WO2023278628A1 - Human placental hematopoietic stem cell derived natural killer cells in acute myeloid leukemia (aml) remission with minimal residual disease (mrd) or relapsed/refractory aml - Google Patents

Human placental hematopoietic stem cell derived natural killer cells in acute myeloid leukemia (aml) remission with minimal residual disease (mrd) or relapsed/refractory aml Download PDF

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WO2023278628A1
WO2023278628A1 PCT/US2022/035596 US2022035596W WO2023278628A1 WO 2023278628 A1 WO2023278628 A1 WO 2023278628A1 US 2022035596 W US2022035596 W US 2022035596W WO 2023278628 A1 WO2023278628 A1 WO 2023278628A1
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Robert J. Hariri
Andrew L. Pecora
William VAN DER TOUW
Mark AWADALLA
Cherie S. DALY
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Celularity Inc.
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)

Definitions

  • NK natural killer
  • ILC3 hematopoietic stem or progenitor cells in media comprising stem cell mobilizing factors
  • three-stage methods of producing NK cells and/or ILC3 cells in media comprising stem cell mobilizing factors starting with hematopoietic stem or progenitor cells from cells of the placenta for example, from placental perfusate (e.g., human placental perfusate) or other tissues, for example, umbilical cord blood or peripheral blood.
  • placental perfusate e.g., human placental perfusate
  • other tissues for example, umbilical cord blood or peripheral blood.
  • Acute myeloid leukemia is a heterogeneous hematologic malignancy of the myeloid precursor cell line, characterized by the clonal expansion of abnormal cells, which accumulate in the bone marrow, peripheral blood and/or other tissues, and interfere with the production of normal blood cells. [National Comprehensive Cancer Network [NCCN], 201939].
  • AML is generally classified as primary or secondary, secondary referring to either exposure to prior cytotoxic chemotherapy or by transformation from an underlying hematologic condition such as myelodysplastic syndromes (MDS) or myeloproliferative neoplasm (MPN). Advances in mutational profiling and gene sequencing have allowed for enhanced risk stratification and prognosis [Dohner, 201716].
  • MDS myelodysplastic syndromes
  • MPN myeloproliferative neoplasm
  • the risk factors associated with poor outcomes include older age (i.e., ⁇ 60 years old), adverse cytogenetics and transformation of existing myelodysplasia, etc. [Dohner, 201015].
  • older age i.e., ⁇ 60 years old
  • adverse cytogenetics and transformation of existing myelodysplasia etc.
  • CR complete remission
  • a widely used remission-induction chemotherapy is the combination of cytarabine and anthracycline, consisting of cytarabine 100 to 200 mg/m2/day for 7 days and daunorubicin 45 to 90 mg/m2/day for 3 days, [Löwenberg, 199935; Tallman, 200552] often referred to as the “7 + 3 protocol.”
  • a retrospective analysis of six Eastern Cooperative Oncology Group studies which included both younger and older adults demonstrated that 26% of patients treated with anthracycline and cytarabine-based induction therapy required a second cycle of identical induction therapy to achieve CR [Mangan, 201136].
  • a consolidation regimen is typically employed, which may consist of additional chemotherapy cycles or stem cell transplant, typically allogeneic hematopoietic stem cell transplantation (aSCT).
  • aSCT allogeneic hematopoietic stem cell transplantation
  • treatment options for subjects who choose not to receive emission-induction standard chemotherapy or are considered ineligible to receive remission-induction chemotherapy include low-dose cytarabine, azacitidine or decitabine [Deschler, 200614].
  • HSCT hematopoietic stem cell therapy
  • challenges include accurately assessing the following: prognosis of disease, whether remission can be achieved, and the ability of patients to tolerate aggressive salvage therapies, choosing a successful salvage therapy, and ultimately identifying suitable patients for HSCT.
  • the prognosis in relapsed AML patients is generally poor but depends largely on the timing of relapse (early versus late) and the possibility of allogeneic hematopoietic stem cell transplantation (HSCT).
  • HSCT allogeneic hematopoietic stem cell transplantation
  • MRD Detection in AML Mounting evidence has shown that risk of relapse in AML following chemotherapy has also been correlated to the detection of MRD. MRD is defined as leukemic cells at levels below morphologic detection [Ravandi, 201845; Ossenkoppele, 201341].
  • MRD multiparameter flow cytometry
  • NK cell infusions may induce and/or prolong remission and ultimately survival in high-risk AML patients.
  • Natural killer cells have demonstrated the ability to kill leukemia stem cells [Langenkamp, 200931], which may explain earlier studies demonstrating longer times to relapse in patients given cytoreductive therapy followed by the adoptive transfer of NK cells.
  • one study demonstrated that adoptively transferred NK cells could expand in vivo, and that induction of remission in 5 of 19 poor-prognosis AML patients was associated with NK cell expansion and killer cell immunoglobulin-like receptor (KIR) ligand-mismatch donors [Miller, 200537; Bachanova, 20143].
  • KIR immunoglobulin-like receptor
  • CYNK-001 in the Treatment of AML is an allogeneic off the shelf cell therapy enriched for CD56+/CD3- NK cells expanded from human placental CD34+ cells. CYNK-001 is manufactured in a cryopreserved formulation that is thawed and diluted at the clinical site prior to dose preparation and direct infusion.
  • CYNK-001 is packaged at 30 x 106 cells/mL in a total volume of 20 mL cryopreservation solution containing 10% (w/v) human serum albumin (HSA), 5.5% (w/v) Dextran 40, 0.21% sodium chloride (NaCl) (w/v), 32% (v/v) Plasma-Lyte A, and 5% (v/v) dimethyl sulfoxide (DMSO). It is filled into the container closure, frozen using a controlled rate freezer, and cryopreserved. Prior to releasing to the site, all release and characterization testing will be complete.
  • HSA human serum albumin
  • Dextran 40 0.21% sodium chloride (NaCl) (w/v)
  • DMSO dimethyl sulfoxide
  • CYNK 001 When required by site, CYNK 001 is shipped in vapor phase LN2 to the designated clinical site where it will be processed for dose preparation in a standardized manner just prior to IV administration.
  • This study is the first study to evaluate the safety and potential efficacy of CYNK- 001 in subjects with primary or secondary AML in morphological CR and MRD positivity and in subjects with R/R AML.
  • the use of a 3 + 3 dose escalating tolerability algorithm with strict dose- limiting toxicity (DLT) criteria will allow detection of serious toxicity associated with the use of CYNK 001 in study subjects.
  • the study will be comprised of Treatment Eligibility Period, Treatment Period and Follow-up Period.
  • the Treatment Period will include a Lymphodepletion Regimen that will be used to help prevent rejection of donor cells and to maintain and augment CYNK 001 cells in study subjects.
  • IL-15 is a cytokine critical for NK cell survival and proliferation.
  • Cy/Flu preparative regimen Cy: 60mg/kg x 2 days, Flu: 25mg/m2 x 5 days
  • Cy/Flu preparative regimen was associated with elevated serum IL-15 levels and expansion of the transferred NK cells.
  • Patients who received lower intensity regimens did not demonstrate in vivo expansion of donor cells and showed low to modest changes in serum IL-15 (Miller et al, Blood 2005).
  • Preclinical data also support the need for exposure to IL-15 in vivo for maintaining CYNK-001 persistence.
  • NCT02781467 evaluating PNK-007 using high-dose Cy/Flu (Cy: 8400mg total, Flu: 230mg total) conditioning confirmed elevated IL- 15 in AML patients
  • data generated for cohorts 1-3 following low dose Cy/Flu (Cy: 1656mg total, Flu: 230mg total) in the current study show negligible change to low baseline levels of serum IL- 15.
  • CYNK-001 cells Administering CYNK-001 cells at days 0, 7, 14, and 21 using enhanced lymphodepletion takes advantage of the IL-15 and Treg changes during the treatment period to maximize the persistence of dosed CYNK-001 cells and their functional impact on eliminating AML blasts
  • HLA matching and KIR mismatching will not be used in the selection of CYNK- 001 for an individual subject. However, these data will be collected for retrospective analysis.
  • rhIL-2 Background The use of rhIL-2 therapy has shown regression of tumors, has known adverse toxic effects, and has been in clinical trials for cancer therapy [Siegel, 199151 ].
  • rhIL-2 has been shown to enhance the expression of maturation markers on the surface of NK cells, making them more potent killers in vitro and in vivo [Trinchieri, 199555].
  • Adoptive transfer of haploidentical NK cells has shown a cytokine- dependent expansion phase after infusion, and a reduction after cytokine withdrawal in both the nonclinical setting [Miller, 201438] and clinical trial setting [Geller, 201122].
  • cytokine-dependent cell expansion followed by reduction upon cytokine withdrawal was observed in the pre-clinical model of adoptively transferred PNK 007 cells into non-obese diabetic (NOD scid gamma [NSG]) immunodeficient mice supplemented with human (h)IL-15.
  • NSG non-obese diabetic
  • h human
  • the rhIL-15 dosing regimen was designed to mimic the human clinical response to chemotherapy [Miller, 200537].
  • Clinical trials have assessed the effects of low-dose rhIL-2 administration on activation of NK cells in subjects with cancer. Miller et al demonstrated the safety and feasibility of daily subcutaneous rhIL-2 injections following high-dose chemotherapy and autologous hematopoietic cell transplantation (HCT).
  • rhIL-2 significantly expanded the number of circulating NK cells in vivo, these NK cells were not maximally cytotoxic as determined by in vitro assays [Miller, 199737].
  • Subsequent studies tested an infusion of rhIL-2–activated NK-cell– enriched populations or intravenous rhIL-2 infusions combined with subcutaneous (SC) rhIL-2.
  • SC subcutaneous
  • IL-15 also noted was an elevation of IL-15 during periods of lymphopenia [Miller, 200537].
  • IL-2DT IL-2 diphteria toxin fusion protein
  • NK-cell expansion resultsed in improved NK-cell expansion in patients who were treated with rhIL-29 M IU QOD x 6 doses compared to those who did not receive IL-2DT pre-treatment, showing the importance of optimizing conditions in which potential inhibitory cells, such as Treg cells, could impact NK cell expansion [Bachanova, 20143].
  • rhIL-2 was administered to facilitate NK cell survival and expansion in the PNK-007-AML-001 study (NCT02781467).
  • IL-2 was administered at a dose of 6 M IU/dose SC every other day (QOD) for up to 6 doses after lymphodepleting chemotherapy with cyclophosphamide and fludarabine followed by a single infusion of PNK-007 cells.
  • QOD QOD
  • the efficacy population included one patient (12.5%) with complete remission with incomplete platelet recovery (CRp) assessed on day 21 and one patient (12.5%) with morphologic leukemia-free state (MLFS) assessed on day 28.
  • CRp complete platelet recovery
  • MLFS morphologic leukemia-free state
  • rhIL-2 was well tolerated in 25 subjects treated within both CCT-PNK-007-AML-001 and a similar study in multiple myeloma patients, CCT-PNK-007-MM- 001, studies. There were no serious adverse events that were suspected to be related to rhIL-2 in both studies. In the CCT-PNK-007-AML-001, treatment-emergent adverse events leading to rhIL-2 discontinuation were reported in 2 subjects.
