripheral blood. Expanded populations of natural killer cells produced by the methods presented here are also provided here. Also provided here are methods for the use of placental perfusate, and its natural killer cells, to suppress tumor cell proliferation. In certain embodiments, natural killer cells are used in combination with, or treated with, one or more immunomodulatory compounds, for example, the immunomodulatory compounds referred to as IMiDs ™. 3. SCENARIO OF THE INVENTION
[0003] Natural killer cells (NK) are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells do not express antigenic T-cell receptors (TCR), CD3 or B-cell surface immunoglobulin (Ig) receptors. NK cells generally express the surface markers CD16 (FcyRIII) and CD56 in humans, but a subclass of NK cells. is CD16. NK cells are cytotoxic; small granules in your cytoplasm contain special proteins like perforin and proteases known as granzymes. As soon as it is released near a cell marked for extermination, perforin forms pores on the cell membrane of the target cell through which granzymes and associated molecules can enter, inducing apoptosis. A granzyme, granzyme B (also known as granzyme 2 and as cytotoxic serine stearase 1 associated with T lymphocytes), is a crucial serine protease for the rapid induction of target cell apoptosis in this cell-mediated immune response.
[0004] NK cells are activated in response to macrophage-derived interferons or cytokines. Activated NK cells are referred to as lymphokine-activated killer cells (LAK). NK cells have two types of surface receptors, labeled "activating receptors" and "inhibiting receptors", which control the cytotoxic activity of the cells.
[0005] Among other activities, NK cells play a role in the rejection of tumors by the host. Because cancer cells have reduced or nonexistent MHC class I expression, they can become targets for NK cells. Accumulated clinical data suggest that haploidentical transplantation of human NK cells isolated from peripheral blood mononuclear cells (PBMC) or bone marrow mediates potent antileukemic effects without causing 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 showing reduced levels, of major histocompatibility complex (MHC) proteins. In addition, the activating receptors expressed on NK cells are known to mediate the detection of “stressed” or transformed cells, with ligands expressed for activating receptors, and thus trigger the activation of NK cells. For example, NCR1 (NKp46) binds to viral hemagglutinins. NKG2D linkers include the CM16 protein with UL16 1 (ULB1), ULB2, ULB3 and A sequence protein related to MHC class I (MICA) and MICB polypeptide. The NK 2B4 protein binds with CD48, and DNAM-1 binds to the Poliovirus receptor (PVR) and Nectin-2, both consistently detected in acute myeloid leukemia (AML). See Penda et al., Blood 105: 2066-2073 (2004). In addition, AML lysis has been described as mainly dependent on the natural cytotoxic receptor (NCR). See Fauriat et al., Blood 109: 323330 (2007). Activated and expanded NK cells and peripheral blood LAK cells have been used in ex vivo therapies and in vivo treatment of patients with advanced cancer, with some success against bone marrow-related diseases, such as leukemia; breast cancer; and certain types of lymphoma. Treatment with LAK cells requires that the patient first receive IL-2, followed by leukopheresis, and then culture and ex vivo incubation of autologous blood cells collected in the presence of IL-2 for a few days. LAK cells must be reinjected with relatively high doses of IL-2 to complete therapy. This purge treatment is expensive and can cause serious side effects. These include fluid retention, pulmonary edema, drop in blood pressure, and high fever.
[0006] Despite the advantageous properties of NK cells in the extermination of tumor cells and virus-infected cells, they remain difficult to work and apply in immunotherapy, mainly due to the difficulty in maintaining their ability to recognize tumors and tumoricide due to culture and to expansion. Thus, there is a need in the art to develop an efficient method to produce and expand natural killer cells that retain tumoricidal functions. 4. SUMMARY OF THE INVENTION
[0007] Methods are provided here for the expansion and differentiation of cells, for example, hematopoietic cells, such as hematopoietic stem cells, for example, CD34 + hematopoietic stem cells, to natural killer cells. In one aspect, a method for producing natural killer cells (NK) comprising culture of hematopoietic stem cells or progenitor cells, for example, CD34 + hematopoietic stem cells or progenitor cells, in a first medium for producing cells is provided here. expanded and differentiated, and the subsequent culture of the expanded cells themselves in a second medium in which said cells expand further and differentiate into natural exterminating cells. The first and second steps comprise the culture of cells in a medium with a unique combination of cellular factors. In certain embodiments, said cellular factors (for example, cytokines) are not comprised in an undefined component of the medium (for example, serum), for example, cellular factors (for example, cytokines) are exogenous to the indefinite component of the medium ( for example, serum). In certain embodiments, the method itself is a two-step method. In certain modalities, the method itself does not comprise any intermediate or tertiary stage in which there is contact with the cells. In a specific embodiment, a method is provided here for the production of a population of activated natural killer cells (NK), comprising: (a) the inoculation of a population of hematopoietic stem cells or progenitor cells in a first medium comprising interleukin- 15 (IL-15) and, optionally, one or more stem cell factors (SCF) and interleukin-7 (IL-7), where IL-15 and the optional SCF and IL-7 itself are not included in an undefined component of the medium itself, so that the population expands, and a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differ in NK cells during said expansion; and (b) expanding the cells of the first step in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells. The natural killer cells produced by the methods provided here (for example, the two-step method) are referred to here as TSNK cells.
[0008] In certain embodiments, the first medium itself comprises one or more of human serum (for example, human serum AB), fetal bovine serum (FBS) or fetal calf serum (FCS), for example, 5% to 20% % v / v, stem cell factor (SCF), for example, 1 ng / ml at 50 ng / ml, tyrosine kinase fms-like ligand (Flt3 ligand), for example, 1 ng / ml at 20 ng / mL; interleukin-7 (IL-7), for example, 1 ng / ml to 50 ng / ml; thrombopoietin (Tpo), for example, 1 ng / ml to 50 ng / ml; interleukin-2 (IL-2), for example, 50 IU / ml to 500 IU / ml; interleukin-15 (IL-15), for example, 1 ng / ml to 50 ng / ml; and / or heparin, for example, 0.1 IU / ml to 10 IU / ml. In a specific modality, the first medium itself comprises stem cell factor, interleukin-7 (IL-7) and interleukin-15 (IL-15). In another specific embodiment, the first medium itself comprises growth medium, human serum (for example, human serum AB), FBS, FCS, SCF, IL-7 and IL-15. In another specific embodiment, the first medium itself further comprises ligand Flt-3 (Flt3-L), Tpo, IL-2, and / or heparin. In another specific embodiment, the first medium itself comprises a culture medium, 10% human serum or fetal bovine serum, 20 ng / mL SCF, 10 ng / mL Flt3-L, 20 ng / mL IL-7 , 20 ng / ml Tpo, 200 IU / ml IL-2, 10 ng / ml IL-5, and 1.5 IU / ml heparin. In another specific modality, the first medium itself does not comprise IL-2. In another specific embodiment, the culture in the first medium itself comprises culture using feeder cells, for example, K562 cells, for example, K562 cells treated with mitomycin C, peripheral blood mononuclear cells (PBMCs), for example, treated PBMCs with mitomycin C, or stem cells adherent to tissue culture, for example, stem cells adherent to tissue culture treated with mitomycin C.
[0009] In certain embodiments, the first medium itself is, or comprises, GBGM®, AIM-V®, X-VIVO ™ 10, X-VIVO ™ 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETE ™, and / or DMEM: F12. In certain embodiments, the medium itself comprises O-acetyl-carnitine (also referred to as acetylcarnitine, O-acetyl-L-carnitine or OAC), for example, about 0.5 mM - 10 mM. In one embodiment, the medium itself comprises Stemspan® H3000, and / or DMEM: F12 and OAC, for example, about 0.5; 1; two; 3; 4; 5; 6; 7; 8; 9; or 10 mM. In a specific modality, the medium itself comprises GBGM®. In another specific modality, the medium itself comprises DMEM: F12 and about 5 mM OAC. In another specific embodiment, the medium comprises STEMSPAN® H3000 and about 5 mM OAC.
[00010] In certain embodiments, the second medium itself comprises cell culture medium comprising one or more of human serum (eg, human serum AB), fetal bovine serum (FBS), or fetal calf serum (FCS), for example example, 5% - 15% FCS v / v; IL-2, for example, 10 IU / ml to 1000 IU / ml; transferrin, for example, 10 μg / mL to 50 μg / mL; insulin, for example, 5 μg / mL to 20 μg / mL; ethanolamine, for example, 5 x 104 to 5 x 105 M; oleic acid, for example, 0.1 μg / mL to 5 μg / mL; linoleic acid, for example, 0.1 μg / mL to 5 μg / mL; palmitic acid, for example, 0.05 μg / mL to 2 μg / mL; bovine serum albumin (BSA), for example, 1 μg / mL to 5 μg / mL; and / or phytohemagglutinin, for example, 0.01 μg / mL to 1 μg / mL. In a specific embodiment, the second medium itself comprises IL-2. In a more specific embodiment, the second medium itself comprises cell culture medium comprising human serum, FBS or FCS (for example, 10% v / v), IL-2, transferrin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid, bovine serum albumin (BSA) and / or phytohemagglutinin. In a more specific embodiment, the second medium itself comprises Iscove's Modified Dulbecco's Medium (IMDM), 10% human serum, FBS or FCS, 400 IU IL-2, 35 μg / mL transferrin, 5 μg / mL insulin, 2 x 105 ethanolamine, 1 μg / mL oleic acid, 1 μg / mL linoleic acid, 0.2 μg / mL palmitic acid, 2.5 μg / mL BSA and 0.1 μg / mL phytohemagglutinin. In another specific embodiment, the culture in the second medium itself comprises the use of feeder cells, for example, K562 cells (for example, K562 cells treated with mitomycin C), or PBMCs (for example, PBMCs treated with mitomycin C), for example example, when the cells are introduced into the second medium itself, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days later. In certain embodiments, the medium itself comprises GBGM®, AIM-V®, X-VIVO ™ 10, X-VIVO ™ 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETE ™, and / or DMEM: F12. In certain embodiments, the medium itself comprises one or more of O-acetyl-carnitine (also referred to as acetylcarnitine, O-acetyl-L-carnitine or OAC), or a compound that affects the cyclization of acetyl-CoA in mitodronia, thiazovivine , Y-27632, pi-integrin, rho-kinase (ROCK) inhibitors, caspase inhibitors or other anti-apoptotic compounds / peptides, NOVA-RS (Sheffield BioScience) or other small molecule growth enhancers. In certain embodiments, the medium itself comprises nicotinamide. In certain embodiments, the medium itself comprises about 0.5 mM - 10 mM OAC. In one embodiment, the medium itself comprises Stemspan® H3000, and / or DMEM: F12 and OAC, for example, about 0.5; 1; two; 3; 4; 5; 6; 7; 8; 9; or 10 mM. In a specific modality, the medium itself comprises GBGM®. In another specific modality, the medium itself comprises DMEM: F12 and about 5 mM OAC. In another specific embodiment, the medium itself comprises Stemspan® H3000 and about 5 mM OAC.
[00011] In another specific embodiment, a method is provided here for the production of a population of activated natural killer cells (NK), comprising: (a) the inoculation of a population of hematopoietic stem cells or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more stem cell factors (SCF) and interleukin-7 (IL-7), where IL-15 and the optional SCF and IL-7 itself do not are comprised of an undefined component of the medium itself, so that the population expands, and a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differ in NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.
[00012] In another specific embodiment, a two-step method is provided here for the production of a population of activated natural killer cells (NK), where a first step of the method itself comprises the expansion of a population of hematopoietic stem cells or progenitor cells in a first medium comprising one or more between SCF, IL-7 and IL-15, and where SCF, IL-7 and IL-15 itself are not comprised in an indefinite component of the medium itself (for example , serum), and where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differ in NK cells during said expansion; and where a second stage of the method itself comprises expanding the cells of the first stage in a second medium comprising IL-2, to produce activated NK cells. In another specific embodiment, the first medium itself further comprises one or more between fms-like tyrosine kinase 3 ligand (Flt3-L), thrombopoietin (Tpo), interleukin-2 (IL-2), and / or heparin. In another specific embodiment, the first medium itself comprises about 5% - 20% of fetal bovine serum or human serum. In another specific embodiment, SCF is present in a concentration of about 1 to about 150 ng / mL in the first medium. In another specific embodiment, IL-2 is present in a concentration of about 50 to about 1500 IU / mL in the first medium. In another specific embodiment, IL-7 is present in a concentration of about 1 to about 150 ng / mL in the first medium. In another specific embodiment, IL-15 is present in a concentration of about 1 to about 150 ng / mL in the first medium. In another specific embodiment, Tpo is present in a concentration of about 1 to about 150 ng / mL in the first medium. In another specific embodiment, heparin is present in a concentration of about 0.1 to about 30 U / mL in the first medium. In another specific embodiment, IL-2 in the second stage is present in a concentration of about 50 to about 1500 IU / mL in the second medium. In another specific embodiment, the second medium itself additionally comprises one or more of fetal calf serum (FCS), transferrin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid, bovine serum albumin (BSA) and phytohemagglutinin.
[00013] In certain specific modalities, hematopoietic stem cells or progenitor cells themselves are cultured in the first medium by 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 before the culture itself in the second medium. In certain other specific modalities, the cells themselves are cultured in the second medium itself by 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 more specific embodiment, hematopoietic stem cells or progenitor cells themselves are grown in said first medium for 21 days, and then grown in said second medium for 21 days.
[00014] Here is also provided a population of natural killer cells produced by the two-step method described above, referred to here as TSNK cells. In a specific embodiment, the NK cells themselves (for example, TSNK cells) are CD3-CD56 +. In a specific embodiment, the NK cells themselves (for example, TSNK cells) are CD3-CD56 + CD16-. In another specific embodiment, the NK cells themselves (for example, TSNK cells) are additionally CD94-CD117 +. In another specific embodiment, the NK cells themselves (for example, TSNK cells) are additionally CD161-. In another specific embodiment, the NK cells themselves (for example, TSNK cells) are additionally NKG2D +. In another specific embodiment, the NK cells themselves are additionally NKp46 +. In another specific embodiment, the NK cells themselves are additionally CD226 +.
[00015] In certain embodiments, more than 90%, 92%, 94%, 96% or 98% of the TSNK cells themselves are CD56 + and CD16-. In some modalities, at least 80%, 82%, 84%, 86%, 88% or 90% of the TSNK cells themselves are CD3- and CD56 +. In other modalities, more than 90%, 92%, 94%, 96% or 98% of the TSNK cells themselves are CD56 +, CD16- and CD3-. In other modalities, at least 50%, 52%, 54%, 56%, 58% or 60% of the TSNK cells themselves are NKG2D +. In other modalities, less than 10%, 9%, 8%, 7%, 6%, 5%, 4% or 3% of the TSNK cells themselves are NKB1 +. In certain other embodiments, less than 10%, 8%, 6%, 4% or 2% of the TSNK cells themselves are NKAT2 +. In certain other modalities, less than 10%, 8%, 6%, 4% or 2% of the TSNK cells themselves are CD56 + and CD16 +. In more specific modalities, at least 50%, 55%, 60%, 65% or 70% of the TSNK CD3-, CD56 + cells themselves are NKp46 +. In other more specific modalities, at least 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the CDN-, CD56 + TSNK cells themselves are CD117 +. In other more specific modalities, at least 20%, 25%, 30%, 35%, 40% or 45% of the TSNK CD3-, CD56 + cells themselves are CD94 +. In other more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the CD3-, CD56 + TSNK cells themselves are CD161-. In other more specific modalities, at least 10%, 12%, 14%, 16%, 18% or 20% of the CDN-, CD56 + TSNK cells themselves are CD226 +. In more specific modalities, at least 20%, 25%, 30%, 35% or 40% of the TSNK CD3-, CD56 + cells themselves are CD7 +. In more specific modalities, at least 30%, 35%, 40%, 45%, 50%, 55% or 60% of the CD3-, CD56 + TSNK cells themselves are CD5 +.
[00016] In another aspect, the use of TSNK cells to suppress tumor cell proliferation, treat viral infection or treat cancer, for example, blood cancer and solid tumors, is provided here. In certain embodiments, TSNK cells are contacted with, or used in combination with, an immunomodulatory compound, for example, an immunomodulatory compound described in Section 6.2.1, below, or thalidomide.
[00017] In a specific modality, the cancer itself is a solid tumor. In another embodiment, the cancer itself is a blood cancer. In specific modalities, cancer is glioblastoma, primary ductal carcinoma, leukemia, acute T leukemia, chronic myeloid lymphoma (CML), acute myeloid leukemia (AML), chronic myeloid leukemia, lung carcinoma, colon adenocarcinoma, histiocytic lymphoma, colorectal carcinoma, colorectal carcinoma prostate cancer, multiple myeloma, or retinoblastoma.
[00018] In another specific modality, hematopoietic cells, for example, hematopoietic stem cells, or progenitor cells, from which TSNK cells are produced, are obtained from placental perfusate, umbilical cord blood or peripheral blood. In another specific embodiment, hematopoietic cells, for example, hematopoietic stem cells, or progenitor cells, from which TSNK cells are produced, are combined cells of placental perfusate and umbilical cord blood, for example, umbilical cord blood from the same placenta as the perfusate. In another specific embodiment, the umbilical cord blood itself is isolated from a placenta other than the placenta from which the perfusate is obtained. In certain embodiments, the combined cells can be obtained by mixing or combining the umbilical cord blood with the placental perfusate. In certain embodiments, cord blood and placental perfusate are combined in a ratio of 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 similar, by volume, to obtain the combined cells. In a specific embodiment, umbilical cord blood and placental perfusate are combined in a ratio of 10: 1 to 1:10, 5: 1 to 1: 5. or from 3: 1 to 1: 3. In another specific modality, umbilical cord blood and placental perfusate are combined in a ratio of 10: 1, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5 or 1:10. In a more specific modality, umbilical cord blood and placental perfusate are combined in a ratio of 8.5: 1.5 (85%: 15%).
[00019] In certain embodiments, umbilical cord blood and placental perfusate are combined in a ratio of 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 similar, by total nucleated cell (TNC) content to obtain the combined cells. In a specific embodiment, umbilical cord blood and placental perfusate are combined in a ratio of 10: 1 to 1:10, 5: 1 to 1: 5. or from 3: 1 to 1: 3. In another specific modality, umbilical cord blood and placental perfusate are combined in a ratio of 10: 1, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5 or 1:10.
[00020] In one embodiment, therefore, a method is provided here for the treatment of an individual with cancer or a viral infection, comprising administering to the individual itself an effective amount of isolated TSNK cells.
[00021] In a specific embodiment, the isolated TSNK cells were treated with an immunomodulatory compound, for example, an immunomodulatory compound described in Section 6.2.1, below, or thalidomide, prior to the actual administration. In another specific embodiment, the method comprises administering to the individual (1) an effective amount of isolated TSNK cells; and (2) an effective amount of an immunomodulatory compound or thalidomide. An "effective amount" in this context means an amount of TSNK cells, and optionally an immunomodulating compound or thalidomide, which results in a detectable improvement in one or more symptoms of the cancer or infection itself, compared to an individual with cancer or infection itself, to which the TSNK cells themselves have not been administered and, optionally, an immunomodulatory compound or thalidomide. In a specific embodiment, the immunomodulating compound itself is lenalidomide or pomalidomide. In another embodiment, the method further comprises administering an anti-cancer compound to the individual, for example, one or more of the anti-cancer compounds described in Section 6.8.3, below.
[00022] In another embodiment, a method is provided here to suppress tumor cell proliferation comprising contacting the tumor cells with a therapeutically effective amount of TSNK cells.
[00023] In a specific embodiment, the isolated TSNK cells were treated with an immunomodulatory compound, for example, an immunomodulatory compound described in Section 6.2.1, below, or thalidomide, prior to contact itself. In another specific embodiment, the method comprises administering to the individual (1) an effective amount of isolated TSNK cells; and (2) an effective amount of an immunomodulatory compound or thalidomide. An "effective amount" in this context means an amount of TSNK cells, and optionally an immunomodulating compound or thalidomide, which results in a detectable improvement in one or more symptoms of the cancer or infection itself, compared to an individual with the cancer or the disease. infection itself to which the TSNK cells themselves have not been administered and, optionally, an immunomodulatory compound or thalidomide. In a specific embodiment, the immunomodulating compound itself is lenalidomide or pomalidomide. In another embodiment, the method further comprises administering an anti-cancer compound to the individual, for example, one or more of the anti-cancer compounds described in Section 6.8.3, below.
[00024] In a specific embodiment of this method, tumor cells are blood cancer cells. In another specific embodiment, the tumor cells are solid tumor cells. In another embodiment, 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, glioblastoma cells, lung carcinoma cells, colon adenocarcinoma cells, histiocytic lymphoma cells, multiple myeloma cells, retinoblastoma cells, colorectal carcinoma cells, prostate cancer cells, or colorectal adenocarcinoma cells. In another specific modality, the contact itself occurs in vitro. In another specific modality, the contact itself occurs in vivo. In a more specific modality, the in vivo contact itself occurs in a human.
[00025] In another aspect, a method is provided here for the treatment of an individual with multiple myeloma, comprising administering to the individual (1) lenalidomide; (2) melphalan; and (3) expanded NK cells, where the NK cells themselves are efficient in the treatment of multiple myeloma in said individual. In a specific embodiment, the NK cells themselves are umbilical cord blood NK cells, or NK cells produced from umbilical cord blood hematopoietic cells, for example, hematopoietic stem cells. In another embodiment, the NK cells themselves were produced by any of the methods described here for the production of NK cells, for example, for the production of TSNK cells. In another embodiment, the NK cells themselves were expanded prior to said administration. In another embodiment, lenalidomide, melphalan, and / or NK cells themselves are administered separately from one another. In certain specific modalities of the method for the treatment of an individual with multiple myeloma, the NK cells themselves are produced by a two-step method for the production of a population of activated natural killer cells (NK), where a first step of the method itself comprises the expansion of a population of hematopoietic stem cells or progenitor cells in a first medium comprising one or more between stem cell factors (SCF), interleukin-7 (IL-7) and interleukin-15 (IL-15 ), and where SCF, IL-7 and IL-15 itself are not comprised in an undefined component of the medium itself, and where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or cells progenitors themselves differ in NK cells during said expansion; and where a second stage of the method itself comprises the expansion of the cells of the first stage in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells.
[00026] In other specific modalities of the method for the treatment of an individual with multiple myeloma, the NK cells themselves are produced by a method comprising: (a) the inoculation of a population of hematopoietic stem cells or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more stem cell factors (SCF) and interleukin-7 (IL-7), where IL-15 and the optional SCF and IL-7 itself are not comprised in an undefined component of the medium itself, so that the population expands, and a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differ in NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.
[00027] In another aspect, a method is provided here for the treatment of an individual with chronic lymphocytic leukemia (CLL), comprising administering to an individual a therapeutically effective dose of (1) lenalidomide; (2) melphalan; (3) fludarabine; and (4) expanded NK cells, for example, TSNK cells, where the NK cells themselves are efficient in treating said CLL in the individual itself. In a specific embodiment, the NK cells themselves are umbilical cord blood NK cells, or NK cells produced from umbilical cord blood hematopoietic cells, for example, hematopoietic stem cells. In another embodiment, the NK cells themselves were produced by any of the methods described here for the production of NK cells, for example, for the production of TSNK cells. In a specific embodiment of any of the above methods, said NK cells were expanded for at least 10 days before the actual administration. In a specific embodiment of any of the above methods, lenalidomide, melphalan, fludarabine, and expanded NK cells are administered to the individual itself separately. In certain specific modalities of the method for the treatment of an individual with CLL, the NK cells themselves are produced by a two-step method for the production of a population of activated natural killer cells (NK), where a first step of the method itself said comprises the expansion of a population of hematopoietic stem cells or progenitor cells in a first medium comprising one or more between stem cell factors (SCF), interleukin-7 (IL-7) and interleukin-15 (IL-15) , and where SCF, IL-7 and IL-15 itself are not comprised in an undefined component of the medium itself, and where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differentiate into NK cells during said expansion; and where a second stage of the method itself comprises the expansion of the cells of the first stage in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells.
[00028] In other specific modalities of the method for the treatment of an individual with CLL, the NK cells themselves are produced by a method comprising: (a) the inoculation of a population of hematopoietic stem cells or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more stem cell factors (SCF) and interleukin-7 (IL-7), where IL-15 and the optional SCF and IL-7 itself do not are comprised of an undefined component of the medium itself, so that the population expands, and a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differ in NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.
[00029] In one aspect, a method is provided here for the cryopreservation of a population of NK cells, for example, TSNK cells. In one embodiment, the method itself comprises: (a) the inoculation of a population of hematopoietic stem cells or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more cell-factor trunk (SCF) and interleukin-7 (IL-7), where IL-15 and the optional SCF and IL-7 itself are not included in an indefinite component of the medium itself, so that the population expands, and a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves will differentiate into NK cells during said expansion; (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells; and (c) the cryopreservation of the NK cells from step (b) in a cryopreservation medium. In a specific embodiment, step (c) itself further comprises (1) the preparation of a solution with cell suspension; (2) adding the cryopreservation medium to the cell suspension solution from step (1) to obtain a cryopreserved cell suspension; (3) cooling the cryopreserved suspension from step (3) to obtain a cryopreserved sample; and (4) the storage of the cryopreserved sample below -80 ° C. In certain embodiments, the method does not include intermediate steps between steps (a) and (b), and between steps (b) and (c).
[00030] In another embodiment, said method for the cryopreservation of a population of NK cells, for example, TSNK cells, comprises: (a) the expansion of a population of hematopoietic stem cells or progenitor cells in a first medium comprising a or more between stem cell factors (SCF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15) and heparin, and where SCF, IL-2, IL-7 and IL-15 themselves are not comprised in an undefined component of the medium itself, and where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differ in NK cells during said expansion; (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells; and (c) the cryopreservation of the NK cells from step (b) in a cryopreservation medium. In a specific embodiment, step (c) itself further comprises (1) the preparation of a solution with cell suspension; (2) adding the cryopreservation medium to the cell suspension solution from step (1) to obtain a cryopreserved cell suspension; (3) cooling the cryopreserved suspension from step (3) to obtain a cryopreserved sample; and (4) the storage of the cryopreserved sample below -80 ° C. In certain embodiments, the method does not include intermediate steps between steps (a) and (b), and between steps (b) and (c), and / or does not include additional culture steps before step (a).
[00031] In another specific embodiment, hematopoietic cells, for example, hematopoietic stem cells or progenitor cells from which TSNK cells are produced express one or more among the hsa-miR-380, hsa-miR-512 microRNAs, hsa-miR-517, hsa-miR-518c, hsa-miR-519b, and hsa-miR-520a at a detectably higher level than in natural peripheral blood killer cells, as determined, for example, by quantitative time PCR real (qRT-PCR).
[00032] In another specific embodiment of the methods above, the TSNK cells themselves are brought into contact with an immunomodulating compound or thalidomide in an amount and time sufficient for the natural exterminating cells themselves to express higher detectable amounts of granzyme B or than an equivalent number of natural killer cells, for example, TSNK cells, which have not been contacted with the immunomodulating compound or thalidomide itself. In another specific embodiment, the TSNK cells themselves are contacted with an immunomodulating compound or thalidomide in an amount and time sufficient for the natural killer cells themselves to express higher cytotoxicity against the tumor cells themselves than an equivalent number of cell cells. natural exterminators, for example, TSNK cells, which have not been contacted with said immunomodulatory compound, for example, lenalidomide or pomalidomide, or with thalidomide. In another specific embodiment, the TSNK cells themselves express one or more of BAX, CCL5, CCR5, CSF2, FAS, GUSB, IL2RA, or TNFRSF18 at a higher level than an equivalent number of natural killer cells, for example, cells TSNK, who have not been contacted with the immunomodulating compound or the thalidomide itself. In another specific embodiment, the TSNK cells themselves express one or more of ACTB, BAX, CCL2, CCL3, CCL5, CCR5, CSFl, CSF2, ECEl, FAS, GNLY, GUSB, GZMB, ILIA, IL2RA, IL8, IL10, LTA , PRF1, PTGS2, SKI, and / or TBX21 at a higher level than an equivalent number of natural killer cells, for example, TSNK cells, which have not been contacted with the immunomodulating compound or thalidomide itself.
[00033] In certain modalities of the methods for treating or suppressing tumors described above, TSNK cells are combined with other natural killer cells, for example, natural killer cells isolated from placental perfusate, umbilical cord blood or peripheral blood, or produced from hematopoietic cells by a different method. In specific embodiments, TSNK cells are combined with natural killer cells obtained from another source, or made by a different method, 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 similar.
[00034] In another aspect, a composition comprising isolated TSNK cells is provided here. In a specific embodiment, the TSNK cells themselves are produced from hematopoietic cells, for example, hematopoietic stem cells or progenitor cells isolated from placental perfusate, umbilical cord blood, and / or peripheral blood. In another specific embodiment, the TSNK cells themselves comprise at least 50% of the cells in the composition. In another specific embodiment, the TSNK cells themselves comprise at least 80%, 85%, 90%, 95%, 98% or 99% of the cells in the composition. In certain embodiments, more than 90%, 92%, 94%, 96% or 98% of the TSNK cells in the composition themselves are CD56 + and CD16-. In other modalities, at least 80%, 82%, 84%, 86%, 88% or 90% of the TSNK cells in the composition itself are CD3- and CD56 +. In other modalities, at least 50%, 52%, 54%, 56%, 58% or 60% of the cells themselves are NKG2D +. In other modalities, less than 10%, 9%, 8%, 7%, 6%, 5%, 4% or 3% of the cells themselves are NKB1 +. In certain other embodiments, less than 10%, 8%, 6%, 4% or 2% of the TSNK cells themselves are NKAT2 +. In certain other modalities, less than 10%, 8%, 6%, 4% or 2% of the TSNK cells themselves are CD56 + and CD16 +. In more specific modalities, at least 50%, 55%, 60%, 65% or 70% of the TSNK CD3-, CD56 + cells themselves are NKp46 +. In other more specific modalities, at least 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the CDN-, CD56 + TSNK cells themselves are CD117 +. In other more specific modalities, at least 20%, 25%, 30%, 35%, 40% or 45% of the CD3-, CD56 + cells themselves are CD94 +. In other more specific modalities, at least 10%, 12%, 14%, 16%, 18% or 20% of the CDN-, CD56 + TSNK cells themselves are CD226 +. In more specific modalities, at least 20%, 25%, 30%, 35% or 40% of the TSNK CD3-, CD56 + cells themselves are CD7 +. In more specific modalities, at least 30%, 35%, 40%, 45%, 50%, 55% or 60% of the CD3-, CD56 + TSNK cells themselves are CD5 +.
[00035] In another specific embodiment, TSNKCD56 +, CD16- cells isolated are from a single individual. In a more specific embodiment, the natural CD56 +, CD16- isolated killer cells themselves comprise natural killer cells from at least two different individuals. In another specific embodiment, the TSNK cells themselves were contacted with an immunomodulating compound or thalidomide in an amount and time sufficient for the TSNK cells themselves to express higher detectable amounts of granzyme B or perforin than an equivalent number of natural killer cells, that is, TSNK cells, which have not been contacted with the immunomodulatory compound or the thalidomide itself. In another specific embodiment, the composition itself further comprises an immunomodulatory compound or thalidomide. In certain embodiments, the immunomodulatory compound is a compound described in Section 6.2.1 below, for example, an aminosubstituted isoindoline compound.
[00036] In another specific embodiment, the composition additionally comprises one or more anticancer compounds, for example, one or more among the anticancer compounds described in Section 6.8.2 below.
[00037] In a more specific embodiment, the composition comprises TSNK cells and natural killer cells from another source, or made by another method. In a specific embodiment, the other source itself is placental blood and / or umbilical cord blood. In another specific modality, the other source itself is peripheral blood. In more specific embodiments, TSNK cells are combined with natural killer cells from another source, or made by another method, 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 similar.
[00038] In another specific embodiment, the composition comprises TSNK cells and isolated placental perfusate or isolated placental perfusate cells. In a more specific modality, the placental perfusate itself is from the same individual as the TSNK cells. In another more specific embodiment, the placental perfusate itself comprises an individual's placental perfusate different from that of the TSNK cells themselves. In another specific embodiment, all, or substantially all (for example, more than 90%, 95%, 98% or 99%) of the cells in the placental perfusate itself are fetal cells. In another specific embodiment, placental perfusate or placental perfusate cells comprise fetal and maternal cells. In a more specific embodiment, the fetal cells in the placental perfusate itself comprise less than about 90%, 80%, 70%, 60% or 50% of the cells in the perfusate itself. In another specific modality, the perfusate itself is obtained by passing a 0.9% NaCl solution through the placental vasculature. In another specific modality, the perfusate itself comprises a culture medium. In another specific modality, the perfusate itself was treated to remove erythrocytes. In another specific embodiment, the composition itself comprises an immunomodulatory compound, for example, an immunomodulatory compound described in Section 5.2.1.1 below, for example, an aminosubstituted isoindoline compound. In another specific embodiment, the composition additionally comprises one or more anticancer compounds, for example, one or more among the anticancer compounds described in Section 6.8.2, below.
[00039] In another specific embodiment, the composition comprises TSNK cells and placental perfusate cells. In a more specific embodiment, the placental perfusate cells themselves are from the same individual as the TSNK cells themselves. In another more specific embodiment, said placental perfusate cells are from an individual different from that of the TSNK cells themselves. In another specific embodiment, the composition comprises isolated placental perfusate and isolated cells of placental perfusate, where the isolated placental perfusate and the isolated cells of placental perfusate themselves are from different individuals. In another more specific embodiment of any of the above embodiments comprising placental perfusate, the placental perfusate itself comprises placental perfusate from at least two individuals. In another more specific embodiment of any of the above embodiments comprising placental perfusate cells, the placental perfusate cells themselves are at least two individuals. In another specific embodiment, the composition itself comprises an immunomodulatory compound. In another specific embodiment, the composition additionally comprises one or more anticancer compounds, for example, one or more among the anticancer compounds described in Section 6.8.2, below. 4.1 Definitions
[00040] As used here, the terms "immunomodulatory compound" and "IMiD ™" do not include thalidomide.
[00041] As used here, “lenalidomide” means 3- (4'-aminoisoindolin-1'-one) -1-piperidine-2,6-dione (name according to the Chemical Abstracts Service) or 2,6-Piperidinedione, 3 - (4-amino-1,3-dihydro-1-oxo-2H-isoindol-2-yl) - (name according to the International Union of Pure and Applied Chemistry, IUPAC). As used herein, "pomalidomide" means 4-amino-2- (2,6-dioxopiperidin-3-yl) isoindole-1,3-dione.
[00042] As used here, "multipotent", when referring to a cell, means that the cell has the ability to differentiate into a cell of another cell type. In certain embodiments, a “multipotent cell” is a cell that has the capacity to generate in any subset of a mammal's body approximately 260 cell types. Unlike pluripotent cells, a multipotent cell does not have the capacity to form any of the cell types.
[00043] As used here, "feed cells" refer to cells of one type that are co-cultured with cells of a second type, to provide an environment in which cells of the second type can be maintained, and perhaps proliferate. Without being limited to any theory, the feed cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules (for example, steroids), nucleic acid molecules, growth factors (for example, bFGF), other factors (for example, cytokines), and metabolic nutrients for the target cells. In certain embodiments, the food cells grow in a monolayer.
[00044] As used here, "natural killer cell" or "NK cells" without further modification, includes natural killer cells from any histological source.
[00045] As used here, "TSNK" and "TSNK cells" refer to natural killer cells produced by the culture / expansion methods (for example, two-step method) described here.
[00046] As used here, “placental perfusate” means the perfusion solution that has passed through at least part of a placenta, for example, a human placenta, for example, through the placental vasculature, including a plurality of cells collected by perfusion solution while passing through the placenta.
[00047] As used herein, "placental perfusate cells" means nucleated cells, for example, total nucleated cells, isolated from, or isolable from, placental perfusate.
[00048] As used herein, "suppression of tumor cells", "suppression of tumor cell proliferation", or the like, includes retarding the growth of a tumor cell population, for example, by exterminating one or more of the tumor cells in the tumor cell population itself, for example, by contact of the tumor cell population with, for example, TSNK cells or with a population of cells comprising TSNK cells.
[00049] As used here, the term "hematopoietic cells" includes hematopoietic stem cells and hematopoietic progenitor cells.
[00050] As used here, the "undefined component" is an art term in the field of culture media that refers to components whose constituents are not generally provided or quantified. Examples of an "undefined component" include, without limitation, human sum (for example, human serum AB) and fetal serum (for example, fetal bovine serum or fetal calf serum).
[00051] As used here, “+”, when used to indicate the presence of a particular cell marker, means that the cell marker is detectably present in the fluorescence-activated cell count on an isotopic control; or it is detectable over the background signal in quantitative or semi-quantitative RT-PCR.
[00052] As used here, "-", when used to indicate the presence of a particular cell marker, means that the cell marker is not detectably present in the fluorescence-activated cell count on an isotopic control; or it is not detectable on the background signal in quantitative or semi-quantitative RT-PCR. 5. BRIEF DESCRIPTION OF THE FIGURES
[00053] FIGURE 1: relative expansion of NK cells differentiated from hematopoietic stem cells (HSCs) with various formulations of the medium. The error bars represent the standard deviation in relation to three donors. X-axis: day (D) of the culture. Y axis: relative expansion compared to day 0 (start of culture).
[00054] FIGURE 2: phenotypic characterization of NK cells cultured with NK2A medium. The cells were triple labeled with PE-anti-CD56, FITC-antiCD3, PerCP-antiCD16. Horizontal and vertical lines: fluorescent marker levels significantly above the background signal.
[00055] FIGURE 3: relative expansion of NK cells cultured with NK2A (FF), NK2A (placental stem cells as feed cells), NK2A (MSC as feed cells) or two-stage NK media. X-axis: day (D) of the culture. Y axis: relative expansion compared to day 0 (start of culture).
[00056] FIGURE 4: cytotoxicity of NK cells cultured with NK2A medium (without feed cells), two-stage NK, NK2A with adherent placental stem cells cultured in tissue (PSC) CD34-, CD10 +, CD105 +, CD200 +, as cells feed, or NK2A with bone marrow-derived mesenchymal stem cells (MSC) as feed cells, on Day 45 after the start of culture. Representative data from 3 donors are shown in FIGURE 4.
[00057] FIGURE 5: phenotypic characterization of NK cells on day 41 (D41) of the culture. Representative data from 3 donors are presented. X-axis: percentage of NK cells, produced by the two-step method, which are CD3-CD56 +, CD16-CD56 +, or CD16 + CD56 +; or that express NKB1, NKG2D, NKp46, CD94, CD117, CD226, CD7 or CD5. The cells were cultured in NK2A medium (without feed cells), two-stage NK, NK2A with adherent placental stem cells grown in tissue (PSC) CD34-, CD10 +, CD105 +, CD200 +, as feed cells, or NK2A with cells - bone marrow-derived mesenchymal stem (MSC) as feed cells, on Day 41 after the start of culture.
[00058] FIGURE 6: expression of CD94 and CD117 in the NK CD56 + CD3- cell population during NK cultivation in NK2A medium. The dominant population of CD56 + CD94 + CD117 + cells was identified from NK cells cultured in NK2A medium, which is distinguishable from NK cells derived from embryonic stem cells (ESC) (CD56 + CD94 + CD117 below / -). Representative data from 3 donors are shown in FIGURE 6. X-axis: fluorescence of anti-CD94 labeled with phycoerythrin (PE). Y-axis: anti-CD117 fluorescence labeled with APC. Horizontal and vertical lines: fluorescent marker level significantly above the background signal. D13, D20, D28, D35: Days 13, 20, 28 and 35 after the start of CD34 + cell culture.
[00059] FIGURE 7: Effects of placental stem cells on cultured NK cells compared to pure MSC and NK2A media. X-axis: NK cells cultured in NK2A medium without a feed layer (FF); NK cells cultured in NK2A medium with bone marrow-derived mesenchymal stem cells (MSC) as feed cells; or NK2A medium with adherent placental stem cells cultured in tissue (PSC) CD34-, CD10 +, CD105 +, CD200 +, as a food layer. Y-axis (left): cytotoxicity, expressed as a percentage of the remaining tumor cells (1.0 = 100%); cytotoxicity indicated by hollow squares. Y-axis (right): relative expansion of NK cells using the two-step method; relative expansion expressed as asterisks.
[00060] FIGURES 8A-8B: effects of the relative ratios of umbilical cord blood (UCB) and human placental perfusate (HPP) on the purity of CD34 + cells after thawing. FIGURE 8A: effects of HPP volumetric content (% v / v) on the purity of CD34 + Lin-. X-axis: volumetric fraction of HPP in the UCB mixed with HPP (combination). Y-axis: percentage of CD34 + Lin- cells. FIGURE 8B: effects of the content of HPP TNC (TNC%) on the purity of CD34 + Lin-. Y-axis: percentage of CD34 + Lin- cells. 6. DETAILED DESCRIPTION
[00061] Here is provided a new method for the production and expansion of NK cells from hematopoietic cells, for example, hematopoietic stem cells or progenitor cells. The hematopoietic cells used to produce the NK cells can be isolated from any source, for example, without limitation, placenta, umbilical cord blood, placental blood, peripheral blood, spleen or liver. In a certain embodiment, NK cells are produced from expanded hematopoietic cells, for example, hematopoietic stem cells and / or hematopoietic progenitor cells. In one embodiment, hematopoietic cells are collected from a source of such cells, for example, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver and / or bone marrow. In a specific modality, hematopoietic cells are expanded and differentiated, continuously, in a first medium without the use of food cells. The cells are then cultured in a second medium in the presence of feed cells. Such isolation, expansion and differentiation can be carried out in a central facility, which provides expanded hematopoietic cells for transport for decentralized expansion and differentiation at points of use, for example, hospitals, military bases, military front lines, or the like. 6.1 Hematopoietic cells
[00062] Hematopoietic cells useful in the methods described here can be any hematopoietic cells capable of differentiating into NK cells, or the like. Hematopoietic cells can be obtained from tissue sources such as bone marrow, umbilical cord blood, placental blood, peripheral blood, liver or the like, or combinations thereof. Hematopoietic cells can be obtained from the placenta. In a specific embodiment, hematopoietic cells are obtained from placental perfusate. Hematopoietic cells of placental perfusate may comprise a mixture of fetal and maternal hematopoietic cells, for example, a mixture in which the maternal cells comprise more than 5% of the total number of hematopoietic cells. Preferably, hematopoietic cells of placental perfusate comprise at least about 90%, 95%, 98%, 99% or 99.5% of fetal cells.
[00063] In another specific embodiment, hematopoietic cells, for example, hematopoietic stem cells or progenitor cells, from which TSNK cells are produced, are obtained from placental perfusate, umbilical cord blood or peripheral blood. In another specific embodiment, hematopoietic cells, for example, hematopoietic stem cells or progenitor cells, from which TSNK cells are produced, are combined cells of placental perfusate and umbilical cord blood, for example, umbilical cord blood. the same placenta as the perfusate. In another specific embodiment, the umbilical cord blood itself is isolated from a placenta other than the placenta from which the placental perfusate itself is obtained. In certain embodiments, the combined cells can be obtained by mixing or combining the umbilical cord blood with the placental perfusate. In certain embodiments, umbilical cord blood and placental perfusate are combined in a ratio of 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 similar, by volume, to obtain the combined cells. In a specific embodiment, umbilical cord blood and placental perfusate are combined in a ratio of 10: 1 to 1:10, 5: 1 to 1: 5, or 3: 1 to 1: 3. In another specific modality, umbilical cord blood and placental perfusate are combined in a ratio of 10: 1, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5 or 1:10. In a more specific modality, umbilical cord blood and placental perfusate are combined in a ratio of 8.5: 1.5 (85%: 15%).
[00064] In certain embodiments, umbilical cord blood and placental perfusate are combined in a ratio of 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 similar by total nucleated cell (TNC) content to obtain the combined cells. In a specific embodiment, umbilical cord blood and placental perfusate are combined in a ratio of 10: 1 to 10: 1, 5: 1 to 1: 5, or 3: 1 to 1: 3. In another specific modality, umbilical cord blood and placental perfusate are combined in a ratio of 10: 1, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5 or 1:10.
[00065] In another specific modality, hematopoietic cells, for example, hematopoietic stem cells or progenitor cells from which TSNK cells themselves are produced, come from both umbilical cord blood and placental perfusate, but in these the umbilical cord blood itself is isolated from a placenta other than the placenta from which the placental perfusate itself is obtained.
[00066] In certain embodiments, hematopoietic cells are CD34 + cells. In specific embodiments, the hematopoietic cells useful in the methods described here are CD34 + CD38 + or CD34 + CD38-. In a more specific embodiment, the hematopoietic cells are CD34 + CD38-Lin-. In another specific embodiment, the hematopoietic cells are one or more among CD2-, CD3-, CD11b-, CD11c-, CD14-, CD16-, CD19-, CD24-, CD56-, CD66b- and / or glycophorin A-. In another specific embodiment, the cells are CD2-, CD3-, CD11b-, CD11c-, CD14-, CD16-, CD19-, CD24-, CD56-, CD66b- and glycophorin A-. In another more specific embodiment, the hematopoietic cells are CD34 + CD38-CD33-CD117- CD235-CD36-.
[00067] In another embodiment, the hematopoietic cells are CD45 +. In another specific modality, the hematopoietic cells are CD34 + CD45 +. In another embodiment, the hematopoietic cell is Thi-1 +. In a specific embodiment, the hematopoietic cell is CD34 + Thy-1 +. In another embodiment, the hematopoietic cells are CD133 +. In specific modalities, the hematopoietic cells are CD34 + CD133 + or CD133 + Thy-1 +. In another specific embodiment, the CD34 + hematopoietic cells are CXCR4 +. In another specific embodiment, the CD34 + hematopoietic cells are CXCR4-. In another embodiment, hematopoietic cells are positive for KDR (vascular growth factor 2 receptor). In specific modalities, the hematopoietic cells are CD34 + KDR +, CD133 + KDR + or Thy-1 + KDR +. In certain other embodiments, hematopoietic cells are positive for aldehyde dehydrogenase (ALDH +), for example, the cells are CD34 + ALDH +.
[00068] In certain other embodiments, the CD34 + cells are CD45-. In specific embodiments, CD34 + cells, for example, CD34 +, CD45- cells, express one or more, or even all, among the miRNAs: hsa-miR-380, hsa-miR-512, hsa-miR-517, hsa- miR-518c, hsa-miR-519b, and / or hsa-miR-520a.
[00069] In certain embodiments, the hematopoietic cells are CD34-.
[00070] Hematopoietic cells may not yet have certain markers that indicate impairment of the lineage, or a lack of naivety in development. For example, in another embodiment, the hematopoietic cells are HLA-DR-. In specific embodiments, the hematopoietic cells are CD34 + HLA-DR-, CD133 + HLA-DR-, Thy-1 + HLA-DR- or ALDH + HLA-DR-. In another embodiment, hematopoietic cells are negative in relation to one or more, preferably more than one, of the lineage markers CD2, CD3, CDl lb, CDl lc, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin A.
[00071] In this way, hematopoietic cells can be selected for use in the methods described here based on the presence of markers that indicate a non-differentiated state, or based on the absence of lineage markers indicating that at least some differentiation of lineage has occurred. Methods for the isolation of cells, including hematopoietic cells, based on the presence or absence of specific markers are discussed in detail, for example, in Section 6.1.2 below.
[00072] The hematopoietic cells used in the methods provided herein can be a substantially homogeneous population, for example, a population comprising at least about 95%, at least about 98% or at least about 99% of hematopoietic cells having the same tissue as a source, or a population comprising hematopoietic cells that exhibit the same cellular markers associated with hematopoietic cells. For example, in various embodiments, hematopoietic cells can comprise at least 95%, 98% or 99% of bone marrow hematopoietic cells, umbilical cord blood, placental blood, peripheral blood, or placenta, for example, placental perfusate.
[00073] The hematopoietic cells used in the methods provided herein can be obtained from a single individual, for example, from a single placenta, or from a plurality of individuals, for example being able to be mixed. In cases where hematopoietic cells are obtained from a plurality of individuals and mixed, hematopoietic cells can be obtained from the same tissue source. Thus, in various modalities, the hematopoietic cells are all placental, for example, placental perfusate, all of placental blood, all of umbilical cord, all of peripheral blood, or the like.
[00074] The hematopoietic cells used in the methods described here may, in certain embodiments, comprise hematopoietic cells having one or more tissues as sources. For example, in certain embodiments, when hematopoietic cells from two or more sources are combined for use in the methods presented here, a plurality of hematopoietic cells used for the production of TSNK cells comprise placental hematopoietic cells, for example, placental perfusate. In various embodiments, the hematopoietic cells used for the production of TSNK cells comprise hematopoietic cells of the placenta and umbilical cord; placenta and peripheral blood; placenta and placental blood, or placenta and bone marrow. In a preferred embodiment, hematopoietic cells comprise hematopoietic cells of placental perfusate in combination with hematopoietic cells of umbilical cord, where the umbilical cord and placenta are from the same individual, that is, where the perfusate and umbilical cord blood coincide. In embodiments in which hematopoietic cells comprise hematopoietic cells having two tissues as sources, the hematopoietic cells of the sources can be combined in a ratio of, for example, 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 9: 1. 6.1.1 Placental hematopoietic stem cells
[00075] In certain embodiments, the hematopoietic cells used in the methods provided herein are placental hematopoietic cells. As used here, “placental hematopoietic cells” means hematopoietic cells obtained from the placenta itself, and not from placental blood or umbilical cord blood. In one embodiment, the hematopoietic placental cells are CD34 +. In a specific embodiment, placental hematopoietic cells are predominantly (for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98 %) CD34 + CD38- cells. In another specific embodiment, placental hematopoietic cells are predominantly (for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98 %) CD34 + CD38 + cells. Placental hematopoietic cells can be obtained from a postpartum mammalian (e.g., human) placenta by any means known to those skilled in the art, for example, by perfusion.
[00076] In another embodiment, the placental hematopoietic cell is CD45-. In a specific embodiment, the hematopoietic cell is CD34 + CD45-. In another specific modality, the placental hematopoietic cells are CD34 + CD45 +. 6.2 Production of natural killer cells
[00077] The production of NK cells by the present method comprises the expansion of a population of hematopoietic cells. During cell expansion, a plurality of hematopoietic cells in the hematopoietic cell population differs into NK cells.
[00078] In one embodiment, a method is provided here for producing a population of activated natural killer cells (NK), comprising (a) inoculating a population of hematopoietic stem cells or progenitor cells in a first medium comprising interleukin -15 (IL-15) and, optionally, one or more stem cell factors (SCF) and interleukin-7 (IL-7), where IL-15 and the optional SCF and IL-7 itself are not understood into an undefined component of the medium itself, so that the population expands, and a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differ in NK cells during said expansion ; and (b) expanding the cells of the first step in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.
[00079] In another embodiment, the NK cells provided here are produced by a two-stage process of expansion / differentiation and maturation of the NK cells. The first and second steps comprise the cultivation of cells in media with a unique combination of cellular factors. In certain embodiments, the process involves (a) culturing and expanding a population of hematopoietic cells in a first medium, where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic cells differentiate into NK cells; and (b) the expansion of the NK cells from step (a) in a second medium, where the NK cells are further expanded and differentiated, and where the NK cells are matured (for example, made active or having cytotoxic activity). In certain embodiments, the method does not include intermediate steps between steps (a) and (b), additional culture steps before step (a), and / or additional steps (for example, maturation step) after step (b ). 6.2.1 First stage of culture
[00080] In certain embodiments, the methods provided here comprise a first stage of culture and expansion of a population of hematopoietic cells in a first medium, where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic cells differ in NK cells.
[00081] Without being bound by any parameter, mechanism or theory, the culture of hematopoietic cells as provided here results in the continuous expansion of hematopoietic cells and their differentiation into NK cells from the cells themselves. In certain embodiments, hematopoietic cells, for example, stem cells or progenitor cells, used in the methods provided here are expanded and differentiated in the first step using a feed layer. In other modalities, hematopoietic cells, for example, stem cells or progenitor cells, are expanded and differentiated in the first step without the use of a food layer.
[00082] The expansion and differentiation of hematopoietic cells independent of feeding cells can occur in any cell compatible with culture and cell expansion, for example, flask, tube, beaker, plate, multi-well plate, pouch, or the like. In a specific embodiment, the expansion and differentiation of hematopoietic cells independent of feeding cells can occur in a bag, for example, a flexible, gas-permeable, fluorocarbon culture bag (for example, from American Fluoroseal). In a specific embodiment, the container in which the hematopoietic cells are expanded is suitable for transport, for example, to a location such as a hospital or military zone where the expanded NK cells are expanded and further differentiated.
[00083] In certain modalities, hematopoietic cells are expanded and differentiated, for example, continuously, in a first culture medium. In one embodiment, the first culture medium is a medium free of animal components. Exemplary means of exemplifying animal components used in the methods provided herein include, but are not limited to, Basic Eagle Medium (BME), Dulbecco's Modified Eagle's Medium (DMEM), Glasgow's Minimum Essential Medium (GMEM), Modified Eagle's Medium by Dulbecco / F-12 Ham Nutritional Mix (DMEM / F-12), Minimal Essential Medium (MEM), Iscove Modified Dulbecco Medium (IMDM), F-10 Ham Nutritional Mix (Ham's F-10), F- Nutritional Mix 12 Ham (Ham's F-12), RPMI-1640 medium, Williams E medium, STEMSPAN® (Cat. No. Stem Cell Technologies, Vancouver, Canada), Glycostem Basic Growth Medium (GBGM®), AIM-V® medium (Invitrogen), X-VIVO ™ 10 (Lonza), X-VIVO ™ 15 (Lonza), OPTMIZER (Invitrogen), STEMSPAN® H3000 (STEMCELL Technologies), CELLGRO COMPLETE ™ (Mediatech), or any modified variants or combinations thereof.
[00084] In preferred embodiments, the first culture medium comprises one or more medium supplements (for example, nutrients, cytokines and / or factors). Supplements of medium suitable for use in the methods provided herein include, for example, without limitation, serum such as human serum AB, fetal bovine serum (FBS) or fetal calf serum (FCS), vitamins, bovine serum albumin (BSA), amino acids (eg, L-glutamine), fatty acids (eg, oleic acid, linoleic acid or palmitic acid), insulin (eg, recombinant human insulin), transferrin (iron-saturated human transferrin), β-mercaptoethanol, factor stem cell (SCF), tyrosine kinase similar to Fms 3 ligand (FK3-L), cytokines such as interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15) , thrombopoietin (Tpo), heparin, or O-acetyl-carnitine (also called acetylcarnitine, O-acetyl-L-carnitine, or OAC). In a specific embodiment, the medium used here comprises AB human serum. In another specific embodiment, the medium used here comprises FBS. In another specific modality, the medium used here comprises OAC.
[00085] In certain embodiments, the first medium does not comprise one or more of: granulocyte colony stimulating factor (G-CSF), granulocyte and macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6 ), Macrophage inflammatory protein 1α (MIP1α), or leukemia inhibiting factor (LIF).
[00086] Therefore, in one aspect, a two-step method for the production of NK cells is provided here, where the first step itself comprises the expansion and differentiation of a population of hematopoietic cells in a first culture medium in the absence of feed cells, where a plurality of hematopoietic cells in the hematopoietic cell population itself differs into NK cells during the expansion itself, and where the medium comprises SCF in a concentration of about 1 to about 150 ng / mL , IL-2 at a concentration of about 50 to about 1500 IU / mL, IL-7 at a concentration of about 1 to about 150 ng / mL, IL-15 at a concentration of about 1 to about 150 ng / mL, and heparin at a concentration of about 0.1 to about 30 IU / mL, and where the SCF, IL-2, IL-7, IL-15 and heparin themselves are not included in one undefined component of the medium itself (for example, serum). In certain embodiments, the medium itself comprises one or more of O-acetyl-carnitine (also called acetylcarnitine, O-acetyl-L-carnitine, or OAC), or a compound that affects the cyclization of acetyl-CoA in mitodronia, thiazovivine , Y-27632, pi-integrin, Rho-kinase (ROCK) inhibitors, caspase inhibitors or other anti-apoptotic compounds / peptides, NOVA-RS (Sheffield Bio-Science) or other small molecule growth enhancers. In certain embodiments, the medium itself comprises nicotinamide. In certain embodiments, the medium itself comprises about 0.5 mM - 10 mM OAC. In one embodiment, the medium itself comprises Stemspan® H3000, and / or DMEM: F12 and about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM OAC. In a specific modality of the method, the medium itself is GBGM®. In another specific embodiment, the medium itself comprises Stemspan® H3000 and about 5 mM OAC. In another specific modality, the medium itself comprises DMEM: F12 and about 5 mM OAC. OAC can be added at any time during the culture methods provided here. In certain modalities, the OAC itself is added to the first medium and / or during the first stage of culture. In some modalities, the OAC itself is added to the first medium on Day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21 of the culture. In a specific modality, the OAC itself is added to the first medium on the 7th Day of the first stage of culture. In a more specific modality, the OAC itself is added to the first medium on the 7th Day of the first stage of culture and is present in the first and second stages of culture. In certain modalities, the OAC itself is added to the second medium and / or during the second stage of culture. In some modalities, the OAC itself is added to the second medium on Day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21 of the culture.
[00087] In another specific embodiment, the medium itself is IMDM supplemented with about 5-20% BSA, about 1-10 μg / mL of recombinant human insulin, about 10-50 μg / mL of saturated human transferrin with iron and about 10-50 μM of β-mercaptoethanol. In another specific embodiment, the medium itself does not comprise one or more, or none, between IL-11, IL-3, homeobox-B4 (HoxB4), and / or methylcellulose.
[00088] In other specific embodiments, the medium itself comprises SCF at a concentration of about 0.1 to about 500 ng / ml; about 5 to about 100 ng / ml; or about 20 ng / ml. In other specific embodiments, the medium itself comprises IL-2 at a concentration of about 10 to about 2000 IU / ml; or about 100 to about 500 IU / ml; or about 200 IU / ml. In other specific embodiments, the medium itself comprises IL-7 at a concentration of about 0.1 to about 500 ng / ml; about 5 to about 100 ng / ml; or about 20 ng / ml. In other specific embodiments, the medium itself comprises IL-15 at a concentration of about 0.1 to about 500 ng / ml; about 5 to about 100 ng / ml; or about 10 ng / ml. In other specific embodiments, the medium itself comprises heparin at a concentration of about 0.05 to about 100 U / ml; or about 0.5 to about 20 U / ml; or about 1.5 U / ml.
[00089] In another specific embodiment of the method, the medium itself further comprises tyrosine kinase similar to the Fms 3 ligand (Flt-3L) at a concentration of about 1 to about 150 ng / mL, thrombopoietin (Tpo) at a concentration of about 1 to about 150 ng / ml, or a combination of both. In other specific embodiments, the medium itself comprises Flt-3L at a concentration of about 0.1 to about 500 ng / ml; about 5 to about 100 ng / ml; or about 20 ng / ml. In other specific embodiments, the medium itself comprises Tpo at a concentration of about 0.1 to about 500 ng / ml; about 5 to about 100 ng / ml; or about 20 ng / ml.
[00090] In a more specific embodiment of the method, the first culture medium is GBGM®, which comprises about 20 ng / ml of SCF, about 20 ng / ml of IL-7, about 10 ng / ml of IL-15. In another more specific method, the first culture medium is GBGM®, which comprises about 20 ng / ml SCF, about 20 ng / ml Flt-3L, about 200 IU / ml IL-2 , about 200 ng / ml IL-7, about 10 ng / ml IL-15, about 20 ng / ml Tpo, and about 1.5 U / ml heparin. In another specific embodiment, the first culture medium itself comprises 10% human serum (for example, human serum AB) or fetal serum (for example, FBS).
[00091] In another embodiment, hematopoietic cells are expanded by culturing the cells themselves, for example, in said first medium, in contact with an immunomodulatory compound, for example, a TNF-a inhibitor compound, for a time and in an amount sufficient to cause a detectable increase in hematopoietic cell proliferation at any given time, compared to an equivalent number of hematopoietic cells not contacted with the immunomodulatory compound. See, for example, US Patent Application Publication No. 2003/0235909, the description of which is incorporated herein by reference in its entirety. In certain embodiments, the immunomodulatory compound is an aminosubstituted isoindoline. In a preferred embodiment, the immunomodulatory compound is 3- (4-amino-1-oxo-1,3-dihydroisoindol-2-yl) -piperidin-2,6-dione; 3- (4'-aminoisolindolin-1'-one) -1-piperidin-2,6-dione; 4- (amino) -2- (2,6-dioxo (3-piperidyl)) - isoindolin-1,3-dione; or 4-amino-2- (2,6-dioxopiperidin-3-yl) isoindole-1,3-dione. In another preferred embodiment, the immunomodulating compound is pomalidomide, or lenalidomide. In another embodiment, the immunomodulatory compound itself is a compound with a