  • PNK-007 Human Placental Hematopoietic Stem Cell Derived Natural Killer Cell Dosing: PNK-007, which has been previously used in an AML study (CCT PNK 007 AML 001, NCT02781467), was produced with a cryopreserved Drug Substance, which was subsequently thawed, cultured, washed, filtered, and reformulated as a fresh Drug Product in Plasma-Lyte®-A solution containing 10% (weight/volume) HSA.
  • the cells were formulated at concentrations of 0.5 x 106 cells/mL, 1.5 x 106 cells/mL, 5 x 106 cells/mL or 15 x 106 cells/mL, which allowed a range of clinical doses with similar infusion volumes.
  • PNK-007 is dosed based on subject weight (e.g., 106 cells/kg) so the volume of the infusion scales with the subject weight (approximately 2 mL/kg). Each unit of PNK-007 was custom filled based on the subject weight, so that a full unit delivered the allocated cell dose.
  • a total of 10 subjects were treated with a single infusion of PNK-007 (range 1 x 106 cells/kg to 10 x 106 cells/kg) followed by 5 or 6 total rhIL-2 injections QOD starting on day of PNK-007 infusion to facilitate PNK-007 expansion.
  • Four subjects were treated in the highest dose administered in the PNK-007-AML-001 study, 10 x106 cells/kg PNK-007, with an actual dose infused ranging from 5.86 x 108 to 8.49 x 108 total cells associated with subject weight ranges from 59.3 kg to 83.1 kg.
  • the CCT-PNK-007-AML-001 study was terminated prior to completion due to a business decision.
  • the present invention provides methods of treating acute myeloid leukemia (AML) in a subject comprising administering to the subject an effective amount of CYNK cells to the subject so as thereby to provide an effective treatment of the AML in the subject.
  • AML acute myeloid leukemia
  • the CYNK cells are placental-derived natural killer (NK) cells.
  • the CYNK cells are placental CD34+ cell- derived natural killer (NK) cells.
  • the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells and / or expression
  • markers selected from the group consisting of
  • the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells.
  • markers selected from the group consisting of FGFBP2,
  • CYNK cells are characterized by expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC
  • markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1
  • the CYNK cells are prepared by the methods presented herein.
  • the acute myeloid leukemia is primary acute myeloid leukemia or secondary acute myeloid leukemia.
  • the acute myeloid leukemia is in remission with minimal residual disease.
  • the acute myeloid leukemia is relapsed/refractory AML.
  • providing an effective treatment comprises reducing the rate of minimal residual disease (MRD) relative to placebo.
  • providing an effective treatment comprises converting the subject to MRD negative.
  • providing an effective treatment comprises converting the subject to MRD positive less than 0.1%.
  • the MRD is measured by flow cytometry.
  • the MRD is measured by nucleic acid sequencing, preferably by next generation sequencing.
  • providing an effective treatment comprises reducing the time to minimal residual disease (MRD) response relative to placebo.
  • providing an effective treatment comprises increasing the duration of minimal residual disease (MRD) response relative to placebo.
  • providing an effective treatment comprises reducing the incidence, severity, or duration of the disease as measured by one or more International Myeloma Working Group (IMWG) response criteria relative to placebo.
  • IMWG International Myeloma Working Group
  • providing an effective treatment comprises reducing the incidence, severity, or duration of the disease as measured by the Eastern Cooperative Oncology Group (ECOG) Performance Status relative to placebo.
  • ECOG Eastern Cooperative Oncology Group
  • providing an effective treatment comprises increasing the duration of clinical response relative to placebo.
  • providing an effective treatment comprises increasing the rate of progression free survival, the rate of front-line progression free survival, or the rate of survival relative to placebo.
  • providing an effective treatment comprises increasing the time to progression, the front-line time to progression or the time to death relative to placebo. [0055] In some embodiments of the invention providing an effective treatment comprises increasing the overall survival or front-line overall survival relative to placebo. [0056] In some embodiments of the invention providing an effective treatment comprises increasing the patient reported outcome relative to placebo or relative to pretreatment. [0057] In some embodiments of the invention administering the cells to the subject is performed intravenously. [0058] In some embodiments of the invention from about 6 x 10 8 to about 3.0 x 10 9 cells are administered per administration. [0059] In some embodiments of the invention from about 9 x 10 8 to about 1.8 x 10 9 cells are administered per administration.
  • the treatment comprises 1 to 5 administrations of cells.
  • the treatment comprises 3 administrations of cells.
  • the treatment comprises 4 administrations of cells.
  • the administrations occur approximately 1 week apart.
  • one administration of cells occurs at approximately day 0 of the treatment. [0069] In some embodiments of the invention one administration of cells occurs at approximately day 7 of the treatment. [0070] In some embodiments of the invention one administration of cells occurs at approximately day 14 of the treatment. [0071] In some embodiments of the invention one administration of cells occurs at approximately day 21 of the treatment. [0072] In some embodiments of the invention the treatment comprises about 3 administrations of cells occurring at about days 0, 7, and 14 of the treatment. [0073] In some embodiments of the invention the treatment comprises about 3 administrations of cells occurring at about days 0, 7, 14, and 21 of the treatment. [0074] In some embodiments of the invention the treatment further comprises a lymphodepletion regimen.
  • the lymphodepletion regimen comprises administering cyclophosphamide and fludarabine to the subject.
  • the lymphodepletion regimen comprises administering about 300 mg/m 2 /day cyclophosphamide and 25 mg/m 2 /day fludarabine to the subject.
  • the lymphodepletion regimen comprises administering about 900 mg/m 2 /day cyclophosphamide and 30 mg/m 2 /day fludarabine to the subject.
  • the cyclophosphamide and fludarabine are administered to the subject on days -5, -4, and -3.
  • the cyclophosphamide and fludarabine are administered to the subject on days -6, -5, -4, and -3.
  • the treatment further comprises administering recombinant human interleukin-2 (rhIL-2) to the subject.
  • rhIL-2 recombinant human interleukin-2
  • the administration of rhIL-2 occurrs on each day that CYNK cells are administered.
  • the administration of rhIL-2 occurrs on days 0, 7, and 14.
  • the administration of rhIL-2 occurrs on days 0, 7, 14, and 21.
  • the rhIL-2 is additionally administered every other day between each administration of CYNK cells. [0085] In some embodiments of the invention about 4M IU to about 8MIU of rhIL-2 is administered to the subject for each administration. [0086] In some embodiments of the invention about 6M IU of rhIL-2 is administered to the subject for each administration. [0087] In some embodiments of the invention the treatment days are measured relative to administration fo the first dose of CYNK cells at day 0 of the treatment. [0088] The present invention provides compositions comprising human CYNK cells for use in the treatment of acute myeloid leukemia (AML) in a subject.
  • AML acute myeloid leukemia
  • the present invention provides uses of compositions comprising human CYNK cells for use in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML) in a subject.
  • AML acute myeloid leukemia
  • the cancer is multiple myeloma.
  • the acute myeloid leukemia is primary acute myeloid leukemia or secondary acute myeloid leukemia.
  • the acute myeloid leukemia is in remission with minimal residual disease.
  • the acute myeloid leukemia is relapsed/refractory AML.
  • the CYNK cells are placental-derived natural killer (NK) cells.
  • NK placental-derived natural killer
  • the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells and / or expression
  • markers selected from the group consisting of
  • the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells.
  • markers selected from the group consisting of FGFBP2,
  • the CYNK cells are characterized by expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB
  • markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1
  • the CYNK cells are prepared by the methods presented herein. Terminology [00102] As used herein, the term CYNK are CD34+ cell-derived NK cells produced by the methods described herein. In specific embodiments, CYNK cells are placental-deived NK cells. In other specific embodiments, CYNK-001 is a specific formulation of CYNK cells. [00103] As used herein, the terms “immunomodulatory compound” and “IMiD TM ” do not encompass thalidomide.
  • “lenalidomide” means 3-(4'aminoisoindoline-1'-one)-1-piperidine- 2,6-dione (Chemical Abstracts Service name) or 2,6-Piperidinedione,3-(4-amino-1,3-dihydro-1- oxo-2H-isoindol-2-yl)- (International Union of Pure and Applied Chemistry (IUPAC) name).
  • “pomalidomide” means 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione.
  • multipotent when referring to a cell, means that the cell has the capacity to differentiate into a cell of another cell type.
  • a multipotent cell is a cell that has the capacity to grow into a subset of the mammalian body's approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all of the cell types.
  • feeder cells refers to cells of one type that are co-cultured with cells of a second type, to provide an environment in which the cells of the second type can be maintained, and perhaps proliferate.
  • feeder cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors (e.g., bFGF), other factors (e.g., cytokines), and metabolic nutrients to target cells.
  • feeder cells grow in a mono-layer.
  • the “natural killer cells” or “NK cells” produced using the methods described herein, without further modification include natural killer cells from any tissue source.
  • the “ILC3 cells” produced using the methods described herein, without further modification include ILC3 cells from any tissue source.
  • placental perfusate means perfusion solution that has been passed through at least part of a placenta, e.g., a human placenta, e.g., through the placental vasculature, and includes a plurality of cells collected by the perfusion solution during passage through the placenta.
  • placental perfusate cells means nucleated cells, e.g., total nucleated cells, isolated from, or isolatable from, placental perfusate.
  • tumor cell suppression includes slowing the growth of a population of tumor cells, e.g., by killing one or more of the tumor cells in said population of tumor cells, for example, by contacting or bringing, e.g., NK cells or an NK cell population produced using a three-stage method described herein into proximity with the population of tumor cells, e.g., contacting the population of tumor cells with NK cells or an NK cell population produced using a three-stage method described herein.
  • said contacting takes place in vitro or ex vivo. In other embodiments, said contacting takes place in vivo.
  • the term “hematopoietic cells” includes hematopoietic stem cells and hematopoietic progenitor cells.
  • the “undefined component” is a term of art in the culture medium field that refers to components whose constituents are not generally provided or quantified. Examples of an “undefined component” include, without limitation, serum, for example, human serum (e.g., human serum AB) and fetal serum (e.g., fetal bovine serum or fetal calf serum).
  • FIG.1 shows expansion of NK cells for compounds CRL1 – CRL11.
  • FIG.2 shows expansion of NK cells for compounds CRL12 – CRL22.
  • FIG.3 shows expansion of NK cells relative to SR1 positive control.
  • FIG.4 shows expansion of CD34+ cells from which the NK cells were derived.
  • FIG.5 shows cytotoxicity of the expanded NK cultures.
  • FIG.6 shows that PNK cells highly express genes encoding the cytotoxic machinery.
  • FIG.6A CYNK cells were combined with peripheral blood derived NK cells (PB- NK) at 1:1 ratio and gene expression analyzed on single cell level using 10X Genomics Chromium platform and Illumina sequencing. Bioinformatics analysis utilized 10X Genomics Cell Ranger analysis pipeline.
  • FIG.6B A representative tSNE plot depicting PNK and PB-NK cells as distinct populations.
  • FIG.6C tSNE plots of selected NK cell-associated genes. The data is representative of two donors.
  • FIG.8 shows the gating strategy for PB-NK and CYNK cells.