[00092] where one between X and Y is C = O, the other between X or YC = O or CH2, and R2 is hydrogen or a short alkyl, or a salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate or a mixture of pharmaceutically acceptable stereoisomers thereof. In another embodiment, the immunomodulatory compound itself is a compound with a
where one between X and Y is C = O and the other is CH2 or C = O; R1 is H, (C1-C8) alkyl, cycloalkyl (C3-C7), alkenyl (C2-C8), alkynyl (C2-C8), benzyl, aryl, alkyl (C0-C4) - heterocycloalkyl (C1-C6), (C0-C4) alkyl-heteroaryl (C2-C5), 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 alkyl (C1-C8) - 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, cycloalkyl (C3 C7), alkenyl (C2-C8), alkynyl (C2-C8), benzyl, aryl, (C0-C4) alkyl-heterocycloalkyl (C1-C6) , (C0-C4) alkyl - (C2-C5) heteroaryl, (C0-C8) alkyl -N (R6) 2, (C1-C8) alkyl -OR5, (C1-C8) alkyl-C (O) OR5, (C1-C8) -O (CO) R5 or C (O) OR5; R4 is alkyl (C1-C8), alkenyl (C2-C8), alkynyl (C2-C8), alkyl (C1-C4) -OR5, benzyl, aryl, alkyl (C0-C4) - heterocycloalkyl (C1-C6), or (C0-C4) alkyl-heteroaryl (C2-C5); R5 is (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, benzyl, aryl or (C2-C5) heteroaryl; each R6 is independently H, (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, benzyl, aryl, heteroaryl (C2-C5) or alkyl (C1- C8) -C (O) OR5 , or the R6 groups can be joined to form a heterocycloalkyl group; n is 0 or 1; e * represents a center with chiral carbon; or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate or mixture of stereoisomers thereof. In another embodiment, the immunomodulatory compound itself is a compound with a
where: one between X and Y is C = O and the other is CH2 or C = O; R is H or CH2OCOR '; each between R1, R2, R3 or R4, regardless of the others, is halogen, alkyl with 1 to 4 carbon atoms, or alkoxy with 1 to 4 carbon atoms or (ii) one between R1, R2, R3 or R4 is nitro or -NHR5 and the rest between R1, R2, R3 or R4 are hydrogen; R5 is hydrogen or alkyl with 1 to 8 carbons; R6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chlorine, or fluorine; 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 between R8 and R9 taken independently of each other is hydrogen or alkyl with 1 to 8 carbon atoms, or R8 and R9 taken together are tetramethylene, pentamethylene, hexamethylene, or -CH2CH2X1CH2CH2- where X1 is -O-, -S -, or -NH-; R10 is hydrogen, alkyl with 1 to 8 carbon atoms, or phenyl; e * represents a center with chiral carbon; or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate or mixture of stereoisomers thereof.
[00093] In a specific embodiment, hematopoietic cell expansion is performed in IMDM supplemented with 20% BITS (bovine serum albumin, recombinant human insulin and transferrin), SCF, Flt-3 ligand, IL-3, and 4- (amino) -2- (2,6-dioxo (3-piperidyl)) - isoindoline-1,3-dione (10 μM in 0.05% DMSO). In a more specific embodiment, about 5 x 107 hematopoietic cells, for example, CD34 + cells, are expanded in the medium to about 5 x 1010 cells to about 5 x 1012 cells, which are resuspended in 100 ml of IMDM to produce a expanded hematopoietic cell population. The population of expanded hematopoietic cells is preferably cryopreserved to facilitate transport.
[00094] In several specific modalities, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the hematopoietic cells are differentiated into NK cells.
[00095] In certain embodiments, the hematopoietic cell expansion and differentiation method, as described here, comprises maintaining the cell population comprising the hematopoietic cells themselves between about 2 x 104 and about 2 x 105 cells per milliliter during expansion and differentiation. In certain other embodiments, the hematopoietic cell expansion and differentiation method, as described herein, comprises maintaining the cell population comprising the hematopoietic cells themselves below about 2 x 105 cells per milliliter.
[00096] The time for expansion and differentiation of hematopoietic cells into NK cells can be, for example, from about 3 days to about 120 days. In one embodiment, the differentiation time is about 7 days to about 75 days. In another modality, the differentiation time is from about 14 days to about 50 days. In a specific modality, the differentiation time is from about 21 days to about 28 days. 6.2.2 Second stage
[00097] In the method provided here, hematopoietic cells, for example, stem cells or progenitor cells, and natural killer cells, resulting from the first stage, are further expanded and differentiated in a second stage, for example, without the use of a feed layer or in the presence of feed cells. Cell culture as provided here results in the continuous expansion, differentiation and maturation of NK cells from the first stage. In the second stage, the NK cells are expanded, differentiated and endured, continuously, in a second culture medium, for example, comprising cytokine and / or bioactive molecules different from those of the first medium itself. In certain embodiments, the second culture medium is a medium free of animal components. Exemplary cell culture media free of animal components are described in Section 6.2.1, above.
[00098] Thus, in one aspect, a method for the production of NK cells is provided here, comprising the expansion of the NK cells of the first stage, in a second medium in the presence of feed cells and in contact with interleukin-2 ( IL-2). In specific embodiments, the second medium itself comprises cell growth medium comprising IL-2, for example, 10 IU / ml to 1000 IU / ml, and one or more of: human serum (for example, human serum AB), serum fetal bovine (FBS) or fetal calf serum (FCS), for example, 5% -15% v / v FCS; transferrin, for example, 10 μg / mL to 50 μg / mL; insulin, for example, 5 μg / mL to 20 μg / mL; ethanolamine, for example, 5 x 10-4 to 5 x 10-5 M; oleic acid, for example, 0.1 μg / mL to 5 μg / mL; linoleic acid, for example, 0.1 μg / mL to 5 μg / mL; palmitic acid, for example, 0.05 μg / mL, at 2 μg / mL; bovine serum albumin (BSA), for example, 1 μg / mL, at 5 μg / mL; and / or phytohemagglutinin, for example, 0.01 μg / mL, at 1 μg / mL. In a more specific embodiment, the second medium itself comprises cell growth medium comprising FBS or FCS, for example, 10% v / v FCS, IL-2, transferrin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid , bovine serum albumin (BSA) and phytohemagglutinin. In a more specific embodiment, the second medium itself comprises Iscove's Modified Dulbecco's Medium (IMDM), 10% FBS or FCS, 400 IU IL-2, 35 μg / mL transferrin, 5 μg / mL insulin, 2 x 10-5 M ethanolamine, 1 μg / mL oleic acid, 1 μg / mL linoleic acid (Sigma-Aldrich), 0.2 μg / mL palmitic acid (Sigma-Aldrich), 2.5 μg / mL of BSA (Sigma-Aldrich) and 0.1 μg / mL of phytohemagglutinin.
[00099] In certain embodiments, the second medium does not comprise one or more of granulocyte colony stimulating factor (G-CSF), granulocyte and macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6) , Macrophage inflammatory protein 1a (MIP1a), or leukemia inhibiting factor (LIF).
[000100] In addition to the method, any means described above as compositions are provided here.
[000101] Feed cells, when used, can be established from various types of cells. Examples of these cell types include, without limitation, fibroblasts, stem cells (eg, tissue culture-adherent placental stem cells), blood cells (eg, peripheral blood mononuclear cells (PBMC)), and cancer cells (for example, example, chronic myelogenous leukemia (CML) cells such as K562). In a specific embodiment, said culture in the second medium itself comprises culture using feed cells, for example, K562 cells and / or peripheral blood mononuclear cells (PBMC), for example, at the time when the cells are introduced in the second medium itself, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days later. In certain embodiments, the feed cells are optionally of a different species from the cells they are supporting. For example, human NK cells can be supported by embryonic mouse fibroblasts (from primary culture or a telomerized strain).
[000102] In certain embodiments, the feed cells are optionally deactivated by irradiation (for example, y-irradiation) or treatment with an antimitotic agent such as mitomycin C, to prevent them from growing more than the cells they are supporting, however allow the synthesis of important factors that support NK cells. For example, cells can be irradiated at a dose that inhibits proliferation, but allows the synthesis of important factors that support human embryonic stem cells (hES) (gamma radiation with about 4000 rads).
[000103] The cultivation of NK cells for the second stage can be done in any container compatible with cell culture and expansion, for example, flask, tube, beaker, plate, multi-well plate, pouch, or the like. In a specific embodiment, the culture of NK cells dependent on feed cells can occur in a bag, for example, a flexible, gas-permeable, fluorocarbon culture bag (for example, from American Fluoroseal). In a specific embodiment, the container in which the NK cells are expanded is suitable for transport, for example, to a location such as a hospital or military zone where the expanded NK cells are further expanded, differentiated and matured.
[000104] The differentiation of cells from step 1 into TSNK cells can be assessed by detecting specific NK cell markers, for example, by flow cytometry. Specific NK cell markers include, but are not limited to, CD56, CD94, CD117 and NKp46. Differentiation can also be assessed by the morphological characteristics of NK cells, for example, large size, high protein synthesis activity in the abundant endoplasmic reticulum (ER), and / or preformed granules.
[000105] The time for expansion and differentiation of cells from step 1 into TSNK cells can be, for example, from about 3 days to about 120 days. In one embodiment, the differentiation time is about 7 days to about 75 days. In another modality, the differentiation time is about 14 days to about 50 days. In a specific modality, the differentiation time is about 10 days to about 21 days.
[000106] The differentiation of hematopoietic cells into NK cells can be assessed by detecting markers, for example, CD56, CD94, CD117, NKG2D, DNAM-1 and NKp46 by, for example, flow cytometry. Differentiation can also be assessed by the morphological characteristics of NK cells, for example, large size, high protein synthesis activity in the abundant endoplasmic reticulum (ER), and / or preformed granules. The maturation of NK cells (for example, TSNK cells) can be assessed by detecting one or more functionally relevant markers, for example, CD94, CD161, NKp44, DNAM-1, 2B4, NKp46, CD94, KIR, and the NKG2 family of activation receivers (for example, NKG2D). The maturation of NK cells (for example, TSNK cells) can also be assessed by detecting specific markers during different stages of development. For example, in one embodiment, the pro-NK cells are CD34 +, CD45RA +, CD10 +, CD117- and / or CD161-. In another embodiment, the pre-NK cells are CD34 +, CD45RA +, CD10 +, CD117 +, and / or CD161-. In another embodiment, immature NK cells are CD34-, CD117 +, CD161 +, NKp46- and / or CD94 / NKG2A-. In another embodiment, the NK CD56bright cells are CD117 +, NKp46 +, CD94 / NKG2A +, CD16- and / or KIR +/-. In another embodiment, the CD56 dark NK cells are CD117-, NKp46 +, CD94 / NKG2A +/-, CD16 +, and / or KIR +. In a specific embodiment, the maturation of NK cells (for example, TSNK cells) is determined by the percentage of NK cells (for example, TSNK cells) that are CD161-, CD94 + and / or NKp46 +. In other more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65% or 70% of mature NK cells (for example, TSNK cells ) are NKp46 +. In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the mature NK cells (for example, TSNK cells) are CD94 +. In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the mature NK cells (for example, TSNK cells) are CD161-.
[000107] In certain embodiments, the differentiation of hematopoietic cells into NK cells is assessed by detecting the level of expression of, for example, CD3, CD7 or CD127, CD10, CD14, CD15, CD16, CD33, CD34, CD56, CD94, CD117, CD161, NKp44, NKp46, NKG2D, DNAM-1, 2B4 or TO-PRO-3, using, for example, antibodies to one or more of these cell markers. Such antibodies can be conjugated to a detectable marker, for example, a fluorescent marker, for example, FITC, R-PE, PerCP, PerCP-Cy5.5, APC, APC-Cy7 or APC-H7. 6.3 Isolation of TSNK cells
[000108] Methods for the isolation of natural killer cells are known in the art and can be used to isolate TSNK cells. Natural killer cells can be isolated or enriched by labeling cells from a tissue source, for example, peripheral blood, with antibodies to CD56 and CD3, and separation for CD56 + CD3- cells. TSNK cells can be isolated using a commercially available kit, for example, the NK Cell Isolation Kit (Milteny Biotec). TSNK cells can also be isolated or enriched by removing cells other than NK in a cell population that comprises TSNK cells. For example, TSNK cells can be isolated or enriched by depleting cells that exhibit non-NK cell markers using, for example, antibodies to one or more among CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA DR and / or CD235a (glycophorin A). Negative isolation can also be performed using a commercially available kit, for example, the NK Cell Negative Isolation Kit (Dynal Biotech). Cells isolated by these methods can be further selected, for example, to separate CD16 + and CD16- cells.
[000109] Cell separation can be performed by, for example, flow cytometry, fluorescence-activated cell separation (FACS), or, preferably, magnetic cell separation using micropellets conjugated to specific antibodies. These cells can be isolated, for example, using a magnetically activated cell separation technique (MACS), a method for separating particles based on their ability to bind to magnetic beads (for example, with a diameter of about 0.5-100 μm) that comprise one or more specific antibodies, for example, anti-CD56 antibodies. Magnetic cell separation can be performed and automated using, for example, an AUTOMACS ™ separator (Miltenyi). A variety of useful modifications can be made to the magnetic microspheres, including the covalent addition of antibodies that specifically recognize a particular surface cell molecule or haptens. The beads are then mixed with the cells to allow binding. The cells are then passed through a magnetic field to separate the cells with the specific surface cell marker. In one embodiment, these cells can be isolated and re-mixed with the magnetic beads coupled to an antibody against additional surface cell markers. The cells are then passed through a magnetic field again, isolating the cells that bind to both antibodies. Such cells can then be diluted on separate plates, such as titration plates for clonal isolation. 6.4 Placental perfusate
[000110] TSNK cells can be produced from hematopoietic cells, for example, stem cells or hematopoietic progenitor cells from any source, for example, placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen , liver, or the like. In certain embodiments, hematopoietic stem cells are combined hematopoietic stem cells of placental perfusate and umbilical cord blood from the same placenta used to generate placental perfusate. Placental perfusate comprising placental perfusate cells can be obtained, for example, by the methods described in U.S. Patent Nos. 7,045,148 and 7,468,276, the descriptions of which are incorporated herein in full. 6.4.1 Composition for collecting cells
[000111] Placental perfusate and perfusate cells, from which stem cells or hematopoietic progenitor cells can be isolated, or useful in suppressing tumors or treating an individual with tumor cells, cancer or a viral infection, for example, in combination with TSNK cells, as provided here, they can be collected by perfusing the postpartum placenta of a mammal, for example, of a human, using a composition for collecting placental cells. Perfusate can be collected from the placenta by perfusing the placenta with any physiologically acceptable solution, for example, a saline solution, culture medium, or a more complex composition for collecting cells. A cell collection composition suitable for the perfusion of a placenta, and for the collection and conservation of perfusate cells is described in detail in the related North American Order Publication number 2007/0190042, which is incorporated herein in its entirety by reference.
[000112] 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 (eg, phosphate buffered brine, Kreb's solution, modified Kreb's solution , Eagle's solution, 0.9% NaCl, etc.), a culture medium (for example, DMEM, H.DMEM, etc.) and the like.
[000113] The cell collection composition may comprise one or more components that have a tendency to conserve placental cells, that is, prevent placental cells from dying, or delay the death of placental cells, reduce the number of placental cells in the population of cells that die, or something similar, from the collection until the moment of the culture. Such components can be, for example, an apoptosis inhibitor (for example, a caspase inhibitor or JNK inhibitor); a vasodilator (eg, magnesium sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin, or a phosphate inhibitor, etc. .); a necrosis inhibitor (for example, 2- (1H-indol-3-yl) -3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); a TNF-a inhibitor; and / or an oxygen-carrying perfluorocarbon (for example, perfluoroctyl bromide, perfluordecyl bromide, etc.).
[000114] The cell collection composition may comprise one or more tissue degrading enzymes, for example, 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 (for example, collagenase I, II, III or IV, a Clostridium histolyticum collagenase, etc.); dispase, thermolysin, elastase, trypsin, liberase, hyaluronidase, and the like.
[000115] The cell collection composition may comprise a bactericidal or bacteriostatically effective amount of an antibiotic. In certain non-limiting modalities, the antibiotic is a macrolid (for example, tobramycin), a cephalosporin (for example, cephalexin, cefradine, cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, an erythromycin, a pnicillin (for example, penicillin V), or a quinolone (for example, ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc. In a particular embodiment, the antibiotic is active against Gram (+) and / or Gram (-) bacteria, for example, Pseudomonas aeruginosa, Staphylococcus aureus, and the like.
[000116] The cell collection composition may further 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 with a molecular mass greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain endothelial integrity and cell viability (for example, a synthetic or naturally occurring colloid, a polysaccharide such as a dextran or a polyethylene glycol present in about 25 g / L to about 100 g / L, or about 40 g / L to about 60 g / L); an antioxidant (for example, butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present in about 25 μM to about 100 μM); a reducing agent (for example, N-acetylcysteine present in about 0.1 mM to about 5 mM); an agent that prevents calcium from entering cells (for example, verapamil present in about 2 μM to about 25 μM); nitroglycerin (for example, about 0.05 g / L to about 0.2 g / L); an anticoagulant, in one embodiment, present in an amount sufficient to prevent clotting of residual blood (for example, heparin or hirudin present in a concentration of about 1000 units / L to about 100,000 units / L); or a compound containing amiloride (for example, amiloride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride present in about 1.0 μM to about 5 μM). 6.4.2 Placenta collection and manipulation
[000117] In general, 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 obtaining the patient's complete medical history, and its association with the placenta. Preferably, the medical history continues after delivery.
[000118] Before recovery of the perfusate, umbilical cord blood and placental blood are removed. In certain modalities, after delivery, the umbilical cord blood in the placenta is recovered. The placenta can undergo a conventional umbilical cord blood recovery process. Typically, a needle or cannula, with the help of gravity, is used to remove blood from the placenta (see, for example, Anderson, U.S. Patent No. 5,372,581; Hessel et al, U.S. Patent No. 5,415,665) . The needle or cannula is usually placed in the umbilical vein and the placenta can be gently massaged to assist in draining the umbilical cord blood from the placenta. Such retrieval of umbilical cord blood can be performed commercially, for example, LifeBank Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell. Preferably, the placenta is drained by gravity without additional manipulation, in order to decrease the rupture of tissues during the recovery of umbilical cord blood.
[000119] Typically, a placenta is transported from the delivery or birth room to another location, for example, a laboratory, for the recovery of umbilical cord blood and perfusate collection. The placenta is preferably transported in a sterile, thermally insulated transport device (maintaining the placenta temperature between 20-28 ° C), for example, by placing the placenta, with stapled proximal umbilical cord, in a sterile plastic bag closed by Zip-lock, which is then placed in an insulated container. In another embodiment, the placenta is transported in an umbilical cord blood collection kit substantially as described in U.S. Patent No. 7,147,626. Preferably, the placenta is delivered to the laboratory four to twenty-four hours after delivery. In certain modalities, the proximal umbilical cord is pleated, preferably 4-5 cm (centimeters) from the insertion into the placental disc, before the recovery of the umbilical cord blood. In other modalities, the proximal umbilical cord is pleated after the recovery of the umbilical cord blood. In other modalities, the proximal umbilical cord is pleated after the recovery of the umbilical cord blood, but before further processing of the placenta.
[000120] Before collecting the perfusate, the placenta can be stored under sterile conditions and either at room temperature or at a temperature of 5 to 25 ° C (centigrade). The placenta can be stored for a period of more than forty-eight hours, and preferably for a period of four to twenty-four hours before the infusion of the placenta to remove any residual umbilical 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 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 heparin solution (for example, 1% w / w in a 1: 1000 solution). The exsanguinated placenta is preferably stored for no more than 36 hours before the placental perfusate is collected. 6.4.3 Placental perfusion
[000121] Methods for perfusing mammalian placentas and obtaining placental perfusate are described, for example, in Hariri, U.S. Patent Numbers 7,045,148 and 7,255,879, and in US Order Publications numbers 2007/0190042 and 20070275362 , the descriptions of which are incorporated herein in full by reference.
[000122] Perfusate can be obtained by passing the perfusion solution, for example, a saline solution, culture medium or compositions for collecting cells described above, through the placental vasculature. In one embodiment, a mammalian placenta is perfused by passing the perfusion solution through one or both between the umbilical artery and the umbilical vein. The flow of the perfusion solution through the placenta can be performed using, for example, gravity flow through the placenta. Preferably, the infusion solution is forcibly passed through the placenta using a pump, for example, a peristaltic pump. The umbilical vein can be, for example, cannulated with a cannula, for example, a TEFLON® cannula or plastic, which is connected to a sterile connection apparatus, such as a sterile tubing. The sterile connection apparatus is connected to a perfusion manifold.
[000123] In preparation for the infusion, the placenta is preferably oriented in a way in which the umbilical artery and umbilical vein are located at the highest point of the placenta. The placenta can be perfused by passing an infusion solution through the placental vasculature, or through the placental vasculature and the surrounding tissue. In one embodiment, the umbilical artery and umbilical vein are connected simultaneously to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The infusion solution is passed through the umbilical vein and artery. The infusion solution oozes from and / or passes through the walls of the blood reservoirs into the tissues around the placenta, and is collected in a suitable open container from the surface of the placenta that was attached to the mother's uterus during pregnancy. The perfusion solution can also be introduced through the opening of the umbilical cord and allowed to flow or percolate out of the openings in the wall of the placenta that interface with the maternal uterine wall. In another modality, the perfusion solution is passed through the umbilical veins and collected through the umbilical artery, or passed through the umbilical artery and collected through the umbilical veins, that is, it passes only through the placental vasculature (fetal tissue).
[000124] In one embodiment, for example, the umbilical artery and umbilical vein are connected simultaneously, for example, to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The perfusion solution is passed through the umbilical vein and artery. The infusion solution oozes from and / or passes through the walls of the blood reservoirs into the tissues around the placenta, and is collected in a suitable open container from the surface of the placenta that was attached to the mother's uterus during pregnancy. The perfusion solution can also be introduced through the opening of the umbilical cord and allowed to flow or percolate out of the openings in the wall of the placenta that interface 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.
[000125] In another modality, the perfusion solution is passed through the umbilical veins and collected through the umbilical artery, or passed through the umbilical artery and collected through the umbilical veins. Placental cells collected by this method, which can be referred to as a “closed loop” method, are typically almost exclusively fetal.
[000126] The closed circuit perfusion method can, in one embodiment, be performed as follows. A postpartum placenta is obtained within about 48 hours after delivery. The umbilical cord is pleated and cut after pleating. The umbilical cord can be discarded, or processed to recover, for example, umbilical cord stem cells, and / or to process the umbilical cord membrane for the production of a biomaterial. The amniotic membrane can be maintained during the infusion, or it can be separated from the chorion, for example, using blunt dissection with the fingers. If the amniotic membrane is separated from the chorion before perfusion, it can, for example, be discarded, or processed, for example, to obtain stem cells by enzymatic digestion, or to produce, for example, an amniotic membrane biomaterial, for example. example, the biomaterial described in US Patent Application Publication No. 2004/0048796. After the placenta is cleared of all visible blood clots and residual blood, for example, using sterile gauze, the umbilical cord vessels are exposed, for example, by partially cutting the umbilical cord membrane to expose a cross section of the cord. The vessels are identified, and opened, for example, by advancing a closed alligator clip through the cut end of each vessel. The apparatus, for example, composed of a plastic tube connected to an infusion device or peristaltic pump, is then inserted into each of the placental arteries. The pump can be any pump suitable for this purpose, for example, a peristaltic pump. The plastic tubing, connected to a sterile collection reservoir, for example, a blood bag like a 250 mL collection bag, is then inserted into the placental vein. Alternatively, the tube connected to the pump is inserted into the placental vein, and tubes for one or more collection reservoirs are inserted in one or both of the placental arteries. The placenta is then infused with a volume of infusion solution, for example, about 750 mL of infusion solution. The cells in the perfusate are then collected, for example, by centrifugation.
[000127] In one embodiment, the proximal umbilical cord is pleated during the perfusion, and more preferably, it is pleated at 4-5 cm (centimeters) from the insertion of the cord in the placental disk.
[000128] The first collection of perfusion fluid from a mammalian placenta during the exsanguination process is usually colored with residual blood cells from umbilical cord blood and / or placental blood. The perfusion fluid becomes more colorless as the infusion occurs and residual umbilical cord blood cells are washed out of the placenta. Generally, 30 to 100 mL of perfusion fluid are suitable for initially flushing blood from the placenta, but more or less perfusion fluid may be used depending on the results observed.
[000129] The volume of perfusion fluid used to infuse the placenta can vary depending on 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 infusion liquid can be 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 fluid after exsanguination.
[000130] The placenta can be infused a plurality of times over several hours or several days. If the placenta is to be infused several times, it should be maintained or cultured under aseptic conditions in a suitable container or other vessel, and perfused with a cell collection composition, or a standard infusion solution (for example, a normal saline solution such as brine phosphate buffered (“PBS”)) with or without an anticoagulant (for example, heparin, warfarin sodium, coumarin, bis-hydroxycoumarin), and / or with or without an antimicrobial agent (for example, β-mercaptoethanol (at 1 mM ); antibiotics such as streptomycin (for example, 40-100 μg / mL), penicillin (for example, 40 U / mL), amphotericin B (for example, 0.5 μg / mL). isolated placenta is maintained or cultured for a period of time without collecting the perfusate, so 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 the perfusion and collection of the perfusate.The perfused placenta can be r maintained for one or more additional periods, for example, 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 infused a second time with, for example, 700-800 ml of perfusion fluid. The placenta can be infused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment, the perfusion of the placenta and the collection of the perfusion solution, for example, the collected composition of placental cells, is repeated until the number of nucleated cells recovered is below 100 cells / mL. Perfusates at different times can be further processed individually to collect time-dependent cell populations, for example, total nucleated cells. Perfusates from different times can also be mixed. 1.1.4. Placental perfusate and placental perfusate cells
[000131] Typically, the placental perfusate of a single placental perfusion comprises about 100 million to about 500 million nucleated cells, including hematopoietic cells from which TSNK cells can be produced by the method described here. In certain embodiments, placental perfusate or placental perfusate cells comprise CD34 + cells, for example, hematopoietic stem or progenitor cells. Such cells may, in a more specific embodiment, comprise CD34 + CD45- stem cells or progenitors, CD34 + CD45 + stem cells or progenitors, or the like. In certain embodiments, the perfusate or the perfusate cells are cryopreserved prior to the isolation of the hematopoietic cells from them. In certain other embodiments, the placental perfusate comprises, or the perfusate cells comprise, only fetal cells, or a combination of fetal cells and maternal cells. 6.5 TSNK cells
[000132] In one aspect, TSNK cells, NK cells produced by the methods described here are provided here (for example, the two-step method). There is also provided here a population of cells comprising the TSNK cells produced by the methods described here (for example, the two-step method). In a specific embodiment, the NK cells themselves (for example, TSNK cells) are CD3-CD56 +. In a specific embodiment, the NK cells themselves (for example, TSNK cells) are CD3-CD56 + CD16-. In another specific embodiment, the NK cells themselves (for example, TSNK cells) are additionally CD94 + CD117 +. In another specific embodiment, the NK cells themselves (for example, TSNK cells) are additionally CD161-. In another specific embodiment, the NK cells themselves (for example, TSNK cells) are additionally NKG2D +. In another specific embodiment, the NK cells themselves (for example, TSNK cells) are additionally NKp46 +. In another specific embodiment, the NK cells themselves (for example, TSNK cells) are additionally CD226 +.
[000133] In certain modalities, more than 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, or 98% of the TSNK cells themselves are CD56 + and CD16-. In other modalities, at least 50%, 60%, 70%, 80%, 82%, 84%, 86%, 88% or 90% of the TSNK cells themselves are CD3- and CD56 +. In other modalities, at least 50%, 52%, 54%, 56%, 58% or 60% of the TSNK cells themselves are NKG2D +. In other modalities, less than 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4% or 3% of the cells themselves are NKB1 +. In certain other embodiments, less than 30%, 20%, 10%, 8%, 6%, 4% or 2% of the TSNK cells themselves are NKAT2 +. In certain other modalities, less than 30%, 20%, 10%, 8%, 6%, 4% or 2% of the TSNK cells themselves are CD56 + and CD16 +. In more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65% or 70% of the TSNK CD3-, CD56 + cells themselves are NKp46 + . In other more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of CD3-, CD56 + TSNK cells themselves are CD117 +. In other more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the CDN-, CD56 + TSNK cells themselves are CD94 +. In other more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the CD3-, CD56 + TSNK cells themselves are CD161-. In other more specific modalities, at least 10%, 12%, 14%, 16%, 18% or 20% of the CDN-, CD56 + TSNK cells themselves are CD226 +. In more specific modalities, at least 20%, 25%, 30%, 35% or 40% of the TSNK CD3-, CD56 + cells themselves are CD7 +. In more specific modalities, at least 30%, 35%, 40%, 45%, 50%, 55% or 60% of the CD3-, CD56 + TSNK cells themselves are CD5 +.
[000134] In several other embodiments, TSNK cells can be combined with, for example, NK cells, where the NK cells themselves have been isolated from a tissue source and have not been expanded; NK cells isolated from a tissue source and expanded, or NK cells produced by a different method, for example, natural exterminating cells CD56 + CD16 +, for example, 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 histological environment.
[000135] TSNK cells can have either a fetal genotype or a maternal genotype. For example, because the postpartum placenta, as a source of hematopoietic cells suitable for the production of TSNK cells, comprises tissue and cells from the fetus and mother, the placental perfusate may comprise only fetal cells, or a substantial majority of cells fetal cells (for example, more than about 90%, 95%, 98% or 99%), or it can comprise a mixture of fetal and maternal cells (for example, fetal cells comprise less than about 90%, 80%, 70%, 60%, or 50% of the total nucleated cells of the perfusate). In one embodiment, TSNK cells are derived only from fetal placental hematopoietic cells, for example, cells obtained by closed-circuit perfusion of the placenta where the perfusion produces perfusate comprising a substantial majority, or only, fetal placental hematopoietic cells. In another embodiment, TSNK cells are derived from maternal and fetal cells, for example, cells obtained by infusion using the pan method (see above), where the infusion produces perfusate comprising a mixture of fetal and maternal placental cells. Thus, in one embodiment, a population of intermediary natural exterminator cells derived from the placenta is provided here, the substantial majority of which have the fetal genotype. In another embodiment, a population of intermediary natural exterminator cells derived from placenta is provided here, comprising natural exterminator cells with the fetal genotype and natural exterminators with the maternal phenotype.
[000136] TSNK cell populations comprising natural killer cells that were not produced by the methods described here are also provided here. For example, in one embodiment, a population of TSNK cells is provided here which also comprises natural killer cells isolated from, for example, umbilical cord blood, peripheral blood, bone marrow, or a combination of two or more of the above, or cells NK expanded by a method different from the methods described here. Such populations of TSNK cells can comprise TSNK cells and other NK cells in, for example, 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 similar.
[000137] In certain embodiments, isolated natural killer cells (for example, TSNK cells) or populations enriched in natural killer cells (for example, TSNK cells) can be evaluated by detecting one or more functionally relevant markers, for example, CD94 , CD161, NKp44, DNAM-1, 2B4, NKp46, CD94, KIR, and the NKG2 family of activation receptors (for example, NKG2D). In some embodiments, the purity of the isolated or enriched natural killer cells can be confirmed by detecting one or more between CD56, CD3 and CD16.
[000138] Optionally, the cytotoxic activity of isolated or enriched natural killer cells can be evaluated, for example, in a cytotoxicity assay using tumor cells, for example, tumor cells K562, LN-18, U937, WERI-RB-1, Ul 18MG, HT-29, HCC2218, KG-1, or U266 in culture, or the like, as target cells. 6.6 TSNK cells in combination with placental perfusate
[000139] Compositions comprising TSNK cells in combination with placental perfusate, placental perfusate cells and / or adherent placental cells, for example, for use in suppressing the proliferation of a tumor cell or a plurality of tumor cells, are further provided herein. 6.6.1 Combinations of TSNK and perfusate cells or perfusate cells
[000140] Compositions comprising TSNK cells in combination with placental perfusate and / or placental perfusate cells are further provided here. In one embodiment, for example, a volume of placental perfusate supplemented with TSNK cells is provided here. In specific embodiments, for example, each milliliter of placental perfusate is supplemented with about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more TSNK cells. In another embodiment, the placental perfusate cells are supplemented with TSNK cells. In certain other embodiments, when placental perfusate cells are combined with TSNK cells, placental perfusate cells generally comprise about, more than about, or less 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 TSNK cells are combined with a plurality of placental perfusate cells and / or combined natural killer cells, NK cells generally comprise more than about, or less 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 TSNK cells are used to supplement placental perfusate, the volume of solution (for example, saline, culture medium or the like) in which the cells are suspended comprises about, more than about, or less of about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total volume of perfusate more cells, where TSNK cells are suspended in about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more cells per milliliter before supplementation.
[000141] In other embodiments, any of the above cell combinations is in turn combined with umbilical cord blood or nucleated cord blood cells.
[000142] Mixed placental perfusate is also provided here which is obtained from two or more sources, for example, two or more placentas, and combined, for example, mixed. Such mixed 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 chosen at random, or they can be based on, for example, a concentration or amount of one or more cellular factors, for example, cytokines, growth factors, hormones, or the like; the number of placental cells in the perfusate from each source; or other characteristics of the perfusate from each source. Multiple perfusion infusions from the same placenta can be mixed in a similar way.
[000143] Similarly, placental perfusate cells and intermediary natural exterminator cells derived from placenta are obtained here, which are obtained from two or more sources, for example, two or more placentas, and mixed. Such mixed cells may comprise approximately equal numbers of cells from the two or more sources, or different cell numbers from one or more of the mixed sources. The relative cell numbers of each source can be chosen based, for example, on the number of one or more specific types of cells among the cells to be mixed, for example, the number of CD34 + cells, etc.
[000144] TSNK cells, and combinations of TSNK cells with placental perfusate and / or placental perfusate cells, which have been subjected to tests to determine the degree or amount of tumor suppression (i.e., potency) at be expected from, for example, a given number of TSNK cells, or a given volume of perfusate. For example, an aliquot or sample with a certain 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 tumor cell proliferation in the presence of placental perfusate, perfusate cells, natural perfusate killer cells, or combinations thereof, over time (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or more) is compared to proliferation of an equivalent number of tumor cells in the absence of perfusate, perfusate cells, natural placental killer cells, or combinations thereof. The potency of the cells can be expressed, for example, as the number of cells or volume of solution required to suppress tumor cell growth, for example, by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or similar.
[000145] In certain embodiments, TSNK cells are provided as manageable units of pharmaceutical grade. Such units may be provided in discrete volumes, for example, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, 50 ml, 55 ml, 60 ml, 65 ml, 70 ml, 75 ml , 80 ml, 85 ml, 90 ml, 95 ml, 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 to contain a specific number of cells, for example, TSNK cells only, or TSNK cells in combination with other NK or perfusate cells, for example, 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more cells per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more cells per unit. In specific embodiments, the units may comprise about, or less than about, or even about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106 or more TSNK cells per milliliter , or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more cells per unit. Such units can be provided to contain specific numbers of TSNK cells, and / or any of the other cells.
[000146] In the above embodiments, TSNK cells or combinations of TSNK cells with other NK cells, perfusate or perfusate cells can be autologous to a recipient (i.e., obtained from the recipient), or allogeneic to a recipient (i.e., obtained at least one individual other than the recipient itself).
[000147] In certain embodiments, each cell unit is labeled to specify one or more between volume, number of cells, cell type, whether or not the unit has been enriched for a particular cell type, and / or the power of a given number of cells in the unit, or a given number of milliliters of the unit, that is, whether the cells in the unit cause a measurable suppression of the proliferation of a particular type or types of tumor cells. 6.6.2 Combinations of TSNK cells and adherent placental stem cells
[000148] In other embodiments, TSNK cells, either alone or in combination with placental perfusate or placental perfusate cells, are supplemented with isolated adherent placental cells, for example, placental stem cells and multipotent placental cells as described, for example, in Hariri, US Patent Nos. 7,045,148 and 7,255,879, and in US Patent Application Publication No. 2007/0275362, the descriptions of which are incorporated herein in full by reference. “Adherent placental cells” means that the cells adhere to a tissue culture surface, for example, tissue culture plastic. Adherent placental cells useful in the compositions and methods described herein are not tropoblasts, embryonic germ cells or embryonic stem cells. In certain embodiments, adherent placental stem cells are used as feed cells during the two-step method) as described above.
[000149] TSNK cells, either alone or in combination with placental perfusate or placental perfusate cells, can be supplemented with, for example, 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more adherent placental cells per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106 , 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 10 10, 5 x 10 10, 1 x 10 11 or more adherent placental cells. Adherent placental cells in the combinations can be, for example, adherent placental cells that have been cultured by, for example, 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 duplications, or more.
[000150] Isolated adherent placental cells, when grown in primary cultures or expanded in cell culture, adhere to the tissue culture substrate, for example, the surface of the tissue culture container (eg, tissue culture plastic). Adherent placental cells in the culture assume a generally fibroblast-like, starry appearance, with a number of cytoplasmic processes extending from the central cell body. Adherent placental cells are, however, morphologically distinguishable from fibroblasts cultured under the same conditions, as adherent placental cells exhibit a greater number of processes than fibroblasts. Morphologically, adherent placental cells are also distinguishable from hematopoietic stem cells, which generally assume a more rounded morphology, such as pebbles, in culture.
[000151] Isolated adherent placental cells, and adherent placental cell populations, useful in the compositions and methods provided herein, express a plurality of markers that can be used to identify and / or isolate the cells, or cell populations, that comprise the adherent placental cells. Adherent placental cells, and adherent placental cell populations, useful in the compositions and methods provided herein, include adherent placental cells and cell populations containing adherent placental cells obtained directly from the placenta, or any part thereof (for example, amnion, chorion, plaque amnio-chorionic, placental cotyledons, umbilical cord, and the like). The population of adherent placental cells, in one embodiment, is a population (that is, two or more) adherent placental stem cells in culture, for example, a population in a container, for example, a bag.
[000152] Adherent placental cells generally express the markers CD734, CD105 and CD200, 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 cells, and to distinguish adherent placental cells from other cell types. Since adherent placental cells can express CD73 and CD105, they may have characteristics similar to those of mesenchymal stem cells. The lack of expression of CD34, CD38 and / or CD45 identifies adherent placental stem cells as non-hematopoietic stem cells.
[000153] In certain embodiments, the isolated adherent placental cells described here detectably suppress the proliferation of cancer cells or the growth of tumors.
[000154] In certain embodiments, the isolated adherent placental cells are isolated placental stem cells. In certain other embodiments, the isolated adherent placental cells are isolated multipotent placental cells. In a specific embodiment, the isolated adherent placental cells are CD34-, CD10 + and CD105 + as detectable by flow cytometry. In a more specific embodiment, the CD34-, CD10 +, CD105 + adherent placental cells isolated are adherent multipotent placental cells. In another more specific modality, CD34-, CD10 +, CD105 + adherent placental cells alone have the potential to differentiate into cells of a neural phenotype, cells of an osteogenic phenotype, or cells of a chondrogenic phenotype. In a more specific embodiment, the CD34-, CD10 +, CD105 + adherent placental cells isolated are additionally CD200 +. In another more specific embodiment, the CD34-, CD10 +, CD105 + adherent placental cells isolated are additionally CD90 + or CD45-, as detected by flow cytometry. In another more specific embodiment, the CD34-, CD10 +, CD105 +, CD200 + adherent placental cells are additionally CD90 + or CD45-, as detected by flow cytometry. In another more specific embodiment, the CD34-, CD10 +, CD105 +, CD200 +, CD90 +, CD45- adherent placental cells are additionally CD80- and CD86-, as detected by flow cytometry.
[000155] In one embodiment, the isolated adherent placental cells are CD200 +, HLA-G +. In a specific embodiment, the isolated adherent placental cells themselves are also CD73 + and CD105 +. In another specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38- or CD45-. In a more specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38- or CD45-, CD73 + and CD105 +. In another embodiment, the isolated adherent placental cells themselves produce one or more embryo-like bodies when grown under conditions that allow the formation of embryo-like bodies.
[000156] In another embodiment, the isolated adherent placental cells are CD73 +, CD105 +, CD200 +. In a specific modality of the populations themselves, the isolated adherent placental cells are also HLA-G +. In another specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38- or CD45-. In another specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38-, CD45- and HLA-G +. In another specific embodiment, the isolated adherent placental cells themselves produce one or more embryo-like bodies when grown under conditions that allow the formation of embryo-like bodies.
[000157] In another embodiment, the isolated adherent placental cells are CD200 +, OCT-4 +. In a specific embodiment, the isolated adherent placental cells themselves are also CD73 + and CD105 +. In another specific embodiment, the isolated adherent placental cells themselves are also HLA-G +. In another specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38-, CD45-, CD73 +, CD105 + and HLA-G +. In another specific embodiment, the isolated adherent placental cells themselves produce one or more embryo-like bodies when grown under conditions that allow the formation of embryo-like bodies.
[000158] In another embodiment, the isolated adherent placental cells are CD105 + and HLA-G +. In a specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38- or CD45-. In another specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38- and CD45-. In another specific embodiment, the isolated adherent placental cells themselves are also OCT-4 +. In another specific modality, the isolated adherent placental cells themselves are also CD200 +. In another specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38-, CD45-, OCT-4 + and CD200 +.
[000159] In another embodiment, the isolated adherent placental cells are CD73 +, CD105 + stem cells, where the cells themselves produce one or more embryo-like bodies when grown under conditions that allow the formation of embryo-like bodies. In a specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38- or CD45-. In another specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38- and CD45-. In another specific embodiment, the isolated adherent placental cells themselves are also OCT-4 +. In a more specific embodiment, the isolated adherent placental cells themselves are also OCT-4 +, CD34-, CD38- and CD45-.
[000160] In another embodiment, the adherent placental stem cells are OCT-4 + stem cells, where the adherent placental stem cells produce one or more embryo-like bodies when grown under conditions that allow the formation of embryo-like bodies , and where the stem cells themselves have been identified as detectably suppressing the proliferation of cancer cells or the growth of tumors.
[000161] In various modalities, 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 the isolated adherent placental cells themselves are OCT-4 +. In a specific modality of the populations above, the isolated adherent placental cells themselves are also CD73 + and CD105 +. In another specific embodiment, the isolated adherent placental cells themselves are also CD34-, CD38- or CD45-. In another specific modality, the stem cells themselves are CD200 +. In a more specific embodiment, the isolated adherent placental cells themselves are also CD73 +, CD105 +, CD200 +, CD34-, CD38- and CD45-. In another specific embodiment, the isolated adherent placental cells were expanded, for example, passed at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.
[000162] In a more specific embodiment of any of the above embodiments, isolated adherent placental cells express ABC-p (a placental-specific ABC carrier protein; see, for example, Allikmets et al, Cancer Res. 58 (23): 5337-9 (1998)).
[000163] In another embodiment, the isolated adherent placental cells are CD29 +, CD44 +, CD73 +, CD90 +, CD105 +, CD200 +, CD34- and CD133-. In another embodiment, isolated adherent placental cells constitutively secrete IL-6, IL-8 and monocyte-chemoprotein protein (MCP-1).
[000164] Each of the isolated adherent placental cells mentioned above may comprise cells obtained and isolated directly from a mammalian placenta, or cells that have been cultured and passed on 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 of these. Suppressor tumor cell pluralities of the isolated adherent placental cells described above may comprise about at least, or no more than, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more isolated adherent placental cells. 6.6.3 Compositions comprising conditioned medium with adherent placental cells
[000165] Also provided here is the use of a composition comprising TSNK cells and, in addition, conditioned medium, where the composition itself is tumor suppressive, or is effective in the treatment of cancer or viral infection. Adherent placental cells such as those described in Section 6.6.2, above, can be used to produce conditioned medium that is a suppressor of tumor cells, anticancer or antiviral, that is, medium comprising one or more biomolecules secreted or excreted by cells that have a detectable tumor cell suppressive effect, an anticancer effect or antiviral effect. In several embodiments, the conditioned medium comprises medium in which the cells have proliferated (i.e., grown) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, the conditioned medium comprises medium in which such 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 cells, for example, placental cells, or cells of another type. In another embodiment, the conditioned medium provided herein comprises medium in which isolated adherent placental cells, for example, isolated adherent placental stem cells or isolated adherent multipotent placental cells, and cells other than isolated adherent placental cells, for example, non-adherent stem cells -placental or multipotent cells, have been cultured.
[000166] Such conditioned medium can be combined with any, or any combination of TSNK cells, placental perfusate, placental perfusate cells, to form a composition that is tumor cell suppressant, anticancer or antiviral. In certain embodiments, the composition comprises less than half of volume-conditioned medium, for example, about, or less than, about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15 %, 10%, 5%, 4%, 3%, 2%, or 1% by volume.
[000167] Thus, in one embodiment, a composition is provided here comprising TSNK cells and culture medium from a culture of isolated adherent placental cells, where the isolated adherent placental cells themselves (a) adhere to the substrate; and (b) are CD34-, CD10 + and CD105 +; where the composition itself suppresses the growth or proliferation of tumor cells, or is anticancer or antiviral. In a specific embodiment, the isolated adherent placental cells are CD34-, CD10 + and CD105 + as detected by flow cytometry. In a more specific modality, the CD34-, CD10 + and CD105 + adherent placental cells isolated are placental stem cells. In another more specific modality, the CD34-, CD10 + and CD105 + adherent placental cells isolated are multipotent adherent placental cells. In another specific modality, isolated CD34-, CD10 + and CD105 + adherent placental cells have the potential to differentiate into cells of a neural phenotype, cells of an osteogenic phenotype, or cells of a chondrogenic phenotype. In a more specific embodiment, the isolated CD34-, CD10 + and CD105 + adherent placental cells are additionally CD200 +. In another more specific embodiment, the CD34-, CD10 + and CD105 + adherent placental cells isolated are additionally CD90 + or CD45-, as detected by flow cytometry. In another more specific embodiment, the CD34-, CD10 + and CD105 + adherent placental cells isolated are additionally CD90 + or CD45-, as detected by flow cytometry. In another more specific embodiment, the adherent placental cells CD34-, CD10 +, CD105 + and CD200 + isolated are additionally CD90 + or CD45-, as detected by flow cytometry. In another more specific embodiment, the adherent placental cells CD34-, CD10 +, CD105 + and CD200 + isolated are additionally CD90 + or CD45-, as detected by flow cytometry. In another more specific embodiment, the CD34-, CD10 +, CD105 +, CD200 +, CD90 +, CD45- adherent placental cells are additionally CD80- and CD86-, as detected by flow cytometry.
[000168] In another embodiment, a composition is provided here comprising TSNK cells and culture medium from a culture of isolated adherent placental cells, where the isolated adherent placental cells themselves (a) adhere to a substrate; and (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 embryo-like bodies in a population of placental cells that comprise placental stem cells where the population itself is grown under conditions that allow the formation of one or more embryo-like bodies, or express OCT-4 and facilitate the formation of an or more embryo-like bodies in a population of placental cells that comprise placental stem cells where the population itself is grown under conditions that allow the formation of one or more embryo-like bodies; where the composition itself detectably suppresses the growth or proliferation of tumor cells, or is anticancer or antiviral. In a specific embodiment, the composition further comprises a plurality of isolated adherent placental cells themselves. In another specific embodiment, the composition comprises a plurality of non-placental cells. In a more specific embodiment, the non-placental cells themselves comprise CD34 + cells, for example, hematopoietic progenitor cells, such as peripheral blood hematopoietic progenitor cells, umbilical cord blood hematopoietic progenitor cells, or hematopoietic progenitor cells of placental blood. Non-placental cells can also comprise stem cells, such as mesenchymal stem cells, for example, bone marrow-derived mesenchymal stem cells. Non-placental cells can also be one or more types of adult cells or cell lines. In another specific embodiment, the composition comprises an antiproliferative agent, for example, an anti-MIP-1α or anti-MIP-1β antibody.
[000169] In a specific embodiment, the culture medium conditioned by one of the cells or cell combinations described above is obtained by a plurality of isolated adherent placental cells co-cultured with a plurality of tumor cells in a ratio of about 1: 1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1 from isolated adherent placental cells to tumor cells. For example, conditioned culture medium or supernatant can be obtained from a culture comprising about 1 x 105 isolated adherent placental cells, about 1 x 106 isolated adherent placental cells, about 1 x 107 isolated adherent placental cells, or about 1 x 108 isolated adherent placental cells, or more. In another specific embodiment, the conditioned culture medium or supernatant is obtained from a co-culture comprising about 1 x 105 to about 5 x 105 isolated adherent placental cells and about 1 x 105 tumor cells; about 1 x 10 6 to about 5 x 10 6 isolated adherent placental cells and about 1 x 10 6 tumor cells; about 1 x 107 to about 5 x 107 isolated adherent placental cells and about 1 x 107 tumor cells; or about 1 x 108 to about 5 x 108 isolated adherent placental cells and about 1 x 108 tumor cells; 6.7. Cell Conservation
[000170] Cells, for example, TSNK cells or placental perfusate cells comprising hematopoietic stem cells or progenitors, can be conserved, that is, kept under conditions that allow long-term storage, or under conditions that inhibit cell death due to to, for example, apoptosis or necrosis.
[000171] Placental perfusate can be produced by passing a cell collection composition through at least part of the placenta, for example, through the placental vasculature. The cell collection composition comprises one or more compounds that act to conserve the cells contained in the perfusate. Such placental cell collection composition may comprise an apoptosis inhibitor, necrosis inhibitor and / or an oxygen-carrying perfluorocarbon, as described in U.S. Patent Application Publication No. 20070190042, the description of which is incorporated herein in its entirety by reference. .
[000172] In one embodiment, the perfusate or population of placental cells is collected from a postpartum mammalian placenta, for example, from a human, by contacting the perfusate or population of cells with a collection composition comprising an inhibitor of apoptosis and an oxygen-carrying perfluorocarbon, where the apoptosis inhibitor itself is present in an amount and for a time sufficient to reduce or prevent apoptosis in the population of placental cells, for example, adherent placental cells, for example, placental stem or multipotent placental cells, when compared to a cell population not contacted with the apoptosis inhibitor. For example, the placenta can be perfused with the cell-collecting composition, and placental cells, for example, total nucleated placental cells, are isolated in this way. In a specific embodiment, the apoptosis inhibitor is a caspase inhibitor. In another specific embodiment, the apoptosis inhibitor itself is a JNK inhibitor. In a more specific embodiment, the JNK inhibitor itself does not modulate the differentiation or proliferation of adherent placental cells, for example, adherent placental stem cells or adherent multipotent placental cells. In another embodiment, the cell collection composition comprises the apoptosis inhibitor itself and the oxygen-carrying perfluorocarbon itself in separate phases. In another embodiment, the cell collection composition further comprises an emulsifier, for example, lecithin. In another embodiment, the apoptosis inhibitor itself and perfluorocarbon itself are between about 0 ° C and about 25 ° C at the time of contact with the placental cells. In another more specific modality, the apoptosis inhibitor itself and perfluorocarbon itself are between about 2 ° C and 10 ° C, or between about 2 ° C and about 5 ° C, at the time of contact with the placental cells. . In another more specific modality, the contact itself is made during the transport of said cell population. In another more specific modality, the contact itself is made during the freezing and thawing of the said cell population.
[000173] In another embodiment, the placental perfusate and / or the placental cells can be collected and preserved by contacting the perfusate and / or the cells with an apoptosis inhibitor and an organ preservative compound, where the apoptosis inhibitor itself is present in an amount and for a time sufficient to reduce or prevent apoptosis of the cells, when compared to perfusate or placental cells not contacted with the apoptosis inhibitor. In a specific embodiment, the organ preservative compound is a UW solution (described in U.S. Patent No. 4,798,824; also known as VIASPAN ™; see also Southard et al., Transplantation 49 (2) 251-257 (1990)) or a solution described in Stern et al., U.S. Patent No. 5,552,267, the descriptions of which are incorporated herein in full by reference. In another embodiment, the organ preservative composition itself is hydroxyethylated starch, lactobionic acid, raffinose, or a combination of these. In another embodiment, the placental cell collection composition additionally comprises a perfluorocarbon, in two stages or as an emulsion.
[000174] In another embodiment of the method, placental cells are contacted with a cell collection composition comprising an apoptosis inhibitor and an oxygen-carrying perfluorocarbon, organ preservative compound, or combination thereof, during the perfusion. In another modality, placental cells are contacted with the collection of cell collection itself after collection by infusion.
[000175] Typically, during the collection, enrichment and isolation of placental cells, it is preferable to minimize or eliminate cellular stress due to hypoxia and mechanical stress. In another modality of the method, therefore, the placental perfusate or a population of placental cells is exposed to a hypoxic condition during collection, enrichment or isolation for less than six hours during conservation itself, where a hypoxic condition is a concentration oxygen that is less than the normal blood oxygen concentration. In a more specific modality, the perfusate or the population of placental cells themselves are exposed to the hypoxic condition itself for less than two hours during the actual conservation. In another more specific modality, the perfusate or the population of placental cells themselves are exposed to the hypoxic condition itself for less than an hour, or less than thirty minutes, or are not exposed to a hypoxic condition, during collection, enrichment or isolation. In another specific modality, the population of placental cells itself is not exposed to shear stress during collection, enrichment or isolation.
[000176] Cells, for example, placental perfusate cells, hematopoietic cells, for example, CD34 + hematopoietic stem cells; NK cells, for example, TSNK cells; isolated adherent placental cells provided herein can be cryopreserved, for example, in a cryopreservation medium in small reservoirs, for example, septum ampoules or flasks. In certain embodiments, the cells provided here are cryopreserved at a concentration of about 1 x 104 - 5 x 108 cells per ml. In specific embodiments, the cells provided here are cryopreserved at a concentration of about 1 x 10 6 - 1.5 x 10 7 cells per ml. In more specific embodiments, the cells provided here are cryopreserved at a concentration of about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 1.5 x 107 cells per ml.
[000177] Suitable cryopreservation media include, but are not limited to, normal brine, culture medium including, for example, growth medium, or cell freezing medium, for example commercially available cell freezing medium, for example, C2695, C2639 or C6039 (Sigma); CryoStor® CS2, CryoStor® CS5 or CryoStor®CS10 (BioLife Solutions). The cryopreservation medium preferably comprises DMSO (dimethylsulfoxide), in a concentration of, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% (v / v). The cryopreservation medium can comprise additional agents, for example, methylcellulose, dextran, albumin (e.g., human serum albumin), trehalose, and / or glycerol. In certain embodiments, the cryopreservation medium comprises about 1% - 10% DMSO, about 25% - 75% dextran and / or about 20% - 60% human serum albumin (HSA). In certain embodiments, the cryopreservation medium comprises about 1% - 10% DMSO, about 25% - 75% trehalose and / or about 20% - 60% human serum albumin (HSA). In a specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% dextran and 40% HSA. In a more specific modality, the cryopreservation medium comprises 5% DMSO, 55% dextran (10% w / v in normal brine) and 40% HSA. In another specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% dextran and 40% HSA. In a more specific modality, the cryopreservation medium comprises 5% DMSO, 55% trehalose (10% w / v in normal brine) and 40% HSA. In another specific embodiment, the cryopreservation medium comprises CryoStor® CS5. In another specific embodiment, the cryopreservation medium comprises CryoStor® CS10.
[000178] The cells provided here can be cryopreserved by any one of a variety of methods, and at any stage of cell culture, expansion and differentiation. For example, the cells provided here can be cryopreserved right after the isolation of the original tissues or organs, for example, placental perfusate or umbilical cord blood, or during, or after the first or second steps of the methods described above. In certain embodiments, hematopoietic cells, for example, stem cells or hematopoietic progenitor cells, are cryopreserved within about 1, 5, 10, 15, 20, 30, 45 minutes or within about 1, 2, 4, 6, 10, 12, 18, 20 or 24 hours after the isolation of the original tissues or organs. In certain embodiments, the cells themselves are cryopreserved within 1, 2 or 3 days after the isolation of the original tissues or organs. In certain embodiments, the cells themselves are cryopreserved after being cultured in a first medium as described in Section 6.2.1 above, for about 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 some embodiments, the cells themselves are cryopreserved after being cultured in a first medium as described in Section 6.2.1 above, for about 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, and in a second half for about 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 as described in Section 6.2.1, above.
[000179] In one aspect, a method of cryopreserving a population of NK cells, for example, TSNK cells, is provided here. In one embodiment, the method itself comprises: (a) the inoculation of a population of stem cells or hematopoietic progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more between factor cells -trunk (SCF) and interleukin-7 (IL-7), where the IL-15 and the optional SCF and IL-7 itself are not included in an undefined component of the medium itself, so that the population expands , and a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves will differentiate into NK cells during said expansion; (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells; and (c) the cryopreservation of the NK cells from step (b) in a cryopreservation medium. In a specific embodiment, step (c) itself further comprises (1) the preparation of a solution with cell suspension; (2) adding the cryopreservation medium to the cell suspension solution from step (1) to obtain a cryopreserved cell suspension; (3) cooling the cryopreserved suspension from step (3) to obtain a cryopreserved sample; and (4) the storage of the cryopreserved sample below -80 ° C. In certain embodiments, the method does not include intermediate steps between steps (a) and (b), and between steps (b) and (c), and / or does not include additional culture steps before step (a).
[000180] In another embodiment, said method for the cryopreservation of a population of NK cells, for example, TSNK cells, comprises: (a) the expansion of a population of hematopoietic stem cells or progenitor cells in a first medium comprising a or more between stem cell factors (SCF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15) and heparin, and where SCF, IL-2, IL-7 and IL-15 themselves are not comprised in an undefined component of the medium itself, and where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells themselves differ in NK cells during said expansion; (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells; and (c) the cryopreservation of the NK cells from step (b) in a cryopreservation medium. In a specific embodiment, step (c) itself further comprises (1) the preparation of a solution with cell suspension; (2) adding the cryopreservation medium to the cell suspension solution from step (1) to obtain a cryopreserved cell suspension; (3) cooling the cryopreserved suspension from step (3) to obtain a cryopreserved sample; and (4) the storage of the cryopreserved sample below -80 ° C. In certain embodiments, the method does not include intermediate steps between steps (a) and (b), and between steps (b) and (c).
[000181] The cells provided here are preferably cooled in a freezer with a controlled rate, for example, about 0.1; 0.3; 0.5; or 1 ° C / min during cryopreservation. A preferred cryopreservation temperature is between about -80 ° C and -180 ° C, preferably about -125 ° C to about -140 ° C. Cryopreserved cells can be transferred to liquid nitrogen before 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. The cryopreserved cells are preferably thawed at a temperature of about 20 ° C to about 40 ° C, preferably at a temperature of about 37 ° C. In certain embodiments, the cryopreserved cells are thawed after being cryopreserved for about 1, 2, 4, 6, 10, 12, 18, 20 or 24 hours, or for about 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 certain embodiments, the cryopreserved cells are thawed after being cryopreserved for about 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 months. In certain embodiments, the cryopreserved cells are thawed after being cryopreserved for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.
[000182] Suitable defrosting media include, but are not limited to, normal brine, plasmalyte culture medium including, for example, growth medium, for example, RPMI medium. In preferred embodiments, the thawing medium comprises one or more of medium supplements (for example, nutrients, cytokines and / or factors). Supplements of medium suitable for thawing the cells provided herein include, for example, and without limitation, serum such as human serum AB, fetal bovine serum (FBS) or fetal calf serum (FCS), vitamins, human serum albumin (HSA), bovine serum albumin (BSA), amino acids (eg L-glutamine), fatty acids (eg oleic acid, linoleic acid or palmitic acid), insulin (eg recombinant human insulin), transferrin (human transferrin saturated with iron), β-mercaptoethanol, stem cell factor (SCF), tyrosine kinase similar to Fms 3 ligand (Flt3-L), cytokines such as interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), thrombopoietin (Tpo) or heparin. In a specific embodiment, the defrosting medium useful in the methods provided here comprises RPMI. In another specific embodiment, the defrosting medium itself comprises plasmalyte. In another specific embodiment, the defrosting medium itself comprises about 0.5 - 20% FBS. In another specific embodiment, the defrosting medium itself comprises about 1, 2, 5, 10, 15 or 20% FBS. In another specific embodiment, the defrosting medium itself comprises about 0.5 - 20% HSA. In another specific embodiment, the defrosting medium itself comprises about 1, 2, 5, 10, 15 or 20% HSA. In a more specific embodiment, the defrosting medium itself comprises about 5% HSA.
[000183] The cryopreservation methods provided here can be optimized to allow long-term storage, or under conditions that inhibit cell death by, for example, apoptosis or necrosis. In one embodiment, post-thaw cells comprise more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% viable cells, as determined by, for example, counting automatic cell phone or the trypan blue method. In another embodiment, the post-thaw cells comprise about 0.5; 1; 5; 10; 15; 20 or 25% of dead cells. In another embodiment, the post-thaw cells comprise about 0.5; 1; 5; 10; 15 or 20% of newly apoptotic cells. In another modality, about 0.5; 1; 5; 10; 15 or 20% of post-thaw cells enter apoptosis after 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 after being thawed, for example, as determined by an apoptosis assay (for example, TO-PR03 or AnnV / PI apoptosis assay kits) . In certain embodiments, post-thaw cells are re-preserved after they have been cultured, expanded or differentiated using the methods provided here. 6.8. Uses of TSNK cells
[000184] The TSNK cells provided here can be used in methods for the treatment of individuals with cancer, for example, individuals with solid tumor cells and / or blood cancer cells, or people with a viral infection. The TSNK cells provided herein can also be used in methods to suppress tumor cell proliferation. 6.8.1. Treatment of individuals with cancer
[000185] In one embodiment, a method is provided here for treating an individual with a cancer, for example a blood cancer or a solid tumor, comprising administering to the individual itself a therapeutically effective amount of TSNK cells. In certain embodiments, the individual has a deficiency of natural killer cells, for example, a deficiency of NK cells active against the individual's cancer. In a specific embodiment, the method further comprises administering to the individual itself isolated placental perfusate or isolated placental perfusate cells, for example, a therapeutically effective amount of isolated placental perfusate or isolated placental perfusate cells. In another specific embodiment, the method further comprises administering to the individual itself an effective amount of an immunomodulatory compound, for example, an immunomodulatory compound described in Section 6.2.1, above, or thalidomide. As used here, an "effective amount" is an amount that, for example, results in a detectable improvement in, or a decrease in the progression of, or elimination of, one or more symptoms of a cancer from which the individual suffers.
[000186] In a specific modality, cancer is a blood cancer, for example, leukemia or lymphoma. In more specific modalities, cancer is an acute leukemia, for example, acute T leukemia, acute myeloid leukemia (AML), acute pro-myelocytic leukemia, acute myeloblastic leukemia, acute megakarioblastic leukemia, precursor acute B lymphoblastic leukemia, acute T lymphoblastic leukemia precursor, Burkitt's leukemia (Burkitt's lymphoma), or acute biphenotypic leukemia; a chronic leukemia, for example, chronic myeloid lymphoma, chronic myeloid leukemia (CML), chronic monocytic leukemia, chronic lymphocytic leukemia (CLL), mild lymphocytic lymphoma, or pro-lymphocytic leukemia of B cells; hair follicle lymphoma, pro-lymphocytic T-cell leukemia; or a lymphoma, for example, histiocytic lymphoma, lymphoplasmic lymphoma (for example, Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasia (for example, plasma cell myeloma, plasmacytoma, a monoclonal immunoglobulin deposition disorder, or a severe chain disorder), extranodal marginal zone B cell lymphoma (MALT lymphoma), marginal zone B cell nodal lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse wide B cell lymphoma, lymphoma B-cell wide mediastinal (thymic), B-cell wide intravascular lymphoma, primary effusion lymphoma, T-cell wide granular lymphocytic leukemia, aggressive NK cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T cell lymphoma , nasal type, enteropathy-type T lymphoma, hepatosplenic T-lymphoma, blastic NK cell lymphoma, fungal mycosis (Sezary syndrome), a cell lymphoproliferative disorder CD30-positive T (for example, primary cutaneous anaplastic broad cell lymphoma or lymphomatoid papulosis), angioimmunobastic T lymphoma, peripheral T lymphoma, unspecified, anaplastic wide cell lymphoma, Hodgkin lymphoma or lymphocyte nodular Hodgkin lymphoma -predominant. In another specific modality, cancer is multiple myeloma or myelodysplastic syndrome.
[000187] In certain other specific embodiments, cancer is a solid tumor, for example, a carcinoma, such as an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a duct cell carcinoma, a lung carcinoma, thyroid carcinoma, nasopharyngeal carcinoma, melanoma (for example, malignant melanoma), non-melanoma skin carcinoma, or unspecified carcinoma; a desmoid tumor; a desmoplastic tumor of small round cells; an endocrine tumor; an Ewing's sarcoma; a germ cell tumor (for example, testicular cancer, ovarian cancer, choriocarcinoma, endodermal bell tumor, germinoma, etc.); a hepatoblastoma; a hepatocellular carcinoma; a neuroblastoma; a non-rhabdomyosarcoma soft tissue sarcoma; an osteosarcoma; a retinoblastoma; a rhabdomyosarcoma; or a Wilms tumor. In another embodiment, the solid tumor is pancreatic cancer or breast cancer. In other modalities, the solid tumor is pancreatic cancer or breast cancer. In other modalities, the solid tumor is an acoustic neuroma; an astrocytoma (for example, a class I pilocytic astrocytoma, a class II low-grade astrocytoma; a class III anaplastic astrocytoma; or a class IV glioblastoma multiforme); a chordoma; a craniopharyngioma; a glioma (for example, a brain stem cell glioma; an ependymoma; a mixed glioma; an optic nerve glioma; or a subependimoma); a glioblastoma; a medulloblastoma; a meningioma; a metastatic brain tumor; an oligodendroglioma; a pineoblastoma; a pituitary tumor; a primitive neuroectodermal tumor; or a schwannoma; In another modality, cancer is prostate cancer.
[000188] In certain embodiments, the individual with cancer, for example, a blood cancer or solid tumor, for example, an individual with a deficiency of natural killer cells, is an individual who received a bone marrow transplant before the actual administration . In certain other modalities, bone marrow transplantation occurred to treat the cancer itself. In certain modalities, bone marrow transplantation occurred to treat a condition other than the cancer itself. In certain modalities, the individual received an immunosuppressant in addition to the bone marrow transplant itself. In certain modalities, the individual who has received a bone marrow transplant exhibits one or more symptoms of graft versus host disease (GVHD) at the time of administration itself. In certain other modalities, the individual who has received a bone marrow transplant receives the administration of the cells themselves before a symptom of graft versus host disease (GVHD) has manifested.
[000189] In certain specific modalities, the individual with cancer, for example, a blood cancer, received at least one dose of a TNFα inhibitor, for example, ETANERCEPT® (Enbrel), prior to the administration itself. In specific modalities, the individual himself received the actual dose of a TNFa inhibitor within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the cancer diagnosis itself said. In a specific embodiment, the individual who received a dose of a TNFα inhibitor exhibits acute myeloid leukemia. In a more specific modality, the individual who received a dose of a TNFα inhibitor and exhibits acute myeloid leukemia also exhibits the deletion of the long arm of chromosome 5 in the blood cells. In another modality, the individual with cancer, for example, a blood cancer, exhibits a Philadelphia chromosome.
[000190] In certain other modalities, cancer, for example, a blood cancer or a solid tumor, in the individual itself, is refractory to one or more anticancer drugs. In a specific modality, the cancer is refractory to GLEEVEC® (imatinib mesylate).
[000191] In certain modalities, cancer, for example, a blood cancer, in the individual itself, responds to at least one anticancer drug; in this embodiment, placental perfusate, isolated placental perfusate cells, isolated natural killer cells, for example, natural placental killer cells, for example, intermediate natural killer cells derived from placenta, isolated combined natural killer cells, and / or combinations of these , and optionally an immunomodulatory compound, are added as adjunct treatments or as a combined therapy with the anti-cancer drug itself. In certain other modalities, the individual with cancer, for example, a blood cancer, was treated with at least one anticancer drug, and had a relapse, prior to the administration itself.
[000192] In one aspect, a method is provided here for the treatment of an individual with multiple myeloma, comprising administering to the individual (1) lenalidomide; (2) melphalan; and (3) expanded NK cells, where the NK cells themselves are efficient in the treatment of multiple myeloma in said individual. In a specific embodiment, the NK cells themselves are umbilical cord blood NK cells, or NK cells produced from umbilical cord blood hematopoietic cells, for example, hematopoietic stem cells. In another embodiment, the NK cells themselves were produced by any of the methods described here for the production of NK cells, for example, for the production of TSNK cells. In another embodiment, the NK cells themselves were expanded prior to said administration. In another embodiment, lenalidomide, melphalan, and / or NK cells themselves are administered separately from each other. In certain specific modalities of the method for the treatment of an individual with multiple myeloma, the NK cells themselves are produced by a two-step method for the production of a population of activated natural killer cells (NK), where a first step of the method itself comprises the expansion of a population of hematopoietic stem cells or progenitor cells in a first medium comprising one or more between stem cell factors (SCF), interleukin-7 (IL-7) and interleukin-15 (IL-15 ), and where SCF, IL-7 and IL-15 itself are not comprised in an undefined component of the medium itself, and where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or cells progenitors themselves differ in NK cells during said expansion; and expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2).
[000193] In another aspect, a method is provided here for the treatment of an individual with chronic lymphocytic leukemia (CLL), comprising administering to an individual a therapeutically effective dose of (1) lenalidomide; (2) melphalan; (3) fludarabine; and (4) expanded NK cells, for example, TSNK cells, where the NK cells themselves are efficient in treating said CLL in the individual itself. In a specific embodiment, the NK cells themselves are umbilical cord blood NK cells, or NK cells produced from umbilical cord blood hematopoietic cells, for example, hematopoietic stem cells. In another embodiment, the NK cells themselves were produced by any of the methods described here for the production of NK cells, for example, for the production of TSNK cells. In a specific embodiment of any of the above methods, said NK cells were expanded for at least 10 days before the actual administration. In a specific embodiment of any of the above methods, lenalidomide, melphalan, fludarabine, and expanded NK cells are administered to the individual itself separately. In certain specific modalities of the method for the treatment of an individual with CLL, the NK cells themselves are produced by a two-step method for the production of a population of activated natural killer cells (NK), where a first step of the method itself said comprises the expansion of a population of hematopoietic stem cells or progenitor cells in a first medium comprising one or more between stem cell factors (SCF), interleukin-7 (IL-7) and interleukin-15 (IL-15) , and where SCF, IL-7 and IL-15 itself are not comprised in an undefined component of the medium itself, and where a plurality of hematopoietic stem cells or progenitor cells within the population of hematopoietic stem cells or progenitor cells properly differentiate into NK cells during said expansion; and where a second step of the method itself comprises the expansion of the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells. 6.8.2. Suppression of tumor cell proliferation
[000194] Here is also provided a method for suppressing the proliferation of tumor cells, comprising the contact of the tumor cells, comprising the contact of the tumor cells with TSNK cells. Optionally, tumor cells and / or TSNK cells are contacted with isolated placental perfusate or isolated placental perfusate cells. In another specific embodiment, tumor cells and / or TSNK cells are additionally contacted with an immunomodulatory compound, for example, an immunomodulatory compound described in Section 6.2.1, above, or thalidomide, so that the proliferation of tumor cells is detectably reduced when compared to tumor cells of the same type not contacted with TSNK cells. Optionally, tumor cells and / or TSNK cells contacted with an immunomodulatory compound are contacted with isolated placental perfusate or with isolated placental perfusate cells.
[000195] As used here, the term "contact", in relation to cells, in a modality encompasses direct physical contact, for example, cell-by-cell, between placental perfusate, placental perfusate cells, natural exterminating cells, for example , TSNK cells, and / or isolated combined natural killer cells and tumor cells. In another modality, "contact" encompasses the presence in the same physical space, for example, placental perfusate, placental perfusate cells, natural killer cells, for example, TSNK cells, and / or isolated combined natural killer cells and tumor cells are placed in the same container (for example, culture plate, multi-well plate) as tumor cells. In another modality, the "contact" between placental perfusate, placental perfusate cells, natural killer cells or natural placental killer cells and tumor cells is carried out, for example, by the injection or infusion of the placental perfusate or cells, for example, placental perfusate cells, combined natural killer cells or natural killer cells, for example, placental intermediate natural killer cells, in an individual, for example, a human comprising tumor cells, for example, a cancer patient.
[000196] In a specific embodiment, tumor cells are blood cancer cells, for example, leukemia cells or lymphoma cells. In more specific embodiments, cancer and acute leukemia, for example, acute T-leukemia cells, acute myeloid leukemia (AML) cells, acute pro-myelocytic leukemia cells, acute myeloblastic leukemia cells, acute megakarioblastic leukemia cells, precursor B acute lymphoblastic leukemia cells, precursor T acute lymphoblastic leukemia cells, Burkitt's leukemia (Burkitt's lymphoma) cells, or acute biphenotypic leukemia cells; a chronic leukemia, cells of, for example, chronic myeloid lymphoma cells, chronic myeloid leukemia (CML) cells, chronic monocytic leukemia cells, chronic lymphocytic leukemia cells (CLL), cells of mild lymphocytic lymphoma, or cells of or leukemia B-cell pro-lymphocyte; hair follicle lymphoma, T-cell pro-lymphocytic leukemia; or lymphoma cells, for example, histiocytic lymphoma cells, lymphoplasmic lymphoma cells (for example, Waldenstrom macroglobulinemia cells), marginal zone splenic lymphoma cells, plasma cell neoplasm cells (for example, myeloma cells of plasma cells, plasmacytoma cells, cells from a monoclonal immunoglobulin deposition disorder, cells from or a severe chain disorder), extranodal marginal zone B cell lymphoma cells (MALT lymphoma), nodal B cell lymphoma cells from marginal zone (NMZL), follicular lymphoma cells, mantle cell lymphoma cells, B-cell broad diffuse lymphoma cells, B-cell broad mediastinal (thymic) lymphoma cells, B-cell broad intravascular lymphoma cells, cells of primary effusion lymphoma, granular T-cell lymphocytic leukemia cells, aggressive NK cell leukemia cells, cell leukemia / lymphoma cells adult T, nasal extranodal NK / T cell lymphoma cells, enteropathy type T lymphoma cells, hepatosplenic T lymphoma cells, NK cell blast lymphoma cells, fungal mycosis cells (Sezary syndrome), a CD30-positive T cell lymphoproliferative disorder (for example, primary cutaneous anaplastic broad cell lymphoma or lymphomatoid papulosis), angioimmunobastic T lymphoma cells, peripheral T lymphoma cells, unspecified cells, anaplastic broad cell lymphoma cells , cells of a lymphocyte-predominant Hodgkin's lymphoma or a lymphocyte-predominant nodular Hodgkin's lymphoma. In another specific embodiment, the tumor cells are multiple myeloma cells or myelodysplasia syndrome cells.
[000197] In specific embodiments, tumor cells are solid tumor cells, for example, carcinoma cells, such as adenocarcinoma cells, adrenocortical carcinoma cells, colon adenocarcinoma cells, colorectal adenocarcinoma cells, colorectal carcinoma cells, cells ductal cell carcinoma, lung carcinoma cells, thyroid carcinoma cells, nasopharyngeal carcinoma cells, melanoma cells (for example, malignant melanoma cells), non-melanoma skin carcinoma cells, or non-melanoma carcinoma cells specified; desmoid tumor cells; rounded small cell desmoplastic tumor cells; endocrine tumor cells; Ewing's sarcoma cells; germ cell tumor cells (for example, testicular cancer, ovarian cancer, choriocarcinoma, endodermal bell tumor, germinoma, etc.); hepatoblastoma cells; hepatocellular carcinoma cells; neuroblastoma cells; non-rhabdomyosarcoma soft tissue sarcoma cells; osteosarcoma cells; retinoblastoma cells; rhabdomyosarcoma cells; or Wilms' tumor cells. In another embodiment, the solid tumor is pancreatic cancer or breast cancer. In other embodiments, solid tumor cells are pancreatic cancer or breast cancer cells. In other embodiments, the solid tumor cells are acoustic neuroma cells; astrocytoma cells (for example, pilocytic astrocytoma class I cells, low grade class II astrocytoma cells; class III anaplastic astrocytoma cells; or class IV glioblastoma multiforme cells); chordoma cells; craniopharyngioma cells; glioma cells (e.g., brain stem cell glioma cells; ependymoma cells; mixed glioma cells; optic nerve glioma cells; or subependimoma cells); glioblastoma cells; medulloblastoma cells; meningioma cells; metastatic brain tumor cells; oligodendroglioma cells; pineoblastoma cells; pituitary tumor cells; primitive neuroectodermal tumor cells; or schwannoma cells; In another embodiment, the tumor cells are prostate cancer cells.
[000198] As used herein, "therapeutically beneficial" and "therapeutic benefits" include, but are not limited to, for example, the reduction in the size of a tumor, decrease or cease the expansion of a tumor; reduction in the number of cancer cells in a tissue sample, for example, a blood sample, per unit volume; the clinical improvement in any particular cancer symptom or tumor that the individual itself has, the decrease or cessation of the worsening of any particular cancer symptom that the individual has, etc. 6.8.3. Cancer treatment using TSNK cells and other anticancer agents
[000199] Treatment of an individual with cancer using the TSNK cells described here may be part of an anti-cancer therapy regimen that includes one or more other anti-cancer agents. Such anti-cancer agents are known in the art. Specific anticancer agents that can be administered to an individual with cancer, for example, an individual with tumor cells, in addition to TSNK cells, and optionally perfusate, perfusate cells, natural killer cells other than TSNK cells, include, but are not limited to a: acivicin; aclarubicin; acodazole chloride; acronin; adozelesin; adrucil; aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperline; avastin (bevacizumab); azacytidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; byzelesin; bleomycin sulfate; brequinar sodium; bropyrimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin chloride; carzelesin; cedefmgol; celecoxib (COX-2 inhibitor); chlorambucil; cirolemycin; cisplatin; cladribine; chrysnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin chloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin chloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomitine hydrochloride; elsamitrucine; enloplatin; enpromate; epipropidine; epirubicin chloride; erbulozole; esorubicin chloride; stramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; ethoprine; fadrozole chloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; sodium fostriecin; gemcitabine; gemcitabine chloride; hydroxyurea; idarubicin chloride; i-phosphamide; ilmofosine; iproplatin; irinotecan; irinotecan chloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole chloride; lometrexol sodium; lomustine; losoxantrone chloride; masoprocol; maytansine; meclorethamine chloride; megestrol acetate; melengestrol acetate; melphalan; menogaryl; mercaptopurine; methotrexate; sodium methotrexate; methoprine; meturedepa; mitindomide; mitocarcin; mitochromine; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone chloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobromano; piposulfan; pyroxantrone chloride; plicamycin; plomestane; porfimer sodium; porphyromycin; prednimustine; procarbazine chloride; puromycin; puromycin chloride; pyrazofurin; riboprin; safmgol; safingol chloride; semustine; simtrazene; sodium sparfosate; sparsomycin; spirogermanium chloride; spiromustina; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; sodium tecogalana; taxotere; tegafur; teloxantrone chloride; temoporfm; teniposide; teroxirone; testolactone; tiamiprine; thioguanine; thiotepa; thiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole chloride; uracil mustard; uredepa; vapreotide; verteporfm; vinblastine sulfate; vincristine sulfate; vindesina; vindesine sulfate; vinepidine sulfate; vinglicinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicin chloride.
[000200] Other anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5- ethinyluracil; abiraterone; aclarubicin; acylfulvene; adecipenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrografolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenotic protein-1; anti androgen prostatic carcinoma; antiestrogen; antinaoplastone; antisense oligonucleotides; aphidicoline glycinate; apoptosis modulating genes; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azathyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisanthrene; bisaziridinylpermine; bisnafide; bistrathene A; byzelesin; breflate; bropyrimine; budotitane; butionin sulfoximine; calcipotriol; calphostin C; camptosar (also called Campto; irinotecan); camptothecin derivatives; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage-derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropine B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogues; clotrimazole; collismicin A; collismicin B; combretastatin A4; combretastatin analogue; conagenin; crambescidine 816; chrysnatol; cryptoficin 8; cryptoficin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cipemicin; cytarabine ocphosphate; citolitic factor; cytostatin; dacliximab; decitabine; dehydrodidenmin B; deslorelin; dexamethasone; dexiphosphamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol; duocarmicina SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; element; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopyridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texafirina; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramanton; ilmofosine; ilomastat; imatinib (for example, GLEEVEC®), imiquimod; immunostimulating peptide; insulin-like growth factor receptor-1 inhibitor; interferon agonists; interferons; interleukins; iobengguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladina; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; alpha leukocyte interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lysoclinamide 7; lobaplatin; lombricinaa; lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; texafirine lutetium; lysophylline; lytic peptides; maytansine; Mannostatin A; marimastat; masoprocol; maspin; matrilisin inhibitors; matrix metalloproteinase inhibitors; menogaryl; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogs; mitonafide; fibroblast growth factor mitotoxin-saporin; mitoxantrone; mopharotene; molgramostim; Erbitux (cetuximab), human chorionic gonadotropin; sk cell wall of monophosphoryl A + lipid mycobacterium; mopidamol; mustard anti-cancer agent; micaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinalin; N-substituted benzamides; afarelin; nagrestip; naloxone + pentazocine; napavine; naphterpine; nartograstim; nedaplatin; nemorubicin; nerhydronic acid; nilutamide; nisamycin; nitric oxide modulators; antioxidant; nitrulin; oblimersen (GENASENSE®); O6-benzylguanine; octreotide; ocicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin (for example, Floxatin); oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrizoxin; pamidronic acid; panaxitriol; panomiphene; parabactin; pazeliptin; pegaspargase; peldesin; sodium polysulfate pentosan; pentostatin; pentrozole; perflubrone; perfosfamide; perilyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine chloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porphyromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; immunity modulator based on protein A; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; glitter; pyrazoloacridine; polyoxyethylene pyridoxylated hemoglobin conjugate; raph antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; demethylated reteliptin; rhenium ethydronate Re 186; rhizoxin; ribozymes; Retinamide RII; roituquine; ruptured; roquinimex; rubiginone Bl; ruboxil; safmgol; saintopine; SarCNU; sarcofitol A; sargramostim; Sdi 1 mimetics; semustine; inhibitor derived from senoscense 1; sensitive oligonucleotides; signal transduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin-binding protein; sonarmine; sparphosic acid; spicamycin D; spiromustina; splenopentin; spongistatin 1; squalamine; stipiamide; stromelysin inhibitors; sulfmosin; superactive vasoactive intestinal peptide antagonist; suradist; suramin; swainsonin; talimustine; methylated tamoxifen; tauromustine; tazarotene; sodium tecogalana; tegafur; tellurapyril; telomerase inhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine; taliblastin; thiocoralin; thrombopoietin; mimetic thrombopoietin; timalfasin; thymopoietin receptor agonist; timotrinan; thyroid stimulating hormone; tin ethylethiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tirfostines; UBC inhibitors; ubenimex; growth inhibitory factor derived from urogenital bell; urokinase receptor antagonists; vapreotide; variolin B; Vectibix (panitumumab) velaresol; veramine; verdinas; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; Welcovorin (leucovorin); Xeloda (capecitabine); zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. 6.8.4. Treatment of viral infection
[000201] In another embodiment, a method is provided here for treating an individual with a viral infection, comprising administering to the individual itself a therapeutically effective amount of TSNK cells. In certain embodiments, the individual has a deficiency of natural killer cells, for example, a deficiency of NK cells active against the individual's viral infection. In certain specific embodiments, the administration itself further comprises administering to the individual one or more of isolated placental perfusate, isolated placental perfusate cells, isolated natural killer cells, for example, natural placental killer cells, for example, natural killer cells placenta-derived intermediates, isolated combined natural killer cells, and / or combinations thereof. In certain specific embodiments, TSNK cells are contacted with an immunomodulatory compound, for example, an immunomodulatory compound described in Section 6.2.1, above, or thalidomide, prior to the actual administration. In certain other specific embodiments, the administration itself comprises the administration of an immunomodulatory compound, for example, an immunomodulatory compound described in Section 6.2.1, above, or thalidomide, to the individual itself in addition to the TSNK cells themselves, where the amount itself is an amount that, for example, results in a detectable improvement in, decrease in the progression of, or elimination of, one or more symptoms of the viral infection itself. In specific modalities, viral infection is an infection by a virus from the families Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papilommaviridae, Rhabdoviridae, or Togaviridae. In more specific ways, the virus itself is the human immunodeficiency virus (HIV), coxsackievirus, hepatitis A virus (HAV), poliovirus, Epstein-Barr virus (EBV), herpes simplex type 1 (HSV1), herpes simplex type 2 (HSV2), human cytomegalovirus (CMV), human herpesvirus type 8 (HHV8), herpes zoster virus (varicella zoster virus (VZV) or shingles virus), hepatitis B virus (HBV), hepatitis C virus ( HCV), hepatitis D virus (HDV), hepatitis D virus (HEV), influenza virus (e.g., influenza A virus, influenza B virus, influenza C virus, or thogotovirus), rubella virus, parotitis virus, parainfluenza virus, papillomavirus, rabies virus, or rubella virus.
[000202] In other more specific modalities, the virus itself is adenovirus species A, serotype 12, 18 or 31; adenovirus species B, serotype 3, 7, 11, 14, 16, 34, 35 or 50; adenovirus species C, serotype 1, 2, 5 or 6; species D, serotype 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, or 51; species E, serotype 4; or species F, serotype 40 or 41.
[000203] In certain other more specific modalities, the virus is the Apoi virus (APOIV), Aroa virus (AROAV), bagaza virus (BAGV), Banzi virus (BANV), Bouboui virus (BOUV), Cacipacore virus (CPCV), Carey Island virus (CIV), Cowbone Ridge virus (CRV), dengue virus (DENV), Edge Hill virus (EHV), Gadgets Gully virus (GGYV), Ilheus virus (ILHV), Israeli Turkish meningoencephalomyelitis virus (ITV) , Japanese encephalitis virus (JEV), Jugra virus (JUGV), Jutiapa virus (JUTV), kadam virus (KADV), Kedougou virus (KEDV), Kokobera virus (KOKV), Koutango virus (KOUV), forest disease virus Kyasanur (KFDV), Langat virus (LGTV), Meaban virus (MEAV), Modoc virus (MODV), Montana myotic leukoencephalitis virus (MMLV), Murray Valley encephalitis virus (MVEV), Ntaya virus (NTAV), virus Omsk hemorrhagic fever (OHFV), Powassan virus (POWV), Rio Bravo virus (RBV), Royal Farm virus (RFV), Saboya virus (SABV), St. Louis encephalitis virus (SLEV), Sal Vieja virus (SVV) , San Perlit virus a (SPV), Saumarez Reef virus (SREV), Sepik virus (SEPV), Tembusu virus (TMUV), thick strain encephalitis virus (TBEV), Tyuleniy virus (TYUV), Uganda S virus (UGSV), Usutu virus (USUV ), Wesselsbron virus (WESSV), West Nile virus (WNV), Yaounde virus (YAOV), yellow fever virus (YFV), Yokose virus (YOKV), Zika virus (ZIKV).
[000204] In other embodiments, TSNK cells, and optionally placental perfusate and / or placental perfusate cells, are administered to an individual with a viral infection as part of an antiviral therapeutic regimen that includes one or more additional antiviral agents. Specific antiviral agents that can be administered to an individual with a viral infection include, but are not limited to, imiquimod, podofilox, podophyllin, interferon alfa (IFNa), reticolos, nonoxynol-9, acyclovir, famciclovir, valacyclovir, ganciclovir, cidofovir; amantadine, rimantadine; ribavirin; zanamavir and oseltaumavir; protease inhibitors such as indinavir, nelfmavir, ritonavir, or saquinavir; nucleoside reverse transcriptase inhibitors such as didanosine, lamivudine, stavudine, zalcitabine, or zidovudine; and non-nucleoside reverse transcriptase inhibitors such as nevirapine, or efavirenz. 6.8.5. Management
[000205] Determining the number of cells, for example, placental perfusate cells, for example, nucleated cells of placental perfusate, combined natural killer cells, and / or isolated natural killer cells, for example, TSNK cells, and the determination of amount of an immunomodulatory compound, for example, an immunomodulatory compound described in Section 6.2.1, above, or thalidomide, can be performed independently of one another. 6.8.5.1. Cell administration
[000206] In certain embodiments, TSNK cells are used, for example, administered to an individual, in any quantity or number that results in a detectable therapeutic benefit to the individual, for example, an effective quantity, where the individual has a viral infection, cancer, or tumor cells, for example, an individual with tumor cells, a solid tumor, or a blood cancer, for example, a cancer patient. Such cells can be administered to the individual itself in about, at least about, or even about, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108 , 5 x 108, 1 x 109, 5 x 109, 1 x 10 10, 5 x 10 10, or 1 x 10 11 TSNK cells. In other embodiments, TSNK cells can be administered to the individual itself by relative numbers of cells, for example, the individual itself can be administered about, at least about, or even about, 1 x 105, 5 x 105 , 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 10 10, 5 x 10 10, or 1 x 10 11 TSNK cells per kilogram of individual. TSNK cells can be administered to the individual itself according to an approximate ratio between a number of TSNK cells, and optionally placental perfusate cells and / or natural killer cells other than TSNK cells, and a number of tumor cells in the individual itself (for example, an estimated number). For example, TSNK cells can be administered to the individual itself in a ratio of about, at least about, or even about 1: 1, 2: 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 in relation to the number of tumor cells in the individual. The number of tumor cells in such an individual can be estimated, for example, by counting the number of tumor cells in a tissue sample of the individual, for example, blood sample, biopsy, or the like. In specific modalities, for example, for solid tumors, the count itself is performed in combination with the image of the tumor or tumors to obtain an approximate volume of tumors. In a specific embodiment, an immunomodulatory compound or thalidomide, for example, an effective sample of an immunomodulatory compound or thalidomide, is administered to the individual in addition to TSNK cells, optionally placental perfusate cells and / or natural killer cells other than TSNK cells.
[000207] In certain embodiments, the method for suppressing the proliferation of tumor cells, for example, in an individual; treating an individual with a deficiency in the individual's natural killer cells; or treating an individual with a viral infection; or treatment of an individual with cancer, for example, an individual with tumor cells, a blood cancer or a solid tumor, comprises contacting the tumor cells, or administering to the individual itself a combination of TSNK cells and one or more among placental perfusate and / or placental perfusate cells. In specific embodiments, the method further comprises contacting the tumor cells with, or administering to the individual, an immunomodulatory compound or thalidomide.
[000208] In a specific embodiment, for example, the treatment of an individual with a deficiency in the individual's natural killer cells (for example, a deficiency in the number of NK cells or in the reactivity of NK cells to a cancer, tumor or infected cells with viruses); or the treatment of an individual with a cancer or a viral infection, or the suppression of the proliferation of tumor cells, comprises the contact of the tumor cells themselves, or the administration to said individual, of TSNK cells supplemented with isolated placental perfusate cells, or placental perfusate. In specific embodiments, about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more TSNK cells, are supplemented with about, or at least about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106 , 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more isolated placental perfusate cells per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106 , 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more placental perfusate cells. In other more specific embodiments, about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more cells TSNK per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more TSNK 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 perfusate unit.
[000209] In another specific modality, the treatment of an individual with a deficiency in the individual's natural killer cells; the treatment of an individual with cancer; treating an individual with a viral infection; or suppressing the proliferation of tumor cells, comprises the contact of the tumor cells with, or the administration to the individual, TSNK cells, where the TSNK cells themselves are supplemented with adherent placental cells, for example, stem cells or placental multipotent cells adherent, for example, placental cells adhering to CD34-, CD10, CD105 +, CD200 + tissue culture plastic. In specific embodiments, TSNK cells are supplemented with about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more adherent placental stem cells per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more adherent placental cells, for example, stem cells or adherent multipotent placental cells,
[000210] In another specific modality, the treatment of an individual with a deficiency in the individual's natural killer cells; the treatment of an individual with cancer; treating an individual with a viral infection; or suppression of tumor cell proliferation, is carried out using an immunomodulating compound or thalidomide in combination with TSNK cells, where the TSNK cells themselves are supplemented with conditioned medium, for example, conditioned medium by placental cells adhering to culture plastic of CD34-, CD10, CD105 +, CD200 + fabrics, for example, 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.1; 0.8; 0.9; 1; two; 3; 4; 5; 6; 7; 8; 9; 10 ml of culture medium conditioned by stem cells per perfusate unit, or by 104, 105, 106, 107, 108, 1010, or 1011 TSNK cells. In certain embodiments, placental cells adhering to tissue culture plastic are the multipotent adherent placental cells described in U.S. Patent No. 7,468,276 and in Patent Application Publication No. 2007/0275362, the descriptions of which are incorporated herein in full by reference. . In another specific embodiment, the method further comprises contacting the tumor cells, or administering to the individual, an immunomodulatory compound or thalidomide.
[000211] In another specific modality, the treatment of an individual with a deficiency in the individual's natural killer cells; the treatment of an individual with cancer; treating an individual with a viral infection; or suppressing the proliferation of tumor cells, in which TSNK cells are supplemented with placental perfusate cells, the perfusate cells are contacted with interleukin-2 (IL-2) for a period of time before the actual contact. In certain embodiments, the time period itself is about at least, or up to 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 hours before the actual contact.
[000212] TSNK cells, and optionally perfusate or perfusate cells, can be administered once to an individual with a viral infection, an individual with cancer, or an individual with tumor cells, during the course of an anticancer therapy; or they can be administered multiple times, for example, 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, 24, 36 or more weeks during therapy. In embodiments in which cells and an immunomodulatory compound or thalidomide are used, the immunomodulatory compound or thalidomide, and the cells or perfusate, can be administered to the individual together, for example, in the same formulation; separately, for example, in separate formulations, at approximately the same time; or they can be administered separately, for example, in different dosing schedules or at different times of the day. Similarly, in modalities in which the cells and an antiviral compound or anticancer compound are used, the antiviral compound or the anticancer compound, and the cells or the perfusate, can be administered to the individual together, for example, in the same formulation; separately, for example, in separate formulations, at approximately the same time; or they can be administered separately, for example, in different dosing schedules or at different times of the day. TSNK cells, and perfusate or perfusate cells, can be administered regardless of whether TSNK cells, perfusate, or perfusate cells have been administered to the individual in the past or not. 7. EXAMPLES 7.1. Example 1: Recovery of hematopoietic stem cells from human placental perfusate and umbilical cord blood
[000213] Human placental perfusate (HPP) and umbilical cord blood (UCB) cells were generally purified using Ficoll or ammonium chloride to obtain total nucleated cells (TNCs). The TNCs were then used in a positive selection procedure to isolate CD34 + cells using anti-CD34 beads and RoboSep according to the manufacturer's protocol (StemCell Technologies, Inc.). In this experiment, CD34 + cells were isolated with purity greater than 90%. Alternatively, an EasySep® human progenitor cell enrichment kit (StemCell Technologies, Inc.) was used in a negative selection procedure to remove streaked cells using human progenitor cell enrichment cocktail with monoclonal antibodies for the following surface antigens of human cells: CD2, CD3, CD11b, CD11c, CD14, CD 16, CD 19, CD24, CD56, CD66b and Glycophorin A. Using the negative selection, 90% of CD34 + cells were recovered from the raw materials. The cellular composition of the recovered HSCs was summarized in Table 1. Table 1. Cellular composition of the enriched hematopoietic stem cells (HSCs). The standard deviation was calculated from the population averages of 3 donors.
7.2. Example 2: Feed cell-free expansion and differentiation
[000214] CD34 + cells were cultured in the following medium formulations for up to 48 days, and aliquots of cells were removed for evaluations of cell count, cell viability, characterization of differentiation of natural killer cells and functional evaluation.
[000215] NK1 medium: GBGM (Glycostem-based growth medium, Glycostem Cat # CCT-SCB500, Clear cell technology) supplemented with 1% pen / strep (Cat # 15140, Gibco), 20 ng / mL SCF (Cat # 255-SC, R&D Systems), 10 ng / mL Flt-3 ligand (Cat # 308-FK, R&D system), 20 ng / mL TPO (Cat # 288-TP, R&D system), 20 ng / mL IL-7 (Cat # 207-IL, R&D Systems), 200 IU / mL IL-2 (Cat # 202-IL, R&D Systems) and 10 ng / mL IL-15 (Cat # 247-IL, R&D Systems).
[000216] NK2 medium: DMEM (Cat # MT-10-013-CV, Fisher); Ham's F12 (Cat # BW12-615F, Fisher) in 1: 2 supplemented with 2 mM L-Glutamine (Cat # 25030, Invitrogen), 1% pen / strep, 20% AB human serum (Cat # 100-512 , Gemcell), 5 ng / mL sodium selenide (Cat # S9133, Sigma), 50 μM ethanolamine (Cat # E0135, Sigma), 25 μM β-mercaptoethanol (Cat # 21985, Invitrogen), 20 mg / mL ascorbic acid (Cat # 47863, Sigma), 5 ng / mL IL-3 (Cat # 203-IL, R&D Systems), 20 ng / mL SCF, 10 ng / mL Flt-3 ligand, 20 ng / mL of IL-7 and 10 ng / ml of IL-15.
[000217] NK3 medium: X-vivo 20 (Cat # BW04-448Q, Fisher) supplemented with 1% pen / strep, 10% human AB serum (Cat # 100-512, Gemcell) and 500 IU / mL IL -two.
[000218] NK2A medium: GBGM supplemented with 10% human serum AB, 1% pen / strep, 20 ng / ml SCF, 10 ng / ml Flt-3 linker, 20 ng / ml Tpo, 20 ng / ml IL-7, 200 IU / ml IL-2, 10 ng / ml IL-15 and 1.5 IU / ml heparin (Cat # H3149, Sigma).
[000219] NK2B1 medium: DMEM: Ham's F12 in 1: 2 supplemented with 2 mM L-glutamine, 1% pen / strep, 20% human serum AB, 5 ng / mL sodium selenide, 50 μM ethanolamine , 25 μM β-mercaptoethanol, 20 μg / mL ascorbic acid, 5 ng / mL IL-3, 20 ng / mL SCF, 10 ng / mL Flt-3 ligand, 20 ng / mL IL-7 and 10 ng / ml IL-15.
[000220] NK2B2 medium: DMEM: Ham's F12 in 1: 2 supplemented with 2mM L-glutamine, 1% pen / strep, 20% human serum AB, 5 ng / mL sodium selenide, 50 μM ethanolamine, 25 μM β-mercaptoethanol, 20 μg / mL ascorbic acid, 200 IU / mL IL-2, 20 ng / mL SCF, 10 ng / mL Flt-3 ligand, 20 ng / mL IL-7 and 10 ng / ml IL-15.
[000221] NK2C medium: RPMI 1640 (Cat # 22400105, Invitrogen) supplemented with 10% FBS (Cat # SH30070.03, Hyclone), 2 mM L-glutamine, 1% pen / strep, 50 ng / mL of SCF, 50 ng / ml Flt-3 linker, 100 IU / ml IL-2, 20 ng / ml IL-7 and 20 ng / ml IL-15.
[000222] NK2D medium: serum-free medium (StemSpan, Cat # 09650, Stem Cell Technologies, Vancouver, Canada) supplemented with 1 μM synthetic glucocorticoid dexametosone (Dex, Cat # D4902, Sigma, St Louis, MO), 40 ng / mL insulin-like growth factor 1 (IGF-1, Cat # 291-G1-250, R&D Systems, Minneapolis, MN), 100 ng / mL SCF, 40 μg / mL lipids (rich lipid mixture in cholesterol; Cat # C7305-1G, Sigma, St Louis, MO), 5 ng / ml IL-3, 200 IU / ml IL-2, 20 ng / ml IL-7 and 20 ng / ml IL -15.
[000223] The cells collected at different times were washed twice with RPMI 1640 (free from phenol) and 5% FBS, stained with fluorescence conjugated antibodies (Tables 2 and 3) for 15 minutes at 4 ° C, and analyzed by flow cytometry (FACSCanto, BD) and FlowJo cytometry software (Tree Star). Table 2. Antibodies used for cell labeling