  • CYNK and PBMC cells were thawed and stained with fluorophore-coupled antibodies targeting NK cell receptors. The figure demonstrates representative dot plots and the gating strategy for the identification of CYNK and PB-NK cells. See FIG.9 for further characterization of the populations.
  • FIG.9 shows differential expression of surface proteins on CYNK and PB-NK cells. CYNK and PB-NK cells were pre-gated as indicated in FIG.8.
  • FIG.10 shows that CYNK cells form a distinct cell population from PB-NK cells based on surface protein expression.
  • tSNE plots demonstrating differential clustering of CYNK and PB-NK cells based on their surface markers. tSNE plots were generated of flow cytometry data using FlowJo software.
  • FIG.11 shows a schematic of the AML cohort dose escalation plan. 5.
  • DETAILED DESCRIPTION Provided herein are novel methods of producing and expanding NK cells and/or ILC3 cells from hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells.
  • NK cell populations and/or ILC3 cell populations from hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells.
  • the hematopoietic cells e.g., CD34+ hematopoietic stem cells
  • NK cell populations and/or ILC3 cell populations may be obtained from any source, for example, without limitation, placenta, umbilical cord blood, placental blood, peripheral blood, spleen or liver.
  • the NK cells and/or ILC3 cells or NK cell populations and/or ILC3 cell populations are produced from expanded hematopoietic cells, e.g., hematopoietic stem cells and/or hematopoietic progenitor cells.
  • hematopoietic cells are collected from a source of such cells, e.g., placenta, for example from placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver (e.g., fetal liver) and/or bone marrow.
  • the hematopoietic cells used to produce the NK cells and/or ILC3 cells, and NK cell populations and/or ILC3 cell populations, may be obtained from any animal species.
  • the hematopoietic stem or progenitor cells are mammalian cells.
  • said hematopoietic stem or progenitor cells are human cells.
  • said hematopoietic stem or progenitor cells are primate cells.
  • said hematopoietic stem or progenitor cells are canine cells.
  • said hematopoietic stem or progenitor cells are rodent cells.
  • Hematopoietic cells useful in the methods disclosed herein can be any hematopoietic cells able to differentiate into NK cells and/or ILC3 cells, e.g., precursor cells, hematopoietic progenitor cells, hematopoietic stem cells, or the like.
  • Hematopoietic cells can be obtained from tissue sources such as, e.g., bone marrow, cord blood, placental blood, peripheral blood, liver or the like, or combinations thereof.
  • Hematopoietic cells can be obtained from placenta. In a specific embodiment, the hematopoietic cells are obtained from placental perfusate.
  • the hematopoietic cells are not obtained from umbilical cord blood. In one embodiment, the hematopoietic cells are not obtained from peripheral blood. Hematopoietic cells from placental perfusate can comprise a mixture of fetal and maternal hematopoietic cells, e.g., a mixture in which maternal cells comprise greater than 5% of the total number of hematopoietic cells. In certain embodiments, hematopoietic cells from placental perfusate comprise at least about 90%, 95%, 98%, 99% or 99.5% fetal cells.
  • the hematopoietic cells e.g., hematopoietic stem cells or progenitor cells, from which the NK cell populations and/or ILC3 cell populations produced using a three-stage method described herein are produced, are obtained from placental perfusate, umbilical cord blood, fetal liver, mobilized peripheral blood, or bone marrow.
  • the hematopoietic cells e.g., hematopoietic stem cells or progenitor cells, from which the NK cell populations and/or ILC3 cell populations produced using a three- stage method described herein are produced, are combined cells from placental perfusate and cord blood, e.g., cord blood from the same placenta as the perfusate.
  • said umbilical cord blood is isolated from a placenta other than the placenta from which said placental perfusate is obtained.
  • the combined cells can be obtained by pooling or combining the cord blood and placental perfusate.
  • the cord blood and placental perfusate are combined at 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 the like by volume to obtain the combined cells.
  • the cord blood and placental perfusate are combined at a ratio of from 10:1 to 1:10, from 5:1 to 1:5, or from 3:1 to 1:3. In another specific embodiment, the cord blood and placental perfusate are combined at a ratio of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5 or 1:10. In a more specific embodiment, the cord blood and placental perfusate are combined at a ratio of 8.5:1.5 (85%:15%).
  • the cord blood and placental perfusate are combined at 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 the like by total nucleated cells (TNC) content to obtain
  • the cord blood and placental perfusate are combined at a ratio of from 10:1 to 10:1, from 5:1 to 1:5, or from 3:1 to 1: 3. In another specific embodiment, the cord blood and placental perfusate are combined at a ratio of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5 or 1:10.
  • the hematopoietic cells e.g., hematopoietic stem cells or progenitor cells from which said NK cell populations and/or ILC3 cell populations produced using a three-stage method described herein are produced, are from both umbilical cord blood and placental perfusate, but wherein said umbilical cord blood is isolated from a placenta other than the placenta from which said placental perfusate is obtained.
  • the hematopoietic cells are CD34 + cells.
  • the hematopoietic cells useful in the methods disclosed herein are CD34 + CD38 + or CD34 + CD38 – .
  • the hematopoietic cells are CD34 + CD38 – Lin – .
  • the hematopoietic cells are one or more of CD2 – , CD3 – , CD11b – , CD11c – , CD14 – , CD16 – , CD19 – , CD24 – , CD56 – , CD66b – and/or glycophorin A – .
  • the hematopoietic cells are CD2 – , CD3 – , CD11b – , CD11c – , CD14 – , CD16 – , CD19 – , CD24 – , CD56 – , CD66b – and glycophorin A – .
  • the hematopoietic cells are CD34 + CD38 – CD33 – CD117 – .
  • the hematopoietic cells are CD34 + CD38 – CD33 – CD117 – CD235 – CD36 – .
  • the hematopoietic cells are CD45 + .
  • the hematopoietic cells are CD34 + CD45 + . In another embodiment, the hematopoietic cell is Thy-1 + . In a specific embodiment, the hematopoietic cell is CD34 + Thy-1 + . In another embodiment, the hematopoietic cells are CD133 + . In specific embodiments, 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 – .
  • the hematopoietic cells are positive for KDR (vascular growth factor receptor 2).
  • the hematopoietic cells are CD34 + KDR + , CD133 + KDR + or Thy-1 + KDR + .
  • the hematopoietic cells are positive for aldehyde dehydrogenase (ALDH + ), e.g., the cells are CD34 + ALDH + .
  • the CD34 + cells are CD45 – .
  • the CD34 + cells express one or more, or all, of the miRNAs hsa-miR-380, hsa-miR-512, hsa-miR-517, hsa-miR-518c, hsa-miR-519b, hsa-miR-520a, hsa-miR-337, hsa-miR-422a, hsa-miR-549, and/or hsa-miR-618.
  • the hematopoietic cells are CD34 – .
  • the hematopoietic cells can also lack certain markers that indicate lineage commitment, or a lack of developmental naiveté.
  • the hematopoietic cells are HLA-DR – .
  • the hematopoietic cells are CD34 + HLA-DR – , CD133 + HLA-DR – , Thy-1 + HLA-DR – or ALDH + HLA-DR –
  • the hematopoietic cells are negative for one or more, or all, of lineage markers CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin A.
  • hematopoietic cells can be selected for use in the methods disclosed herein on the basis of the presence of markers that indicate an undifferentiated state, or on the basis of the absence of lineage markers indicating that at least some lineage differentiation has taken place. Methods of isolating cells, including hematopoietic cells, on the basis of the presence or absence of specific markers is discussed in detail below.
  • Hematopoietic cells used in the methods provided herein can be a substantially homogeneous population, e.g., a population comprising at least about 95%, at least about 98% or at least about 99% hematopoietic cells from a single tissue source, or a population comprising hematopoietic cells exhibiting the same hematopoietic cell-associated cellular markers.
  • the hematopoietic cells can comprise at least about 95%, 98% or 99% hematopoietic cells from bone marrow, cord blood, placental blood, peripheral blood, or placenta, e.g., placenta perfusate.
  • Hematopoietic cells used in the methods provided herein can be obtained from a single individual, e.g., from a single placenta, or from a plurality of individuals, e.g., can be pooled. Where the hematopoietic cells are obtained from a plurality of individuals and pooled, the hematopoietic cells may be obtained from the same tissue source. Thus, in various embodiments, the pooled hematopoietic cells are all from placenta, e.g., placental perfusate, all from placental blood, all from umbilical cord blood, all from peripheral blood, and the like.
  • placenta e.g., placental perfusate, all from placental blood, all from umbilical cord blood, all from peripheral blood, and the like.
  • Hematopoietic cells used in the methods disclosed herein can, in certain embodiments, comprise hematopoietic cells from two or more tissue sources.
  • a plurality of the hematopoietic cells used to produce natural killer cells using a three-stage method described herein comprise hematopoietic cells from placenta, e.g., placenta perfusate.
  • the hematopoietic cells used to produce NK cell populations and/or ILC3 cell populations produced using a three-stage method described herein comprise hematopoietic cells from placenta and from cord blood; from placenta and peripheral blood; from placenta and placental blood, or placenta and bone marrow.
  • the hematopoietic cells comprise hematopoietic cells from placental perfusate in combination with hematopoietic cells from cord blood, wherein the cord blood and placenta are from the same individual, i.e., wherein the perfusate and cord blood are matched.
  • the hematopoietic cells from 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.
  • the hematopoietic cells used in the methods provided herein are placental hematopoietic cells.
  • placental hematopoietic cells are CD34 + .
  • the placental hematopoietic cells are predominantly (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) CD34 + CD38 – cells.
  • the placental hematopoietic cells are predominantly (e.g., 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 post-partum mammalian (e.g., human) placenta by any means known to those of skill in the art, e.g., by perfusion.
  • the placental hematopoietic cell is CD45 – .
  • the hematopoietic cell is CD34 + CD45 – .
  • the placental hematopoietic cells are CD34 + CD45 + . 5.2.
  • Production of Natural Killer and/or ILC3 Cells and Natural Killer Cell and/or ILC3 Cell Populations comprises expanding a population of hematopoietic cells. During cell expansion, a plurality of hematopoietic cells within the hematopoietic cell population differentiate into NK cells and/or ILC3 cells.
  • a method of producing NK cells comprising culturing hematopoietic stem cells or progenitor cells, e.g., CD34 + stem cells or progenitor cells, in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells, subsequently culturing said first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells, and subsequently culturing said second population of cells in a third medium comprising IL-2 and IL-15, and lacking a stem cell mobilizing agent and LMWH, to produce a third population of cells, wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and wherein at least 70%, for example at least 80%, of the natural killer cells are viable.
  • Tpo stem cell mobilizing agent and thrombopoietin
  • such natural killer cells comprise natural killer cells that are CD16-. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94+. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94+ or CD16+. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94- or CD16-. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94+ and CD16+. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94- and CD16-. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • LIF leukemia inhibiting factor
  • MIP-1 ⁇ macrophage inflammatory protein-1 alpha
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • a method of producing NK cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and CD11a+.
  • Tpo thrombopoietin
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇ , and said third medium lacks LIF, MIP- 1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • a method of producing NK cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of stem cell factor (SCF) and LMWH, to produce a third population of cells; wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and CD11a+.
  • Tpo thrombopoietin
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • a method of producing NK cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of SCF, a stem cell mobilizing agent, and LMWH, to produce a third population of cells; wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and CD11a+.