[000224] Cytotoxicity assay using PKH26 / TO-PRO-3 labeling. The target tumor cells were labeled with PKH26 (Cat # PKH26GL, Sigma-Aldrich), a dye that inserts into the plasma membrane of the cells through its lipophilic aliphatic residue, and then placed in tissue culture plates with a U-shaped bottom. 96 wells and incubated with expanded NK cells, in various target agent ratios (E: T) in 200 μL of RPMI 1640 supplemented with 10% FBS. The cultures were incubated for 4 hours at 37 ° C in 5% CO2. After incubation, cells were collected and TO-PRO-3 (Invitrogen Cat # T3605), a membrane-impervious DNA dye, was added to the cultures (1 μM final concentration) followed by FACS analysis. Cytotoxicity was expressed as the percentage of cells (PKH26 + TO-PRO-3 +) among the total PKH26 + target tumor cells.
[000225] Optimization of expansion and differentiation of CD34 + cells into NK cells. Among the tested media formulations, the NK1, NK2 and NK3 media, the NK1 media showed a 500-fold expansion on the 21st Day (D21). Both the NK2 and NK3 media maintained cell proliferation and differentiation. The further optimization of the medium was carried out for the NK1 medium and the subsequent media were named NK2A, NK2B, NK2C and NK2D. CD34 + cells cultured with the NK2A medium showed 105-fold expansion on Day 55 (D55). Based on the results of the relative expansion, differentiation, and cytotoxicity of NK1, 2 and 3 media, the second batch of NK2A, NK2B, NK2C and NK2D media formulations was made and in D55, a 105-fold expansion was achieved (as shown in Figure 1). The NK2B medium showed an expansion of approximately 3 x 104. Culture in the NK2C medium resulted in an expansion of 3 x 102 times in 21 days, followed by declining cell viability. The NK2D medium did not maintain the cells for the duration of the experiment.
[000226] On the 48th Day (D48), about 90% of the NK cells in the NK2A medium were CD56 + CD3-. Within the CD56 + CD3- population, over 98% of the cells were CD56 + CD16- (as shown in Figure 2), while 58% expressed the NKG2D activation receptor, 68% were NKp46 + and 17% were CD226 + (as shown in Tables 4A and 4B). Tables 4A, 4B. Phenotypic characterization of NK cells expanded in D48

[000227] Additionally, 97.8% of the cells cultured in the NK2A medium and 93.1% of the cells cultured in the NK2B medium were CD56 + CD16- on day 21 of culture. 7.3. Example 3: Culture of NK cells in CNK medium improves the expansion and cytotoxicity of NK cells
[000228] On the 27th Day (D27), CD34 + cells grown in the NK2A medium were further grown in one of the following media:
[000229] Two-stage medium, comprising CNK half and maintenance half
[000230] CNK medium is composed of IMDM (Invitrogen) supplemented with 10% FCS (Hyclone), 200 IU / mL IL-2 (R&D Systems), 35 μg / mL transferrin (Sigma-Aldrich), 5 μg / mL insulin (Sigma-Aldrich), 2 x 10-5 M ethanolamine (Sigma-Aldrich), 1 μg / mL oleic acid (Sigma-Aldrich), 1 μg / mL linoleic acid (Sigma-Aldrich), 0.2 μg / mL of palmitic acid (Sigma-Aldrich), 2.5 μg / mL of BSA (Sigma-Aldrich) and 0.1 μg / mL of phytohemagglutinin (PHA-P, Sigma-Aldrich).
[000231] NK CD56 + CD3 cells cultured in NK2A medium were resuspended at 2.5 x 105 live cells / mL CNK medium in 24-well plates treated with cell culture or T flasks. Allogeneic PBMC and K562 cells treated with mitomycin C (cell line with chronic myeloid leukemia) were both added to the CNK medium as feed cells, until a final concentration of 1 x 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 serum AB, antibiotics, L-glutamine and 400 units of IL-2 per ml) was added to the culture.
[000232] NK2A medium (PDAC) with placental stem cells adhering to cell culture plastic, CD34-, CD10 +, CD105 +, CD200 +, treated with mitomycin C, as feed cells;
[000233] NK2A medium (MSC) with mesenchymal stem cells (MSC) treated with mitomycin C as feed cells; or
[000234] Feed cell-free (FF) NK2A medium as a control.
[000235] The two-step medium improved the relative expansion of CD34 + cells when compared to the NK2A (FF), NK2A (PDAC) and NK2A (MSC) media, particularly between the 27th Day (D27) and the 48th Day (D48 ). See Figure 3.
[000236] On the 35th Day, the proportion of CD34 + cells had already decreased to approximately 4% while the proportion of CD56 + CD3- cells increased to approximately 80% in the middle of two stages. On Day 45 (D45), cells cultured in the middle of two stages showed greater toxicity when compared to NK cells cultured in the media NK2A (FF), NK2A (PDAC) and NK2A (MSC) (as shown in Figure 3). Phenotypic characterization on Day 41 (as shown in Figure 5) showed increased expression of NKp46 and CD226 in cells, indicating a possible explanation for the increase in cytotoxicity. In D41 the proportion of CD226 + cells increased from 0.9% ± 0.8% in the NK2A medium to 13% ± 4% in the two-stage medium; the proportion of NKp46 cells increased from 55.4% ± 8.7% in the NK2A medium to 80% ± 7.85% in the two-stage medium. At D48 the proportion of CD226 + cells increased from 17.3% ± 14.3% in the NK2A medium to 52.3% ± 11.64% in the middle of two stages; the proportion of NKp46 + cells increased from 67.9% ± 5.4% in the NK2A medium to 86% ± 4% in the two-stage medium. There was no significant difference in NKG2D expression between the conditions tested. The changes in CD226 and NKp46 expressions are shown in Table 5, below. Table 5. Expression of CD226 and NKp46 in NK cells cultured in NK2A (FF) and two-stage media on Day 41 (D41) and Day 48 (D48). The standard deviation was calculated for 3 donors.
7.4. Example 4: Comparison between natural killer cells grown in two-stage medium and natural cells derived from embryonic stem cells (ESCs)
[000237] NK cells cultured in the middle of two stages were compared with Nk cells derived from embryonic stem cells (ESCs), which were produced by the method of Woll et al., Blood 113 (4), 6094-6101 (2009 ). Specifically, a difference in the expression levels of CD94 and CD117 was observed during the culture process of both cell types. Figure 6 shows that CD117 expression was higher in two-stage NK cells, or “+”, from Day 7 (D7) to Day 35 (D35), while CD94 expression increased gradually. On Day 35 (D35), about 44% of CD56 + CD3- cells (two steps) were CD94 + CD117 + cells, 37.6% of CD56 + CD3- cells were CD94-CD117 + and 14.7% of CD56 cells + CD3- were CD94 + CD117-. In this way, NK cells produced by the two-step method are distinguishable from NK cells derived from ESCs, 78% of which remained CD117 low / - from the 14th to the 35th day of cultivation. This difference in CD117 expression is useful because NK CD117 + cells are cytotoxic against tumor cells from various tissues, as described in Example 6, below.
[000238] These results suggest that the progression of differentiation of TSNK cells is different from that of ESC-derived NK cells, and that TSNK cells are distinguishable from ESC-derived NK cells. 7.5. Example 5: PDACs improve the relative expansion of natural killer cells grown in NK2A medium
[000239] To assess the effect of placental stem cells adhering to CD10 +, CD34-, CD105 +, CD200 + tissue culture plastics (referred to in this example as PDACs) on the differentiation of hematopoietic stem cells (HSC) into natural killer cells, HSCs were stimulated with PDACs treated with mitomycin C or bone marrow-derived mesenchymal stem cells (MSC) in a 10: 1 ratio PDACs / MSC: HSC on Day 0 and Day 21, while a culture free of feed cells was used as a control. NK2A medium was used as a culture medium. PDACs have been shown to increase the relative expansion of cultured NK cells when compared to the medium alone. However, no significant difference in toxicity was found between cells cultured with or without the food layer. The cells treated with MSC showed the greatest relative expansion, but the least toxicity, as shown in Figure 7. 7.6. Example 6: Cytotoxic activity of expanded NK cells using two-stage medium
[000240] This example demonstrates that NK cells produced from expanded and differentiated CD34 + cells using the two-stage process described above are cytotoxic to tumor cell lines.
[000241] Lactate dehydrogenase (LDH) release assay. The LDH release assay was performed using the CYTOTOX 96® non-radioactive cytotoxicity assay kit (Promega, Cat # G1780). In this assay, cultured NK cells derived from human donor-compatible placental perfusate (HPP) and umbilical cord blood units (Combos units) were used as agent cells, while cells from a certain tumor cell line were used as target cells. Of the three units used in this study, the percentage of HPP cells was 56.6% ± 28.3%. The agent cells and the target cells were placed in 96-well U-bottom tissue culture plates and incubated in various target agent (E: T) ratios in 100 μL of RPMI 1640 without phenol red (Invitrogen, Cat # 11835-030) supplemented with 2% AB human serum (Gemini, Cat # 100-512). The cultures were incubated for 4 hours at 37 ° C in 5% CO2. After incubation, 50 μL of the supernatant was transferred to an enzyme assay plate, LDH activity was detected as provided by the manufacturer, and absorption was measured at 490 nm on an ELISA reader (Synergy HT, Biotek). Cytotoxicity was calculated according to the following equation:% Cytotoxicity = (Sample - Spontaneous agent - Spontaneous target) / (Maximum target - Spontaneous target) * 100, where "Spontaneous agent" is a control for spontaneous release of LDH from agent cells; "Spontaneous target" is a control for spontaneous release of LDH from target cells; and "Maximum target" is a control for maximum LDH release when essentially 100% of the cells undergo lysis.
[000242] Isolation of CD34 + cells from human placental perfusate (HPP) and CD34 + cells from umbilical cord blood (UCB). HPP and UCB cells were purified using Ficoll or ammonium chloride to obtain total nucleated cells (TNCs). The TNCs were then used in a positive selection procedure to isolate CD34 + cells using anti-CD34 beads and RoboSep following the protocol provided by the manufacturer (StemCell Technologies, Inc.). In this experiment, CD34 + cells were isolated with purity greater than 90%.
[000243] Study of the susceptibility of tumor cells to NK cells cultured in two stages. Tumor cell lines (Table 1), including human breast cancer (HCC2218), human colorectal adenocarcinoma (HT-29), human chronic myeloid leukemia (CML), human acute myeloid leukemia (AML), human glioblastoma (LN-18 and U-118MG), human multiple myeloma (U266), human histiocytic lymphoma (U937), and human retinoblastoma (WERI-RB-1) were co-cultured with two-stage NK cells. NK cells grown in two stages included those grown in NK2A medium for 21 days, then grown in CNK medium for 21 days, and those grown in NK2A medium for 28 days and then in CNK medium for 14 days. The cytotoxicity of NK cells was measured by the lactate dehydrogenase (LDH) release test after co-cultivation for 4 hours. At target agent (E: T) ratios of 10: 1, the latter showed, in general, greater cytotoxicity than the former (Table 6). Of the tumor cell lines, LN-18 was the most susceptible to NK-mediated extermination, followed by K562, U937, WERI-RB-1, U-1 18MG, HT-29, HCC2218, KG-1 and U266.
[000244] TSNK cells, therefore, showed significant cytotoxicity against several cancer cell lines. It is also apparent that the cytotoxicity of NK can be increased by extending the culture period in the NK2A medium from 21 days to 28 days. Table 6. Cytotoxicity of TSNK cells cultured targeting tumor cell lines
1. n: number of donors
[000245] MicroRNA characterization of human placental perfusate (HPP) CD34 + cells and umbilical cord blood (UCB) CD34 + cells. CD34 + HPP and UCB cells purified from a compatible donor were subjected to microRNA (miRNA) preparation using a MIRVANA ™ miRNA isolation kit (Ambion, Cat # 1560). CD34 + cells (0.5 to 1.5 x 10 6 cells) were disrupted in a denaturing lysis buffer. The samples were then subjected to acid extraction with phenol + chloroform to isolate highly enriched RNA for species with short RNA. 100% ethanol was added to bring the samples to 25% ethanol. When this lysate / ethanol mixture was passed through a glass fiber filter, species with long RNA were immobilized, and species with short RNA 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 short RNAs were immobilized. This RNA was washed a few times, and eluted in a solution with low ionic strength. The concentration and purity of the short RNA was determined by measuring its absorption at 260 and 280 nm. MiRNAs, which were shown to be unique for HPP CD34 + cells in all donors tested (n = 3), included hsa-miR-380, hsa-miR-512, hsa-miR-517, hsa-miR-518c, hsa-miR -519b, and hsa-miR-520a. 7.7. Example 7: Isolation of CD34 + cells from mixed UCB and HPP
[000246] This example demonstrates the isolation of CD34 + cells from umbilical cord blood (UCB) and human placental perfusate (HPP) mixed (Combo). To assess the mixing ratio of UCB: HPP, side-by-side comparisons of 3 different mixing ratios were performed as follows: (1) Total mixing: 1 x UCB (total volume) + 1 x HPP (total volume); (2) Partial mixture of HPP (33%): 1 x UCB (total volume) + 0.33 x HPP (1/3 of the HPP volume); and (3) Partial mixing of the HPP (10%): 1 x UCB (total volume) + 0.10 x HPP (1/10 of the HPP volume). A total of N = 3 experimental replicates were performed. The TNC and the initial volumes were checked. The mixed samples were then purified for CD34 + cells and CD34 + purity was determined after thawing. The optimal mixing ratio was then determined graphically from the CD34 + purity graphs after thawing as a function of volumetric fractions (as a% Combo) or cell count (TNC). As shown in Figure 8, the CD34 + purity at the end point correlates well with the HPP volumetric content, but not as much as the HPP TNC content. In general, UCB 85% v / v, HPP 15% v / v proved to be the optimal mixing ratio to obtain CD34 + cells with a purity equal to or greater than 80%. 7.8. Example 8: Comparison of NK cell culture using GBGM®-based medium
[000247] This example demonstrates the comparison of NK cell culture processes using two GBGM-based media (the three-stage process and the two-stage process). The two processes are summarized in Table 7. Both processes used GBGM as the baseline for the differentiation of NK cells and CD34 + cells from placenta. Table 7. Summary of three-stage and two-stage processes