  • Tpo thrombopoietin
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • a method of producing NK cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of a stem cell mobilizing agent and LMWH, to produce a third population of cells; and (d) isolating CD11a+ cells from the third population of cells to produce a fourth population of cells; wherein the fourth population of cells comprises natural killer cells that are CD56+, CD3-, and CD11a+.
  • Tpo thrombopoietin
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • said natural killer cells express perforin and EOMES. In certain embodiments, said natural killer cells do not express either ROR ⁇ t or IL1R1.
  • a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising a stem cell mobilizing agent, IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-.
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising SCF, IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-.
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising a stem cell mobilizing agent, SCF, IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-.
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of a stem cell mobilizing agent and LMWH, to produce a third population of cells; and (d) isolating CD11a- cells, or removing CD11a+ cells, from the third population of cells to produce a fourth population of cells; wherein the fourth population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-.
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇
  • said third medium lacks LIF, MIP-1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • said ILC3 cells express ROR ⁇ t and IL1R1.
  • said ILC3 cells do not express either perforin or EOMES. 5.2.1. Production of NK Cell and/or ILC3 Cell Populations Using a Three- Stage Method [00156]
  • a three-stage method of producing NK cell and/or ILC3 cell populations comprises maintaining the cell population comprising said hematopoietic cells at between about 2 x 10 4 and about 6 x 10 6 cells per milliliter.
  • said hematopoietic stem or progenitor cells are initially inoculated into said first medium from 1 x 10 4 to 1 x 10 5 cells/mL. In a specific aspect, said hematopoietic stem or progenitor cells are initially inoculated into said first medium at about 3 x 10 4 cells/mL. [00157] In certain aspects, said first population of cells are initially inoculated into said second medium from 5 x 10 4 to 5 x 10 5 cells/mL. In a specific aspect, said first population of cells is initially inoculated into said second medium at about 1 x 10 5 cells/mL.
  • said second population of cells is initially inoculated into said third medium from 1 x 10 5 to 5 x 10 6 cells/mL. In certain aspects, said second population of cells is initially inoculated into said third medium from 1 x 10 5 to 1 x 10 6 cells/mL. In a specific aspect, said second population of cells is initially inoculated into said third medium at about 5 x 10 5 cells/mL. In a more specific aspect, said second population of cells is initially inoculated into said third medium at about 5 x 10 5 cells/mL in a spinner flask. In a specific aspect, said second population of cells is initially inoculated into said third medium at about 3 x 10 5 cells/mL.
  • the three-stage method comprises a first stage (“stage 1”) comprising culturing hematopoietic stem cells or progenitor cells, e.g., CD34 + stem cells or progenitor cells, in a first medium for a specified time period, e.g., as described herein, to produce a first population of cells.
  • the first medium comprises a stem cell mobilizing agent and thrombopoietin (Tpo).
  • the first medium comprises in addition to a stem cell mobilizing agent and Tpo, one or more of LMWH, Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the first medium comprises in addition to a stem cell mobilizing agent and Tpo, each of LMWH, Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM- CSF.
  • the first medium lacks added LMWH.
  • the first medium lacks added desulphated glycosaminoglycans.
  • the first medium lacks LMWH.
  • the first medium lacks desulphated glycosaminoglycans.
  • each of Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF in addition to a stem cell mobilizing agent and Tpo, each of Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the first medium lacks leukemia inhibiting factor (LIF), macrophage inhibitory protein-1alpha (MIP-1 ⁇ ) or both.
  • LIF leukemia inhibiting factor
  • MIP-1 ⁇ macrophage inhibitory protein-1alpha
  • the second medium comprises a stem cell mobilizing agent and interleukin-15 (IL-15) and lacks Tpo.
  • the second medium comprises, in addition to a stem cell mobilizing agent and IL-15, one or more of LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the second medium comprises, in addition to a stem cell mobilizing agent and IL-15, each of LMWH, Flt-3, SCF, IL-6, IL-7, G- CSF, and GM-CSF.
  • the second medium lacks added LMWH.
  • the second medium lacks added desulphated glycosaminoglycans.
  • the second medium lacks heparin, e.g., LMWH. In a specific embodiment, the second medium lacks desulphated glycosaminoglycans. In certain embodiments, the second medium comprises, in addition to a stem cell mobilizing agent and IL-15, each of Flt-3, SCF, IL- 6, IL-7, G-CSF, and GM-CSF. In specific embodiments, the second medium lacks leukemia inhibiting factor (LIF), macrophage inhibitory protein-1alpha (MIP-1 ⁇ ) or both.
  • LIF leukemia inhibiting factor
  • MIP-1 ⁇ macrophage inhibitory protein-1alpha
  • the third medium comprises IL-2 and IL-15, and lacks a stem cell mobilizing agent and LMWH.
  • the third medium comprises in addition to IL-2 and IL-15, one or more of SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the third medium comprises, in addition to IL-2 and IL-15, each of SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the first medium lacks one, two, or all three of LIF, MIP-1 ⁇ , and Flt3L.
  • the third medium lacks added desulphated glycosaminoglycans.
  • the third medium lacks desulphated glycosaminoglycans.
  • the third medium lacks heparin, e.g., LMWH.
  • the three-stage method is used to produce NK cell and/or ILC3 cell populations. In certain embodiments, the three-stage method is conducted in the absence of stromal feeder cell support.
  • the three-stage method is conducted in the absence of exogenously added steroids (e.g., cortisone, hydrocortisone, or derivatives thereof).
  • said first medium used in the three-stage method comprises a stem cell mobilizing agent and thrombopoietin (Tpo).
  • the first medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and Tpo, one or more of Low Molecular Weight Heparin (LMWH), Flt-3 Ligand (Flt-3L), stem cell factor (SCF), IL-6, IL-7, granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage- stimulating factor (GM-CSF).
  • LMWH Low Molecular Weight Heparin
  • Flt-3L Flt-3 Ligand
  • SCF stem cell factor
  • IL-6 IL-6
  • IL-7 granulocyte colony-stimulating factor
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage- stimulating factor
  • the first medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and Tpo, each of Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the first medium lacks added LMWH.
  • the first medium lacks added desulphated glycosaminoglycans.
  • the first medium lacks LMWH.
  • the first medium lacks desulphated glycosaminoglycans.
  • said Tpo is present in the first medium at a concentration of from 1 ng/mL to 100 ng/mL, from 1 ng/mL to 50 ng/mL, from 20 ng/mL to 30 ng/mL, or about 25 ng/mL. In other aspects, said Tpo is present in the first medium at a concentration of from 100 ng/mL to 500 ng/mL, from 200 ng/mL to 300 ng/mL, or about 250 ng/mL.
  • the LMWH when LMWH is present in the first medium, the LMWH is present at a concentration of from 1U/mL to 10U/mL; the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL; the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL.
  • the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL;
  • the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL;
  • the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL;
  • the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL;
  • the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL;
  • the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL.
  • the LMWH when LMWH is present in the first medium, the LMWH is present at a concentration of from 4U/mL to 5U/mL; the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL.
  • the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL;
  • the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL;
  • the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL.
  • the LMWH when LMWH is present in the first medium, the LMWH is present at a concentration of about 4.5U/mL; the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 25 ng/mL; the G-CSF is present at a concentration of about .25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL.
  • the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 25 ng/mL; the G-CSF is present at a concentration of about .25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL.
  • said first medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite; ascorbic acid; ethanolamine; and glutathione.
  • antibiotics such as gentamycin
  • antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol
  • sodium selenite sodium selenite
  • ascorbic acid ethanolamine
  • glutathione glutathione
  • the medium that provides the base for the first medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially available cell/tissue culture medium such as SCGM TM , STEMMACS TM , GBGM®, AIM-V®, X-VIVO TM 10, X-VIVO TM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETE TM , DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI-1640; or is a medium that comprises components generally included in known cell/tissue culture media, such as the components included in GBGM®, AIM-V®, X-VIVO TM 10, X-VIVO TM 15, OPTMIZER, STEMSPAN® H3000,
  • F12
  • said first medium is not GBGM®. In specific embodiments of any of the above embodiments, the first medium lacks LIF, MIP-1 ⁇ , or both.
  • said second medium used in the three-stage method comprises a stem cell mobilizing agent and interleukin-15 (IL-15), and lacks Tpo. In certain aspects, the second medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and IL-15, one or more of LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the second medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and IL-15, each of LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the second medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and IL-15, each of Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • the second medium lacks added LMWH.
  • the second medium lacks added desulphated glycosaminoglycans.
  • the second medium lacks LMWH.
  • the second medium lacks desulphated glycosaminoglycans.
  • said IL-15 is present in said second medium at a concentration of from 1 ng/mL to 50 ng/mL, from 10 ng/mL to 30 ng/mL, or about 20 ng/mL.
  • the LMWH when LMWH is present in said second medium, the LMWH is present at a concentration of from 1U/mL to 10U/mL; the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL; the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL.
  • the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL;
  • the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL;
  • the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL;
  • the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL;
  • the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL;
  • the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL.
  • the LMWH when LMWH is present in the second medium, the LMWH is present in the second medium at a concentration of from 4U/mL to 5U/mL; the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL.
  • the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL;
  • the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL.
  • the LMWH when LMWH is present in the second medium, the LMWH is present in the second medium at a concentration of from 4U/mL to 5U/mL; the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL.
  • the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL;
  • the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL;
  • the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL.
  • the LMWH when LMWH is present in the second medium, the LMWH is present in the second medium at a concentration of about 4.5U/mL; the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 25 ng/mL; the G-CSF is present at a concentration of about 0.25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL.
  • the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 25 ng/mL; the G-CSF is present at a concentration of about 0.25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL.
  • said second medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite; ascorbic acid; ethanolamine; and glutathione.
  • antibiotics such as gentamycin
  • antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol
  • sodium selenite sodium selenite
  • ascorbic acid ethanolamine
  • glutathione glutathione
  • the medium that provides the base for the second medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially available cell/tissue culture medium such as SCGM TM , STEMMACS TM , GBGM®, AIM-V®, X-VIVO TM 10, X-VIVO TM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETE TM , DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI-1640; or is a medium that comprises components generally included in known cell/tissue culture media, such as the components included in GBGM®, AIM-V®, X-VIVO TM 10, X-VIVO TM 15, OPTMIZER, STEMSPAN® H3000,
  • F12
  • said second medium is not GBGM®.
  • the first medium lacks LIF, MIP-1 ⁇ , or both.
  • said third medium used in the three-stage method comprises IL- 2 and IL-15, and lacks a stem cell mobilizing agent and LMWH.
  • said third medium used in the three-stage method comprises IL-2 and IL-15, and lacks LMWH.
  • said third medium used in the three-stage method comprises IL-2 and IL-15, and lacks SCF and LMWH.
  • said third medium used in the three-stage method comprises IL-2 and IL-15, and lacks SCF, a stem cell mobilizing agent and LMWH.
  • said third medium used in the three-stage method comprises a stem cell mobilizing agent, IL-2 and IL- 15, and lacks LMWH.
  • said third medium used in the three-stage method comprises SCF, IL-2 and IL-15, and lacks LMWH.