[000248] The experimental parameters are summarized as follows: Lots of donors: CD34 + cells of fresh UCB: N = 6 CD34 + cells of fresh "Combo": N = 2 CD34 + cells of cryopreserved "Combo": N = 8 Scale: multi-well plates for T-25, up to multiple T-75 flasks, 1 to 80 by volume of culture.
[000249] Processing methods: Two-stage process Three-stage process Use of feed cells: Without feed cells With inactivated K562 & PBMC feed cells, added to the culture on Day 21 Inoculation density: 20000 - 50000 cells / mL
[000250] CD34 + cells from fresh UCB or fresh combo were generated and cryopreserved with methods described in Examples 7, 10 and 11. Cultures were kept in an incubator at 37 ° C,> 90% humidity, and 5% humidity. CO2. Cell growth was monitored throughout the process (by cell count) and medium changes were performed twice a week to maintain cell concentrations within the range of 5 x 104 - 1 x 106 per mL. Differentiation was monitored by phenotypic analysis on the 21st and 35th days. When feed cells were used, on Day 21 of the NK culture, fresh K562 cells and post-freeze alo-PBMC, cultured for 3 days, were inactivated with mitomycin-C (16 μg / mL, 2 hours, 37 ° C) and added to the conditions of the two-stage process at 1 x 106 per mL. Differentiated NKs were normalized to 0.5 x 106 per ml. On Day 35, after final cell counts were performed, a cytotoxicity assay based on flow cytometry using freshly cultured K562 cells and labeled with PKH (10: 1 E: T ratio, 4 hours, 37 ° C) was performed to assess NK functionality.
[000251] Results:
[000252] The median relative expansion of TNCs for fresh UCB-derived CD34 + cells using both processes was comparable on Day 35. The two-stage process appeared to result in a greater relative expansion of TNC than the three-stage process for fresh combo derived CD34 + cells. The relative expansions of TNC, in general, were comparable for CD34 + cells derived from post-thaw combo using both processes, but were significantly smaller than those derived from fresh UCB or fresh combo. In all cases, both the three-stage and two-stage processes resulted in similar cell contents at the end of cultivation.
[000253] On Day 21, there were no notable phenotype differences in cells originating from UCB or combo. The two-stage process resulted in a higher percentage of CD56 + CD3- NK cells (on average, 14.7%) than the three-stage process (on average, 6.1%). The extent of differentiation (for example, CD56 + CD3- level) appeared to be donor dependent. The percentage of CD3 + CD56- (T cells) and CD3 + CD56 + (NKT-like cells) populations were minimal in all cases.
[000254] On the 35th Day, both processes proved to be effective for the differentiation of NK cells, as evidenced by the final purity levels of CD56 + CD3- high (87.2% and 90.1% for the two and three-stage, respectively). The addition of feed cells in the two-stage process appeared to increase NK phenotypic purity (75.3% without feed cells; 87.2% with feed cells), while no benefit from feed cells in NK purity was observed ( 90.1% without feed cells; 84.7% with feed cells) with the three-stage process.
[000255] Cultured NK cells maintained their CD16- phenotype throughout the process. The two-step process appeared to result in slightly more CD56 + CD3 + cells in the absence of feed cells (on average, 11.2%) than in the other conditions (<2%). The presence of PBMC and K562 feed cells in both processes regulated / significantly activated certain functional NK markers (NKp46, DNAM-1, CD94) in the population of NK cells. In general, the NK purity and the expression profiles of functional markers proved to be comparable between the two processes, when the feeding conditions are identical.
[000256] The functionality of the cultured NK cells, as determined by the 4-hour K562 cytotoxicity assay in vitro, proved to be comparable between the two processes when the feeding conditions are identical. NK cells activated by feed cells were highly effective in exterminating K562 cells in vitro, with average specific lysis of 93.2% and 93.6% of specific lysis for the two and three stage processes, respectively.
[000257] In short, the two- and three-stage processes have been shown to result in growth, phenotype (purity and activation markers), and in vitro functionality for comparable NK cells when the same feeding condition was used. The two-step process offers ease and convenience in the cultivation of NK cells when compared to the three-step process. 7.9. Example 9: Comparison of NK cell culture using various baseline media
[000258] This study aimed to evaluate the differentiation and expansion of CD34 +-derived NK cells using different basal media.
[000259] The experimental conditions are summarized in Table 8. The cells were cultured as described in Example 11. All experiments were set up on the multi-well plate / T flask scale and kept in an incubator at 37 ° C,> 90% humidity , and 5% CO2. Cell growth was monitored throughout the process (by cell count) and medium changes were performed twice a week to maintain cell concentrations within the range of 5 x 104 - 1 x 106 per mL. Differentiation was monitored by phenotypic analysis on Days 21 and 35. On Day 21, K562 and post-thaw allo-PBMC grown for 3 days were inactivated and added to the developing NK cell culture at 1 x 106 per ml. NK cells were normalized to 0.5 x 10 6 per ml. On Day 35, after final cell counts were performed, a cytotoxicity assay based on flow cytometry using freshly cultured K562 cells and labeled with PKH (10: 1 E: T ratio, 4 hours, 37 ° C) was performed to assess NK functionality. Table 8. Summary of experimental conditions for evaluating various baseline media


[000260] Growth results
[000261] Stemspan H3000 and OpTmizer showed growth results comparable (relative TNC expansion) to GBGM on Day 35. Cellgro, X-VIVO 15, AIM-V, X-VIVO 10, DMEM: F12, DMEM: F12 with 5 mM of OAC showed lower growth results than GBGM.
[000262] Phenotypic analysis
[000263] On the 35th Day (end point), GBGM resulted in about 80% purity for CD56 + CD3- cells. OpTmizer and Stemspan H3000 resulted in about 50% purity for CD56 + CD3- cells. DMEM: F12 produced about 35% purity for CD56 + CD3- cells. The media AIM-V, X-VIVO 10, X-VIVO 15 and Cellgro produced about <30% purity for CD56 + CD3- cells. The addition of OAC to the DMDM: F12 basal medium during culture has been shown to greatly increase the final NK purity; the percentage of CD56 + CD3- cells on Day 35 of the culture increased from 35% to 72%. Cytotoxicity / Functionality
[000264] The addition of 5 mM OAC to the baseline DMDM: F12 medium during culture has been shown to greatly increase the activation state and in vitro functionality of NK cells. The addition of OAC also significantly increased the level of activation markers NK NKp46, NKG2D, DNAM-1 and CD94. The in vitro functionality (cytotoxicity at K562) of NK cells on Day 35 also increased substantially, from 21.4% to 97.1%.
[000265] In general, the properties of NK cells have been significantly improved with the addition of 5 mM OAC. 7.10. Example 10: Storage and cryopreservation of NK cells
[000266] This example demonstrates the methods of storing and cryopreserving NK cells. CD34 + hematopoietic stem cells isolated from human placental perfusate (HPP) and umbilical cord blood cells were expanded and differentiated into NK cells using the protocols described in the previous examples. The cells were cryopreserved shortly after being isolated from HPP and umbilical cord blood (on Day 0) or during the first phase of NK cell growth (on the 9th, 14th, 21st or 35th after isolation).
[000267] The cells were cryopreserved in the following cryopreservation formulations:
[000268] Formulation 1- Dextran cryogenic medium: 5% DMSO (Sigma Aldrich, D2650), 55% dextran (10% w / v in normal brine) (10% LMD in injection with 0.9% chloride sodium, Hospira), 40% SAH (Octapharma);
[000269] Formulation 2 - Cryogenic medium of trehalose: 5% DMSO, 55% trehalose (10% w / v in normal brine), 40% HSA;
[000270] Formulation 3 - CryoStor® CS2 (BioLife Solutions);
[000271] Formulation 4 - CryoStor® CS5 (BioLife Solutions);
[000272] Formulation 5 - CryoStor®CS10 (BioLife Solutions);
[000273] Formulation 6 - Serum-free cryogenic medium (Sigma-Aldrich, Cat # 6295);
[000274] Formulation 7 - Glycerol freezing medium (Sigma-Aldrich, CAT # C6039); or
[000275] Formulation 8 - Freezing medium free of DMSO and serum (Sigma-Aldrich, CAT # 2639).
[000276] Cells collected at different times were washed several times with culture media or saline. The cells were then centrifuged to obtain cell pellets. The supernatant was removed, and the cell pellets were suspended in cryopreservation medium at about 1 x 106 - 1.5 x 107 or more cells per milliliter. Aliquots of the cell suspension were removed into septic vials of 1 mL or 2 mL and incubated at 2 - 8 ° C for approximately 10 minutes. Subsequently, the cells were frozen in a controlled rate freezer (Thermo) at 0.5 ° C; min. The frozen vials were transferred to a cryogenic freezer for storage in liquid nitrogen vapor. The cryopreserved NK cells can be thawed quickly in a 37 ° C bath with gentle swirling of the samples until all visible ice has melted. Cell samples can be diluted with pre-heated culture medium. 7.11. Example 11: Storage and cryopreservation of NK cells
[000277] This Example demonstrates another method for storing and cryopreserving NK cells. CD34 + hematopoietic stem cells isolated from human placental perfusate (HPP) and umbilical cord blood cells were expanded and differentiated into NK cells using the protocols described above. The cells were cryopreserved shortly after being isolated from HPP and umbilical cord blood (on the NK (on the 9th, 14th, 21st or 35th day after isolation).
[000278] A cell suspension was prepared by combining Dextran-40 and HSA in the ratio of 60% Dextran-40 v / v, 40% HSA (from a 25% solution) v / v.
[000279] A 2x freezing solution was prepared with 50% Dextran-40 v / v, 40% HSA (from a 25% solution) v / v, 10% DMSO v / v. DMSO was first added slowly to Dextran-40 and mixed well. Subsequently a 25% HSA solution was added to the solution slowly with mixing. The resulting solution was mixed well and brought to room temperature before use. Cryopreservation procedure
[000280] The number of cells was estimated and normalized as a cell suspension to 15 x 106 in the cell suspension solution. The volume of the cell suspension was determined and an equal volume of freshly prepared 2x freezing solution was slowly added and mixed. The final volume of cell suspension in the freezing solution was noted and then distributed to a number of vials. MycoAlert tests were performed on the culture's reserved supernatants to detect mycoplasma contamination. A post-thaw test was performed in a flask to determine post-thaw viability, cell recovery and cell characterization. 7.12. Example 12: Analysis of cryopreserved / thawed NK cells
[000281] Feasibility test. The cryopreserved NK cells in various formulations as in Examples 10 or 11 were thawed. The cells were frozen at a density of 2 x 10 6 - 3 x 10 7 cells / ml. Thawed NK cells were evaluated for cell viability using the automated cell counter Countless® (Invitrogen) at 0, 3 ° and 18 ° days after thawing and compared to fresh cells or pre-freezing cells. Quickly, 10 μL of cell samples were mixed with 10 μL of trypan blue. Cell mixtures were pipetted into the slide of the Countless® chamber. The slide was inserted into the instrument and cells were counted. The post-freeze-thaw cells showed cell viability of about 80% to> 90% viability, depending on the different cryopreservation formulations.
[000282] Apoptosis assay. The thawed NK cells were further evaluated for apoptosis using BD Annc / PI apoptosis assay kit on Days 0, 3 and 18 after thawing. The cells were quickly washed twice with 1x cold PBS and resuspended in 1x binding buffer (BD Annexin V / PI apoptosis kit number 556547, Composition of the binding buffer number 51-66121E 0.1M hepes / NaOH (pH 7, 4), 1.4 M NaCl, 25 mM CaCl2 (for 1x diluted solution, 10x buffer for 9 parts of distilled water). 100 μL of cell suspension containing approximately 100,000 cells were transferred to a FACS tube. 100 μL of 1x binding buffer, 5 μL of AnnV-FITC, and 1.1. μL of PI-PE was added to the tube. The tube was then slowly vortexed and incubated in the dark for 15 minutes. Subsequently, 400 μL of 1x binding buffer was added. The samples were analyzed within 1 hour. The controls used to configure quadrants and gates were non-stained cells, cells stained with AnnV-FITC only and not PI, cells stained with PI only and not AnnV. Apoptotic cells were quantified as a% of the population of events labeled "cells" on the size chart (FSC vs SSC). The post-freeze-thaw cells showed about 5 - 25% of dead / advanced apoptotic cells and 10 - 25% of recent apoptotic cells, depending on the different cryopreservation formulations. In general, formulation 1 (5% DMSO, 55% dextran (10% w / v in normal brine), 40% SAH), formulation 2 (5% DMSO, 55% trehalose (10% m / v in normal brine), 40% SAH), formulation 4 (CryoStor® CS5 (BioLife Solutions), and formulation 5 (CryoStor®CS10 (BioLife Solutions)) 7.13.
[000283] This example demonstrates the evaluation of the storage of cryopreserved cells in the process. Cell culture was initiated with UCB CD34 + cells using the method described by Spanholtz et al, PLoS One. 5 (2): e9221 (2010) using HS-AB or FBS as serum sources. The cell concentration was determined and adjusted, and the medium was replaced as needed. On Days 7, 9, 10 or 14, approximately 106 - 3 x 106 cells were removed from the cell culture, centrifuged and resuspended with cryopreservation medium (5.5% v / v Dextran-40, 10% v / v HSA, 5% v / v DMSO). The cells were frozen in a controlled-rate freezer and transferred to nitrogen storage in liquid phase for cryopreservation. Approximately 1 mL of cells were cryopreserved in each flask, at a concentration ranging from 106 - 107 per mL. The remaining culture was continued until the end point (Day 35), referred to as “Fresh (without cryopreservation in the process)”. Phenotypic analyzes were performed on Days 21, 28 and 35 of the cell culture. In vitro functionality (cytotoxicity at K562, 10: 1 E: T) was assessed on Day 35 (end point) of the culture.
[000284] The post-freezing performance of culture samples cryopreserved in the process (Days 9 and 14) was evaluated as follows. Cell storage vials were quickly thawed in the 37 ° C water bath. The cells were then diluted with RPMI-FBS medium, centrifuged, resuspended, and inoculated into the culture medium. Each of the culture conditions was then continued until Day 35, cumulative from the beginning of the culture process. The analyzes (cell count, viability, phenotypic analysis, evaluation of functionality) were carried out in the same way as their analogues with “fresh” cells.
[000285] Results
[000286] Samples cryopreserved in the process on the 9th, 10th and 14th Days resulted in excellent post-thaw viability, regardless of the time or cell concentration in the process (96.2% - 97.3% Negative Trypan Blue, 86, 1% - negative 93.2% Annexin-V / negative TO-PRO-3). In-process storage had no negative effects on loss of post-culture yield (35 ° Day). Final cell yields between Days 9 and 14 post-thaw and in “fresh” conditions are comparable, with either HS-AB or HS-AB as serum sources.
[000287] The phenotypic profiles of the maturation of NK cells on Days 21/28/35 were shown to be comparable between the "fresh" and "post-thaw" cultures, respectively. Phenotypic purity (CD56 + CD3-) proved to be comparable in the same way. The expression of certain functional NK markers (CD94, NKG2D) was slightly different due to variations from race to race.
[000288] In the K562 cytotoxicity assay, post-frozen NK cells on Days 9/14 showed ~ 0 - 20% lower results in specific K562 lysis compared to cultured NK cells not cryopreserved in the process. However, because the expression levels of surface markers relevant to NK cytotoxic function (DNAM-1, NKp46, NKG2D, etc.) have not been reduced concurrently, the trend would need to be confirmed with additional donors and repeat trials. In general, the cryopreservation in process on Days 9/14 of the cultivation had a minimal impact on the result of the process. 7.14. Example 14: Development of post-thaw medium for measuring NK cells
[000289] This example demonstrates the development of post-thaw medium for dosing NK cells in animals. The effects of the injection medium and cell density were tested on viability, cytotoxicity, cell recovery and lump formation in NK cells. Viability was assessed by dyeing with trypan blue; cytotoxicity was assessed by FACS (10: 1 ratio of NK: K562) and lump formation was assessed by microscopic assay. The results are shown in Tables 9 - 12 for the various types of injection medium and cell density. Table 9. Cell recovery, viability, cytotoxicity and lumps formation under the specific conditions tested