  • said third medium used in the three-stage method comprises a stem cell mobilizing agent, SCF, IL-2 and IL-15, and lacks LMWH.
  • said third medium used in the three-stage method comprises IL-2 and IL-15, and lacks a stem cell mobilizing agent and LMWH.
  • the third medium used in the three-stage method comprises, in addition to IL-2 and IL-15, one or more of SCF, IL- 6, IL-7, G-CSF, or GM-CSF.
  • the third medium used in the three-stage method comprises, in addition to IL-2 and IL-15, each of SCF, IL-6, IL-7, G-CSF, and GM-CSF.
  • said IL-2 is present in said third medium at a concentration of from 10 U/mL to 10,000 U/mL and said IL-15 is present in said third medium at a concentration of from 1 ng/mL to 50 ng/mL.
  • said IL-2 is present in said third medium at a concentration of from 100 U/mL to 10,000 U/mL and said IL-15 is present in said third medium at a concentration of from 1 ng/mL to 50 ng/mL. In certain aspects, said IL-2 is present in said third medium at a concentration of from 300 U/mL to 3,000 U/mL and said IL-15 is present in said third medium at a concentration of from 10 ng/mL to 30 ng/mL. In certain aspects, said IL-2 is present in said third medium at a concentration of about 1,000 U/mL and said IL-15 is present in said third medium at a concentration of about 20 ng/mL.
  • the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM- CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL.
  • the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL.
  • the SCF is present at a concentration of about 22 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 20 ng/mL; the G-CSF is present at a concentration of about 0.25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL.
  • the third medium comprises 100 ng/mL IL-7, 1000 ng/mL IL-2, 20 ng/mL IL-15, and 10 stem cell mobilizing agent and lacks SCF.
  • the third medium comprises 20 ng/mL IL-7, 1000 ng/mL IL-2, 20 ng/mL IL-15, and stem cell mobilizing agent and lacks SCF. In certain aspects, the third medium comprises 20 ng/mL IL-7, 20 ng/mL IL-15, and stem cell mobilizing agent and lacks SCF. In certain aspects, the third medium comprises 100 ng/mL IL-7, 22 ng/mL SCF, 1000 ng/mL IL-2, and 20 ng/mL IL-15 and lacks stem cell mobilizing agent. In certain aspects, the third medium comprises 22 ng/mL SCF, 1000 ng/mL IL-2, and 20 ng/mL IL-15 and lacks stem cell mobilizing agent.
  • the third medium comprises 20 ng/mL IL-7, 22 ng/mL SCF, 1000 ng/mL IL-2, and 20 ng/mL IL-15 and lacks stem cell mobilizing agent. In certain aspects, the third medium comprises 20 ng/mL IL-7, 22 ng/mL SCF, and 1000 ng/mL IL-2 and lacks stem cell mobilizing agent. In specific embodiments of any of the above embodiments, the first medium lacks one, two, or all three of LIF, MIP-1 ⁇ , Flt-3L.
  • said third medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite; ascorbic acid; ethanolamine; and glutathione.
  • antibiotics such as gentamycin
  • antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol
  • sodium selenite sodium selenite
  • ascorbic acid ethanolamine
  • glutathione glutathione
  • the medium that provides the base for the third medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially available cell/tissue culture medium such as SCGM TM , STEMMACS TM , GBGM®, AIM-V®, X-VIVO TM 10, X-VIVO TM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETE TM , DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI-1640; or is a medium that comprises components generally included in known cell/tissue culture media, such as the components included in GBGM®, AIM-V®, X-VIVO TM 10, X-VIVO TM 15, OPTMIZER, STEMSPAN® H3000,
  • F12
  • said third medium is not GBGM®.
  • the particularly recited medium components do not refer to possible constituents in an undefined component of said medium.
  • said Tpo, IL-2, and IL-15 are not comprised within an undefined component of the first medium, second medium or third medium, e.g., said Tpo, IL-2, and IL-15 are not comprised within serum.
  • said LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and/or GM-CSF are not comprised within an undefined component of the first medium, second medium or third medium, e.g., said LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and/or GM-CSF are not comprised within serum.
  • said first medium, second medium or third medium comprises human serum-AB.
  • any of said first medium, second medium or third medium comprises 1% to 20% human serum-AB, 5% to 15% human serum-AB, or about 2, 5, or 10% human serum-AB.
  • said hematopoietic stem or progenitor cells are cultured in said first medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.
  • cells are cultured in said second medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.
  • cells are cultured in said third medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or for more than 30 days.
  • said hematopoietic stem or progenitor cells are cultured in said first medium for 7-13 days to produce a first population of cells, before said culturing in said second medium; said first population of cells are cultured in said second medium for 2-6 days to produce a second population of cells before said culturing in said third medium; and said second population of cells are cultured in said third medium for 10-30 days, i.e., the cells are cultured a total of 19-49 days.
  • said hematopoietic stem or progenitor cells are cultured in said first medium for 8-12 days to produce a first population of cells, before said culturing in said second medium; said first population of cells are cultured in said second medium for 3-5 days to produce a second population of cells before said culturing in said third medium; and said second population of cells are cultured in said third medium for 15-25 days, i.e., the cells are cultured a total of 26-42 days.
  • said hematopoietic stem or progenitor cells are cultured in said first medium for about 10 days to produce a first population of cells, before said culturing in said second medium; said first population of cells are cultured in said second medium for about 4 days to produce a second population of cells before said culturing in said third medium; and said second population of cells are cultured in said third medium for about 21 days, i.e., the cells are cultured a total of about 35 days.
  • the three-stage method disclosed herein produces at least 5000- fold more natural killer cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium.
  • said three-stage method produces at least 10,000-fold more natural killer cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 50,000-fold more natural killer cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 75,000-fold more natural killer cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, the viability of said natural killer cells is determined by 7-aminoactinomycin D (7AAD) staining.
  • 7AAD 7-aminoactinomycin D
  • the viability of said natural killer cells is determined by annexin-V staining. In specific aspects, the viability of said natural killer cells is determined by both 7-AAD staining and annexin-V staining. In certain aspects, the viability of said natural killer cells is determined by trypan blue staining. [00174] In certain aspects, the three-stage method disclosed herein produces at least 5000- fold more ILC3 cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 10,000-fold more ILC3 cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium.
  • said three-stage method produces at least 50,000-fold more ILC3 cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 75,000-fold more ILC3 cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. [00175] In certain aspects, the three-stage method produces natural killer cells that comprise at least 20% CD56+CD3– natural killer cells. In certain aspects, the three-stage method produces natural killer cells that comprise at least 40% CD56+CD3– natural killer cells. In certain aspects, the three-stage method produces natural killer cells that comprise at least 60% CD56+CD3– natural killer cells.
  • the three-stage method produces natural killer cells that comprise at least 70% CD56+CD3– natural killer cells. In certain aspects, the three- stage method produces natural killer cells that comprise at least 80% CD56+CD3– natural killer cells. [00176] In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 20% CD56+CD3–CD11a+ natural killer cells. In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 40% CD56+CD3– CD11a+ natural killer cells. In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 60% CD56+CD3– CD11a+ natural killer cells.
  • the three-stage method disclosed herein produces natural killer cells that comprise at least 80% CD56+CD3– CD11a+ natural killer cells. [00177] In certain aspects, the three-stage method disclosed herein produces ILC3 cells that comprise at least 20% CD56+CD3– CD11a– ILC3 cells. In certain aspects, the three-stage method disclosed herein produces ILC3 cells that comprise at least 40% CD56+CD3– CD11a– ILC3 cells. In certain aspects, the three-stage method disclosed herein produces ILC3 cells that comprise at least 60% CD56+CD3– CD11a– ILC3 cells. In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 80% CD56+CD3– CD11a– ILC3 cells.
  • the three-stage method produces natural killer cells that exhibit at least 20% cytotoxicity against K562 cells when said natural killer cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces natural killer cells that exhibit at least 35% cytotoxicity against the K562 cells when said natural killer cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces natural killer cells that exhibit at least 45% cytotoxicity against the K562 cells when said natural killer cells and said K562 cells are co- cultured in vitro or ex vivo at a ratio of 10:1.
  • the three-stage method produces natural killer cells that exhibit at least 60% cytotoxicity against the K562 cells when said natural killer cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces natural killer cells that exhibit at least 75% cytotoxicity against the K562 cells when said natural killer cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. [00179] In certain aspects, the three-stage method produces ILC3 cells that exhibit at least 20% cytotoxicity against K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1.
  • the three-stage method produces ILC3 cells that exhibit at least 35% cytotoxicity against the K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces ILC3 cells that exhibit at least 45% cytotoxicity against the K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces ILC3 cells that exhibit at least 60% cytotoxicity against the K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1.
  • the three-stage method produces ILC3 cells that exhibit at least 75% cytotoxicity against the K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1.
  • said third population of cells e.g., said population of natural killer cells and/or ILC3 cells
  • said fourth population of cells e.g., said population of natural killer cells and/or ILC3 cells
  • populations of cells comprising natural killer cells, i.e., natural killers cells produced by a three-stage method described herein. Accordingly, provided herein is an isolated natural killer cell population produced by a three- stage method described herein. In a specific embodiment, said natural killer cell population comprises at least 20% CD56+CD3– natural killer cells. In a specific embodiment, said natural killer cell population comprises at least 40% CD56+CD3– natural killer cells. In a specific embodiment, said natural killer cell population comprises at least 60% CD56+CD3– natural killer cells. In a specific embodiment, said natural killer cell population comprises at least 80% CD56+CD3– natural killer cells. In a specific embodiment, said natural killer cell population comprises at least 60% CD16- cells.
  • said natural killer cell population comprises at least 80% CD16- cells. In a specific embodiment, said natural killer cell population comprises at least 20% CD94+ cells. In a specific embodiment, said natural killer cell population comprises at least 40% CD94+ cells.
  • a population of natural killer cells that is CD56+CD3– CD117+CD11a+, wherein said natural killer cells express perforin and/or EOMES, and do not express one or more of ROR ⁇ t, aryl hydrocarbon receptor (AHR), and IL1R1. In certain aspects, said natural killer cells express perforin and EOMES, and do not express any of ROR ⁇ t, aryl hydrocarbon receptor, or IL1R1.
  • said natural killer cells additionally express T-bet, GZMB, NKp46, NKp30, and NKG2D. In certain aspects, said natural killer cells express CD94. In certain aspects, said natural killer cells do not express CD94. [00183] In certain aspects, provided herein is a population of ILC3 cells that is CD56+CD3– CD117+CD11a-, wherein said ILC3 cells express one or more of ROR ⁇ t, aryl hydrocarbon receptor, and IL1R1, and do not express one or more of CD94, perforin, and EOMES.
  • said ILC3 cells express ROR ⁇ t, aryl hydrocarbon receptor, and IL1R1, and do not express any of CD94, perforin, or EOMES.
  • said ILC3 cells additionally express CD226 and/or 2B4.
  • said ILC3 cells additionally express one or more of IL-22, TNF ⁇ , and DNAM-1.
  • said ILC3 cells express CD226, 2B4, IL-22, TNF ⁇ , and DNAM-1.