[000290] The results showed that the Plasmalyte + 1% HSA injection medium maintained the NK cells with better viability and cytotoxicity than the PBS + 1% FBS injection medium. Cytotoxicity also decreased over time after the cells were suspended in the injection medium. No effect of cell density on viability and cytotoxicity was observed when cells were suspended in Plasmalyte + 1% HSA. NK cells also adapted to PBS + 1% FBS and Plasmalyte + 1% HSA media after 1 hour; however, the cells did not show aggregation. Finally, there was no obvious loss in cell recovery and cell viability with the freeze - thaw process. 7.15. Example 16: Development of post-thaw medium for measuring NK cells
[000291] The effects of various concentrations of HSA have also been tested on viability, cytotoxicity, cell recovery and lump formation of NK cells. The same methods as in Example 14 were used. The results are shown in Tables 13 - 16 for the various types of injection medium and cell density. Table 13. Cell recovery, viability, cytotoxicity and lumps formation under the specific conditions tested



[000292] The results show that viability was successfully maintained over 4 hours post-thaw in all three injection media tested. In addition, cytotoxicity decreased over time in all three injection media. However, the level of reduction was lower at higher concentrations of HSA while Plasmalyte + 5% HSA maintained the highest cytotoxicity. It was also observed that NK cells did not aggregate in all three injection media. Overall, Plasmalyte appeared to be a better candidate for injection medium than FBS. 7.16 Example 17: Cultivation of NK cells without IL-2
[000293] This example demonstrates the cultivation of NK cells in the absence of IL-2. Cell culture was performed by the two-stage process described in Example 11. Five different concentrations of IL-2 were tested in the first medium: 0, 200, 500, 1000, 2000 U / ml.
[000294] The results indicate that the development of NK cells in culture does not appear to respond to IL-2 in growth. The purity of NK cells did not appear to depend on IL-2: the cells differ in the CD56 + CD3- phenotype in the absence of IL-2. The combination of IL-7, IL-15 and SCF was sufficient for the development of NK cells in vitro. Equivalents:
[000295] The present invention should not be limited in scope by the specific modalities described thus. In fact, various modifications to the invention in addition to those described will be apparent to those skilled in the art from the foregoing description and the accompanying figures. It is intended that such changes are within the scope of the appended claims.
[000296] All references cited here are incorporated herein in full by reference and for all purposes to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated in full by reference for all purposes. The citation of any order publication relates to its description before the date of completion and should not be seen as an admission that the present invention is not capable of foreseeing such publication due to previous inventions.

权利要求:
Claims (15)
[0001]
1. Two-step in vitro method for the production of a population of activated natural killer cells (NK) characterized by comprising: (a) the inoculation of a population of hematopoietic stem cells or progenitor cells in a first medium comprising interleukin-15 (IL-15) tyrosine kinase ligand similar to Fms 3 (Flt3-L), thrombopoietin (Tpo), stem cell factors (SCF) and interleukin-7 (IL-7), where said IL-15, SCF and IL-7 are not comprised in an indefinite component of said medium, so that the population expands, and a plurality of hematopoietic stem cells or progenitor cells within said population of hematopoietic stem cells or said progenitor cells differ in NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells; where hematopoietic stem cells or progenitor cells are CD34 +.
[0002]
Method according to claim 1, characterized in that the first medium further comprises interleukin-2 (IL-2) or heparin.
[0003]
Method according to claim 1 or 2, characterized in that the first medium further comprises fetal bovine serum or human serum.
[0004]
Method according to claim 1 or 2, characterized in that the SCF is present in a concentration of 1 to 150 ng / ml in the first medium.
[0005]
Method according to claim 1 or 2, characterized in that Flt3-L is present in a concentration of 1 to 150 ng / ml in the first medium.
[0006]
Method according to claim 2, characterized in that IL-2 is present in a concentration of 50 to 1500 IU / ml in the first medium.
[0007]
Method according to claim 1 or 2, characterized in that IL-7 is present in a concentration of 1 to 150 ng / ml in the first medium.
[0008]
Method according to claim 1 or 2, characterized in that IL-15 is present in a concentration of 1 to 150 ng / ml in the first medium.
[0009]
Method according to claim 1 or 3, characterized in that Tpo is present in a concentration of 1 to 150 ng / ml in the first medium.
[0010]
Method according to claim 1, characterized in that said IL-2 in step (b) is present in a concentration of 50 to 1500 IU / ml in the second medium.
[0011]
Method according to claim 1 or 2, characterized in that the second medium further comprises feeder cells.
[0012]
Method according to claim 1, characterized in that said second medium additionally comprises one or more of fetal calf serum (FCS), transferrin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid, bovine serum albumin ( BSA) and phytohemagglutinin.
[0013]
Method according to claim 1, characterized in that the hematopoietic stem cells or progenitor cells comprise hematopoietic stem cells or human placental perfusate progenitor cells and hematopoietic stem cells or umbilical cord progenitor cells, where said placental perfusate and said umbilical cord blood are from the same placenta.
[0014]
Method according to claim 1, characterized in that the NK cells are CD3-CD56 + CD16-.
[0015]
Method according to claim 14, characterized in that the NK cells are additionally CD94 + CD117 +, CD161-, NKG2D +, NKp46 +, or CD226 +.

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法律状态:
2019-06-04| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2020-05-19| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-06-02| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-12-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-23| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/07/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-07-27| B25A| Requested transfer of rights approved|Owner name: CELULARITY INC. (US) |

优先权:

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申请号 | 申请日 | 专利标题

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US36398110P| true| 2010-07-13|2010-07-13|

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US61/363,981|2010-07-13|

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US201161497897P| true| 2011-06-16|2011-06-16|

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US61/497,897|2011-06-16|

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PCT/US2011/043831|WO2012009422A1|2010-07-13|2011-07-13|Methods of generating natural killer cells|

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