  • a method of producing a cell population comprising natural killer cells and ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of a stem cell mobilizing agent and LMWH, to produce a third population of cells; and (d) separating CD11a+ cells and CD11a– cells from the third population of cells; and (e) combining the CD11a+ cells with the CD11a– cells in a ratio of 50:1, 40:1, 30:1, 20:
  • said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1 ⁇ ).
  • said third medium lacks LIF, MIP-1 ⁇ , and FMS-like tyrosine kinase-3 ligand (Flt-3L).
  • said first medium and said second medium lack LIF and MIP-1 ⁇ , and said third medium lacks LIF, MIP- 1 ⁇ , and Flt3L.
  • none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.
  • the CD11a+ cells and CD11a– cells are combined in a ratio of 50:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 20:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 10:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 5:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:1.
  • the CD11a+ cells and CD11a– cells are combined in a ratio of 1:5. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:10. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:20. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:50.
  • any "R" group(s) such as, without limitation, R a , R b , R c , R d , R e , R f , R g , R h , R m , R G , R J , R K , R U , R V , R Y , and R Z represent substituents that can be attached to the indicated atom.
  • R group may be substituted or unsubstituted. If two "R” groups are described as being “taken together” the R groups and the atoms they are attached to can form a cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocycle. For example, without limitation, if R a and R b of an NR a R b group are indicated to be “taken together,” it means that they are covalently bonded to one another to form a ring: [ [00190] In addition, if two “R” groups are described as being “taken together” with the atom(s) to which they are attached to form a ring as an alternative, the R groups are not limited to the variables or substituents defined previously.
  • the indicated “optionally substituted” or “substituted” group may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, acylalkyl, hydroxy, alkoxy, alkoxyalkyl, aminoalkyl, amino acid, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxyalkyl, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyana
  • Ca to Cb in which “a” and “b” are integers refer to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl, aryl, heteroaryl or heteroalicyclyl group.
  • the alkyl, alkenyl, alkynyl, ring(s) of the cycloalkyl, ring(s) of the cycloalkenyl, ring(s) of the aryl, ring(s) of the heteroaryl or ring(s) of the heteroalicyclyl can contain from “a” to “b”, inclusive, carbon atoms.
  • a “C 1 to C 4 alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that is, CH 3 -, CH 3 CH 2 -, CH 3 CH 2 CH 2 -, (CH 3 ) 2 CH-, CH 3 CH 2 CH 2 CH 2 -, CH 3 CH 2 CH(CH 3 )- and (CH 3 ) 3 C-. If no “a” and “b” are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl cycloalkenyl, aryl, heteroaryl or heteroalicyclyl group, the broadest range described in these definitions is to be assumed.
  • alkyl refers to a straight or branched hydrocarbon chain that comprises a fully saturated (no double or triple bonds) hydrocarbon group.
  • the alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated).
  • the alkyl group may also be a medium size alkyl having 1 to 10 carbon atoms.
  • the alkyl group could also be a lower alkyl having 1 to 6 carbon atoms.
  • the alkyl group of the compounds may be designated as “C 1 -C 4 alkyl” or similar designations.
  • “C 1 -C 4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.
  • alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexyl.
  • the alkyl group may be substituted or unsubstituted.
  • alkenyl refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more double bonds. Examples of alkenyl groups include allenyl, vinylmethyl and ethenyl. An alkenyl group may be unsubstituted or substituted.
  • alkynyl refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more triple bonds. Examples of alkynyls include ethynyl and propynyl. An alkynyl group may be unsubstituted or substituted.
  • cycloalkyl refers to a completely saturated (no double or triple bonds) mono- or multi- cyclic hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused fashion. Cycloalkyl groups can contain 3 to 10 atoms in the ring(s) or 3 to 8 atoms in the ring(s).
  • a cycloalkyl group may be unsubstituted or substituted.
  • Typical cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • cycloalkenyl refers to a mono- or multi- cyclic hydrocarbon ring system that contains one or more double bonds in at least one ring; although, if there is more than one, the double bonds cannot form a fully delocalized pi-electron system throughout all the rings (otherwise the group would be “aryl,” as defined herein).
  • Cycloalkenyl groups can contain 3 to 10 atoms in the ring(s) or 3 to 8 atoms in the ring(s). When composed of two or more rings, the rings may be connected together in a fused fashion. A cycloalkenyl group may be unsubstituted or substituted.
  • aryl refers to a carbocyclic (all carbon) monocyclic or multicyclic aromatic ring system (including fused ring systems where two carbocyclic rings share a chemical bond) that has a fully delocalized pi-electron system throughout all the rings. The number of carbon atoms in an aryl group can vary.
  • the aryl group can be a C 6 -C 14 aryl group, a C 6 -C 10 aryl group, or a C 6 aryl group.
  • aryl groups include, but are not limited to, benzene, naphthalene and azulene.
  • An aryl group may be substituted or unsubstituted.
  • heteroaryl refers to a monocyclic or multicyclic aromatic ring system (a ring system with fully delocalized pi-electron system) that contain(s) one, two, three or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur.
  • heteroaryl group can contain 4 to 14 atoms in the ring(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s).
  • heteroaryl includes fused ring systems where two rings, such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond.
  • heteroaryl rings include, but are not limited to, those described herein and the following: furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline and triazine.
  • heteroaryl group may be substituted or unsubstituted.
  • heterocyclyl or “heteroalicyclyl” refers to three-, four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-membered monocyclic, bicyclic, and tricyclic ring system wherein carbon atoms together with from 1 to 5 heteroatoms constitute said ring system.
  • a heterocycle may optionally contain one or more unsaturated bonds situated in such a way, however, that a fully delocalized pi-electron system does not occur throughout all the rings.
  • the heteroatom(s) is an element other than carbon including, but not limited to, oxygen, sulfur, and nitrogen.
  • a heterocycle may further contain one or more carbonyl or thiocarbonyl functionalities, so as to make the definition include oxo-systems and thio-systems such as lactams, lactones, cyclic imides, cyclic thioimides and cyclic carbamates. When composed of two or more rings, the rings may be joined together in a fused fashion. Additionally, any nitrogens in a heterocyclyl may be quaternized. Heterocyclyl or heteroalicyclic groups may be unsubstituted or substituted.
  • heterocyclyl or “heteroalicyclyl” groups include, but are not limited to, those described herein and the following: 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3- dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole, 1,3- dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 1,3-thiazinane, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazoline, imid
  • aralkyl and “aryl(alkyl)” refer to an aryl group connected, as a substituent, via a lower alkylene group.
  • the lower alkylene and aryl group of an aralkyl may be substituted or unsubstituted. Examples include but are not limited to benzyl, 2-phenylalkyl, 3- phenylalkyl and naphthylalkyl.
  • heteroarylkyl and “heteroaryl(alkyl)” refer to a heteroaryl group connected, as a substituent, via a lower alkylene group.
  • heteroaralkyl may be substituted or unsubstituted. Examples include but are not limited to 2- thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, imidazolylalkyl and their benzo-fused analogs.
  • a “heteroalicyclyl(alkyl)” and “heterocyclyl(alkyl)” refer to a heterocyclic or a heteroalicyclylic group connected, as a substituent, via a lower alkylene group.
  • the lower alkylene and heterocyclyl of a heteroalicyclyl(alkyl) may be substituted or unsubstituted. Examples include but are not limited tetrahydro-2H-pyran-4-yl(methyl), piperidin-4-yl(ethyl), piperidin-4-yl(propyl), tetrahydro-2H-thiopyran-4-yl(methyl), and 1,3-thiazinan-4-yl(methyl).
  • “Lower alkylene groups” are straight-chained -CH 2 - tethering groups, forming bonds to connect molecular fragments via their terminal carbon atoms.
  • alkoxy refers to the formula –OR wherein R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) is defined herein.
  • alkoxys are methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, phenoxy and benzoxy.
  • An alkoxy may be substituted or unsubstituted.
  • acyl refers to a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) connected, as substituents, via a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl and acryl. An acyl may be substituted or unsubstituted.
  • alkoxyalkyl refers to an alkoxy group connected, as a substituent, via a lower alkylene group. Examples include C 1-4 alkyl-O-(CH 2 ) n - ,wherein n is an integer in the range of 1 to 6.
  • aminoalkyl refers to an optionally substituted amino group connected, as a substituent, via a lower alkylene group. Examples include H2N(CH 2 ) n - ,wherein n is an integer in the range of 1 to 6.
  • hydroxyalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a hydroxy group. Exemplary hydroxyalkyl groups include but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. A hydroxyalkyl may be substituted or unsubstituted.
  • haloalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkyl, di-haloalkyl and tri-haloalkyl).
  • a halogen e.g., mono-haloalkyl, di-haloalkyl and tri-haloalkyl.
  • groups include but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloro-fluoroalkyl, chloro-difluoroalkyl and 2-fluoroisobutyl.
  • a haloalkyl may be substituted or unsubstituted.
  • haloalkoxy refers to an alkoxy group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkoxy, di- haloalkoxy and tri- haloalkoxy).
  • a halogen e.g., mono-haloalkoxy, di- haloalkoxy and tri- haloalkoxy.
  • groups include but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloro-fluoroalkyl, chloro-difluoroalkoxy and 2- fluoroisobutoxy.
  • a haloalkoxy may be substituted or unsubstituted.
  • a “sulfenyl” group refers to an “-SR” group in which R can be hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • a sulfenyl may be substituted or unsubstituted.
  • a sulfinyl may be substituted or unsubstituted.
  • a “sulfonyl” group refers to an “SO 2 R” group in which R can be the same as defined with respect to sulfenyl. A sulfonyl may be substituted or unsubstituted.
  • An O- carboxy may be substituted or unsubstituted.
  • a “trihalomethanesulfonyl” group refers to an “X 3 CSO 2 -” group wherein each X is a halogen.
  • a “trihalomethanesulfonamido” group refers to an “X 3 CS(O) 2 N(RA)-” group wherein each X is a halogen, and R A hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • the term “amino” as used herein refers to a –NH 2 group.
  • hydroxy refers to a –OH group.
  • a “cyano” group refers to a “-CN” group.
  • the term “azido” as used herein refers to a –N3 group.
  • An “isocyanato” group refers to a “-NCO” group.
  • a “thiocyanato” group refers to a “-CNS” group.
  • An “isothiocyanato” group refers to an “ -NCS” group.
  • S-sulfonamido refers to a “-SO 2 N(R A R B )” group in which R A and R B can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • An S-sulfonamido may be substituted or unsubstituted.
  • N-sulfonamido refers to a “RSO 2 N(R A )-” group in which R and R A can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • R and R A can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • An N-sulfonamido may be substituted or unsubstituted.
  • An O-carbamyl may be substituted or unsubstituted.
  • An N-carbamyl may be substituted or unsubstituted.
  • An O-thiocarbamyl may be substituted or unsubstituted.
  • An N-thiocarbamyl may be substituted or unsubstituted.
  • a C-amido may be substituted or unsubstituted.
  • R and R A can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • An N-amido may be substituted or unsubstituted.
  • a urea may be substituted or unsubstituted.
  • halogen atom or “halogen” as used herein, means any one of the radio- stable atoms of column 7 of the Periodic Table of the Elements, such as, fluorine, chlorine, bromine and iodine.
  • -------- indicates a single or double bond, unless stated otherwise.
  • substituents e.g. haloalkyl
  • substituents there may be one or more substituents present.
  • haloalkyl may include one or more of the same or different halogens.
  • C 1 -C 3 alkoxyphenyl may include one or more of the same or different alkoxy groups containing one, two or three atoms.
  • the abbreviations for any protective groups, amino acids and other compounds are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (See, Biochem. 11:942-944 (1972)).
  • optically active and ”enantiomerically active refer to a collection of molecules, which has an enantiomeric excess of no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, no less than about 91%, no less than about 92%, no less than about 93%, no less than about 94%, no less than about 95%, no less than about 96%, no less than about 97%, no less than about 98%, no less than about 99%, no less than about 99.5%, or no less than about 99.8%.
  • the compound comprises about 95% or more of the desired enantiomer and about 5% or less of the less preferred enantiomer based on the total weight of the two enantiomers in question.
  • R and S are used to denote the absolute configuration of the optically active compound about its chiral center(s).
  • the (+) and (-) are used to denote the optical rotation of an optically active compound, that is, the direction in which a plane of polarized light is rotated by the optically active compound.
  • the (-) prefix indicates that an optically active compound is levorotatory, that is, the compound rotates the plane of polarized light to the left or counterclockwise.
  • the (+) prefix indicates that an optically active compound is dextrorotatory, that is, the compound rotates the plane of polarized light to the right or clockwise.
  • the sign of optical rotation, (+) and (-) is not related to the absolute configuration of a compound, R and S.
  • isotopic variant refers to a compound that contains an unnatural proportion of an isotope at one or more of the atoms that constitute such a compound.
  • an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen ( 1 H), deuterium ( 2 H), tritium ( 3 H), carbon-11 ( 11 C), carbon-12 ( 12 C), carbon-13 ( 13 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen- 18 ( 18 O), fluorine-17 ( 17 F), fluorine-18 ( 18 F), phosphorus-31 ( 31 P), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-35 ( 35 S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-36 ( 36 Cl), chlorine-37 ( 37 Cl), bromine-79 ( 79 Br), bromine-81 ( 81 Br), iodine-123 (
  • an “isotopic variant” of a compound is in a stable form, that is, non-radioactive.
  • an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen ( 1 H), deuterium ( 2 H), carbon-12 ( 12 C), carbon-13 ( 13 C), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-16 ( 16 O), oxygen- 17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), phosphorus-31 ( 31 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-37 ( 37 Cl), bromine-79 ( 79 Br), bromine- 81 ( 81 Br), and iodine-127 ( 127 I).
  • an “isotopic variant” of a compound is in an unstable form, that is, radioactive.
  • an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, tritium ( 3 H), carbon-11 ( 11 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), fluorine-18 ( 18 F), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-35 ( 35 S), chlorine-36 ( 36 Cl), iodine-123 ( 123 I), iodine-125 ( 125 I), iodine-129 ( 129 I), and iodine-131 ( 131 I).
  • any hydrogen can be 2 H, for example, or any carbon can be 13 C, for example, or any nitrogen can be 15 N, for example, or any oxygen can be 18 O, for example, where feasible according to the judgment of one of skill.
  • an “isotopic variant” of a compound contains unnatural proportions of deuterium (D).
  • solvate refers to a complex or aggregate formed by one or more molecules of a solute, e.g., a compound provided herein, and one or more molecules of a solvent, which present in a stoichiometric or non-stoichiometric amount.
  • Suitable solvents include, but are not limited to, water, methanol, ethanol, n-propanol, isopropanol, and acetic acid.
  • the solvent is pharmaceutically acceptable.
  • the complex or aggregate is in a crystalline form.
  • the complex or aggregate is in a noncrystalline form.
  • the solvent is water
  • the solvate is a hydrate. Examples of hydrates include, but are not limited to, a hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate.
  • an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof has the same meaning as the phrase “(i) an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, or an isotopic variant of the compound referenced therein; (ii) a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of the compound referenced therein; or (iii) a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, or an isotopic variant of the compound referenced therein.” 5.3.2.
  • the stem cell mobilizing factor is a compound having Formula (I), (I-A), (I-B), (I-C), or (I-D), as described below.
  • Formula (I) [00248]
  • R a can be hydrogen or C 1 -C 4 alkyl;
  • R b can be R c or -(C 1 -C 4 alkyl)-R c ;
  • R c can be selected from the group consisting of: -OH,
  • joining Y and Z can represent a single bond. In other embodiments, can represent a double bond. In some embodiments, joining Y and Z can represent a single bond. In other embodiments, joining Y and Z can represent a double bond. In some embodiments, when joining G and J representes a single bond, G can be N and the N is substituted with R G . In other embodiments, when joining G and J represents a double bond, G can be N. In some embodiments, when joining G and J representes a double bond, then joining J and R J can be a single bond. In some embodiments, when joining G and J representes a double bond, then joining J and R J can not be a double bond.
  • joining G and J when joining J and R J representes a double bond, then joining G and J can be a single bond. In some embodiments, when joining J and R J representes a double bond, then joining G and J can not be a double bond.
  • R a can be hydrogen. In some embodiments, R a can be C 1 - C 4 alkyl. For example, R a can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl. [00252] In some embodiments, R b can be R c . In some embodiments, R b can be -(C 1 -C 4 alkyl)-R c .
  • R b can be -CH 2 -R c , -CH 2 CH 2 -R c , -CH 2 CH 2 CH 2 -R c , or -CH 2 CH 2 CH 2 CH 2 -R c .
  • R c can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.
  • the moiety when a R c moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents E.
  • E can be –OH.
  • E can be C 1 -C 4 alkyl.
  • E can be C 1 -C 4 haloalkyl.
  • E can be -O(C 1 -C 4 alkyl).
  • E can be -O(C 1 -C 4 haloalkyl).
  • R c when R b is -CH 2 CH 2 -R c , R c can be unsubstituted C 6-10 aryl. In other embodiments, when R b is -CH 2 CH 2 -R c , R c can be substituted C 6-10 aryl. In still other embodiments, when R b is -CH 2 CH 2 -R c , R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.
  • R b can be -(C 1 -C 4 alkyl)-R c and R c can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.
  • R c moiety When a R c moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents E.
  • E can be –OH.
  • E can be C 1 -C 4 alkyl.
  • E can be C 1 -C 4 haloalkyl.
  • E can be -O(C 1 -C 4 alkyl).
  • E can be -O(C 1 -C 4 haloalkyl).
  • R c when R b is -CH 2 CH 2 -R c , R c can be phenyl. In other embodiments, when R b is -CH 2 CH 2 -R c , R c can be naphthyl. In still other embodiments, when R b is -CH 2 CH 2 -R c , R c can be hydroxyphenyl. In still other embodiments, when R b is -CH 2 CH 2 -R c , R c can be indolyl. [00256] In some embodiments, R K can be hydrogen.
  • R K can be unsubstituted C 1-6 alkyl.
  • R K can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, pentyl (branched and straight-chained), or hexyl (branched and straight-chained).
  • R K can be substituted C 1-6 alkyl.
  • R K can be -NH(C 1-4 alkyl).
  • R K can be - NH(CH 3 ), -NH(CH 2 CH 3 ), -NH(isopropyl), or -NH(sec-butyl). In other embodiments, R K can be - N(C 1-4 alkyl) 2 . [00257] In some embodiments, R K can be unsubstituted C 6-10 aryl. In other embodiments, R K can be substituted C 6-10 aryl. In other embodiments, R K can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.
  • R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.
  • the moiety can be substituted with one or more, for example, one, two, three, or four substituents substituents Q.
  • Q can be -OH.
  • Q can be C 1-4 alkyl.
  • Q can be C 1-4 haloalkyl.
  • Q can be halo.
  • Q can be cyano.
  • Q can be -O-(C 1-4 alkyl).
  • Q can be -O-(C 1-4 haloalkyl).
  • R K can be phenyl or naphthyl. In other embodiments, R K can be benzothiophenyl. In other embodiments, R K can be benzothiophenyl. In other embodiments, R K can be benzothiophenyl. In still other embodiments, R K can be pyridinyl. In yet still other embodiments, R K can be pyridinyl substituted with one or more substituents Q.
  • R K can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl.
  • R G can be hydrogen.
  • R G can be C 1- 4 alkyl.
  • R Y and R Z can independently be absent.
  • R Y and R Z can independently be hydrogen.
  • R Y and R Z can independently be halo.
  • R Y and R Z can independently be C 1-6 alkyl. In other embodiments, R Y and R Z can independently be –OH. In still other embodiments, R Y and R Z can independently be -O-(C 1-4 alkyl). In other embodiments, R Y and R Z can independently be -NH(C 1-4 alkyl). For example, R Y and R Z can independently be -NH(CH 3 ), -NH(CH 2 CH 3 ), -NH(isopropyl), or -NH(sec-butyl). In other embodiments, R Y and R Z can independently be - N(C 1-4 alkyl)2.
  • R Y and R Z taken together with the atoms to which they are attached can be joined together to form a ring. In some embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In still other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form .
  • R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In yet other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In yet still other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form .
  • R Y and R Z taken together with the atoms to which they are attached can be joined together to form and .
  • the ring can be substituted with one, two, or three groups independently selected from C 1 -C 4 alkyl, -N(C 1 -C 4 alkyl) 2 , cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms.
  • R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form .
  • R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form . In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be joined together to form .
  • R Y and R Z taken together with the atoms to which they are attached can be joined together to form a ring
  • the ring can be substituted with one, two, or three groups independently selected from C 1 -C 4 alkyl, -N(C 1 -C 4 alkyl) 2 , cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms.
  • R Y and R Z taken together with the atoms to which they are attached can be .
  • R Y and R Z taken together with the atoms to which they are attached can b .
  • R Y and R Z taken together with the atoms to which they are attached can be .
  • R Y and R Z taken together with the atoms to which they are attached can be In other embodiments, R Y and R Z taken together with the atoms to which they are attached can be .
  • R d can be hydrogen.
  • R d can be C1- C 4 alkyl.
  • R d can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl.
  • R d can be halo.
  • R d can be cyano.
  • R m can be hydrogen.
  • R m can be C1- C 4 alkyl.
  • R m can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl.
  • R m can be halo.
  • R m can be fluoro, chloro, bromo, or iodo.
  • R m can be cyano.
  • X, Y, and Z can each be independently N or C, wherein the valency of any carbon atom is filled as needed with hydrogen atoms.
  • X can be N, Y can be N, and Z can be N.
  • X can be N, Y can be N, and Z can be CH.
  • X can be N, Y can be CH, and Z can be N.
  • X can be CH, Y can be N, and Z can be N.
  • X can be CH, Y can be CH, and Z can be N.
  • X can be CH, Y can be CH, and Z can be N.
  • X can be CH, Y can be CH, and Z can be N.
  • X can be CH, Y can be N, and Z can be CH.
  • X can be N, Y can be CH, and Z can be CH. In other embodiments, X can be CH, Y can be CH, and Z can be CH.
  • R a can be hydrogen;
  • R b can be -CH 2 CH 2 -R c ;
  • R K can be selected from the group consisting of: hydrogen, methyl, substituted pyridinyl, unsubstituted benzothiophenyl, and -NH(C 1 -C 4 alkyl);
  • R Y can be -NH(C 1 -C 4 alkyl);
  • R Z can be absent or hydrogen; or R Y and R Z taken together with the atoms to which they are attached can be joined together to form a ring selected from: ,
  • ring can be optionally substituted with one, two, or three groups independently selected from C 1 -C 4 alkyl, -N(C 1 -C 4 alkyl) 2 , cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms;
  • R d can be C 1 -C 4 alkyl;
  • R m can be cyano; and
  • X can be N or CH.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; or R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; or R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; substituted with one or more Q, wherein Q can be selected from cyano, halo, or C 1 -C 4 alkyl; R
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond, R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be substituted C 6-10 aryl; substituted with one or more E, wherein E can be –OH; R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R Y can be -NH(C 1-4 alkyl); R Z can be hydrogen; J can be C; X can be N; Y can be C; Z can be C; and joining Y and Z can be a double bond.
  • the compound of Formula (I) can be 4-(2-((2-(benzo[b]thiophen-3-yl)-6- (isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c , R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E can be –OH; R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R Y and R Z taken together is wherein the ring is substituted with C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be 4-(2-((2-(benzo[b]thiophen-3-yl)-7- isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c , R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E can be –OH; R K can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R Y and R Z taken together is can be C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be 4-(2-((2- (benzo[b]thiophen-3-yl)-7-isopropyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4- yl)amino)ethyl)phenol.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c , R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E can be –OH; R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R Y and R Z taken together is can be C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one.
  • the compound of Formula (I) can be 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- yl)oxy)propanamide.
  • R J when R J is is –NR a R b ; G can be N; joining G and J can be a double bond; R b can be –CH 2 CH 2 -R c ; R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K is unsubstituted five- to ten-membered heteroaryl having 1- 4 atoms selected from the group consisting of O, N, and S; R Y and R Z taken together can be wherein said ring is substituted with -N(C 1-4 alkyl)2; J can be C; X can be N; Y can be C; and Z is C.
  • the compound of Formula (I) can be 4-(2-((2- (benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4-yl)amino)ethyl)phenol.
  • R J when R J is is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; R Y can be -NH(C 1-4 alkyl); R Z can be absent; J can be C; X can be C; Y can be C; Z can be N; and joining Y and Z can be a double bond.
  • the compound of Formula (I) can be 5-(2-((2-(1H-indol-3-yl)ethyl)amino)-6-(sec- butylamino)pyrimidin-4-yl)nicotinonitrile.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be unsubstituted C 1-6 alkyl; R Y and R Z taken together can herein the ring is substituted with unsubstituted C6-C10 aryl; J can be C; X can be N; Y can be C; Z can be C. .
  • the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6- phenylthieno[2,3-d]pyrimidin-4-amine
  • R J when R J can be –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be hydrogen; R Y and R Z taken together can b wherein the ring is substituted with substituted C6-C10 aryl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine [00281]
  • R J O
  • G can be N substituted with R G ; joining G and J can be a single bond;
  • R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S;
  • R Y and R Z taken together can b can be C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be 3-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond R a can be hydrogen R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q can be halo; R Y and R Z taken together can b an be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine.
  • R J when R J is –NR a R b ; G is N; joining G and J can be a double bond; R a can be hydrogen R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q can be cyano; R Y and R Z taken together is an be C; X can be N;
  • the compound of Formula (I) can be 5-(4- ((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be -NH(C 1-4 alkyl); R Y and R Z taken together can b an be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be N 4 -(2-(1H- indol-3-yl)ethyl)-N 2 -(sec-butyl)quinazoline-2,4-diamine.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R Y and R Z taken together can b wherein the ring is substituted with cyano; R d can be C 1 -C 4 alkyl; J can be C; X can be N; Y can be C;
  • the compound of Formula (I) can be 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H- pyrrolo[2,3-d]pyrimidine-5-carbonitrile.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R Y and R Z taken together can b wherein the ring is substituted with C 1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms.
  • the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3- isopropylimidazo[1,5-a]pyrazin-8-amine.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R Y and R Z taken together can b wherein the ring can be substituted with C 1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms.
  • the compound of Formula (I) can be 4-(2- ((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-yl)amino)ethyl)phenol.
  • R J when R J is –NR a R b ; G can be N; joining G and J represents a double bond; R a can be hydrogen R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; R Y and R Z taken together is wherein the ring is substituted with C 1 -C 4 alkyl;J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7- isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile.
  • R J when R J is –NR a R b ; G can be N; joining G and J represents a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; R Y and R Z taken together can be wherein the ring is substituted with C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin- 3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; R Y and R Z taken together can b an be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be N-(2- (1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)furo[3,2-d]pyrimidin-4-amine.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is C 1 -C 4 alkyl; R Y and R Z taken together can b an be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is C 1 -C 4 alkyl; R Y and R Z taken together can b herein the ring is substituted with C 1 -C 4 alkyl J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5- methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine.
  • R J when R J is –NR a R b ; G is N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; R Y and R Z taken together can b an be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I) can be 5-(4- ((2-(1H-indol-3-yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile.
  • compound of Formula (I) wherein the compound can be selected from: 4-(2-((2-(benzo[b]thiophen-3-yl)-6-(isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol; 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol; 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4- yl)amino)ethyl)phenol; 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-5,7-dihydro-6H-
  • the compound of Formula (I) can have the structure of Formula (I- ncluding pharmaceutically acceptable salts thereof, wherein: R J can be –NR a R b ; R a can be hydrogen or C 1 -C 4 alkyl; R b can be R c or - (C 1 -C 4 alkyl)-R c ; R c can be selected from the group consisting of: unsubstituted C 6-10 aryl; substituted C 6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R c moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting
  • R a can be hydrogen. In other embodiments, R a can be C 1 - C4 alkyl.
  • R b can be -(C 1 -C 4 alkyl)-R c .
  • R b can be -CH 2 - R c , -CH 2 CH 2 -R c , -CH 2 CH 2 CH 2 -R c , or -CH 2 CH 2 CH 2 CH 2 -R c .
  • R c can be –OH. In some embodiments, R c can be -O(C 1 -C 4 alkyl).
  • R c when a R c moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents E.
  • E can be –OH.
  • E can be C 1 -C 4 alkyl.
  • E can be C 1 -C 4 haloalkyl.
  • E can be -O(C 1 -C 4 alkyl).
  • E can be - O(C 1 -C 4 haloalkyl).
  • R c can be phenyl.
  • R c can be hydroxyphenyl.
  • R c can be indolyl.
  • R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.
  • R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein the substituted heteroaryl can substituted with one or more substituents Q, wherein each Q can independently selected from the group consisting of: -OH, C1- 4 alkyl, C 1-4 haloalkyl, halo, cyano, -O-(C 1-4 alkyl), and -O-(C 1-4 haloalkyl).
  • R K can be pyridinyl. In other embodiments, R K can be pyridinyl substituted with one or more substituents Q. For example, R K can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl.
  • R e can be hydrogen. In some embodiments, R e can be C 1 - C4 alkyl. For example, R e can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl.
  • R a can be hydrogen;
  • R b can be -(C 1 -C 4 alkyl)-R c ;
  • R c can be selected from the group consisting of: unsubstituted C 6-10 aryl; substituted C 6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R c moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, -O(C 1 -C 4 alkyl), and -O(C 1 -C 4 haloalkyl);
  • R K can be selected from the group consisting
  • R a can be hydrogen;
  • R b can be -(CH 2 -CH 2 )-R c ;
  • R c can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E can be -OH;
  • R K can be selected from the group consisting of: unsubstituted benzothiophenyl and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one substituent Q, wherein Q can be selected from the group consisting of: C 1-4 alkyl, halo, and cyano; and
  • R e can be isopropyl.
  • R J when W is O, R J can be –NR a R b ; R a can be hydrogen; R b can be -CH 2 CH 2 -R c ; R c can be selected from the group consisting of: unsubstituted C 6-10 aryl; substituted C 6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R c moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C 1 -C 4 alkyl, and -O(C 1 -C 4 alkyl); R K can be selected from the group consisting of unsubstituted five- to ten-membere
  • R J when W is S, R J can be –NR a R b ; R a can be hydrogen; R b can be -CH 2 CH 2 -R c ; R c can be selected from the group consisting of: unsubstituted C 6-10 aryl; substituted C 6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R c moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C 1 -C 4 alkyl, and -O(C 1 -C 4 alkyl); R K can be selected from the group consisting of unsubstituted five- to ten-membere
  • R J when R J is –NR a R b ; G can be N; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is C 1 -C 4 alkyl; W can be S; R e can be C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-A) can be N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2- (5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine.
  • R J when R J is –NR a R b ; G can be N; R a can be hydrogen R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; W can be S; R e can be C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-A) can be 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7- isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile.
  • R J when R J is –NR a R b ; G can be N; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; W can be S; R e can be C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-A) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5- fluoropyridin-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine.
  • R J when R J is –NR a R b ; G can be N; R a can be hydrogen; R b can be –CH 2 CH 2 -R c , R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E can be –OH; R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; W can be S; R e can be C 1 -C 4 alkyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-A) can be 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol.
  • R J when R J is –NR a R b ; G can be N; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; W can be O; R e can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-A) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3- yl)furo[3,2-d]pyrimidin-4-amine.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is C 1 -C 4 alkyl; W can be O; R e can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-A) can be N-(2- (1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine.
  • R J when R J is –NR a R b ; G is NR a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; W can be O; R e can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-A) can be 5-(4-((2-(1H-indol-3- yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile.
  • the compound of Formula (I-A), or a pharmaceutically acceptable salt thereof can selected from the group consisting of: N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine; 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7-isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile; N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine; 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine;
  • R c can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, R c can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, when a R c moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents E. In some embodiments, E can be –OH. In some embodiments, E can be C 1 -C 4 alkyl. In some embodiments, E can be C 1 -C 4 haloalkyl.
  • E can be -O(C 1 -C 4 alkyl). In some embodiments, E can be - O(C 1 -C 4 haloalkyl). [00317]
  • R K can be hydrogen. In other embodiments, R K can be C1- C 4 alkyl. For example, R K can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl.
  • R K can be selected from the group consisting of: unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein the substituted heteroaryl can substituted with one or more substituents Q, wherein each Q can independently selected from the group consisting of: -OH, C 1- 4 alkyl, C 1-4 haloalkyl, halo, cyano, -O-(C 1-4 alkyl), and -O-(C 1-4 haloalkyl).
  • R K can be benzothiophenyl.
  • R K can be pyridinyl substituted with one or more substituents Q.
  • R K can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl.
  • R f can be hydrogen. In other embodiments, R f can be C 1-4 alkyl.
  • R f can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl.
  • R f can be unsubstituted C 6 -C 10 aryl.
  • R f can be C 6 - C10 aryl substituted with 1-5 halo atoms.
  • R f can be phenyl substituted with 1-5 halo atoms.
  • R f can be fluorophenyl.
  • U can be N. In other embodiments, U can be CR U .
  • V can be S. In other embodiments, V can be NR V .
  • R U can be hydrogen. In some embodiments, R U can be C 1-4 alkyl. In other embodiments R U can be halo. For example, R U can be fluoro, chloro, bromo, or iodo. In still other embodiments, R U can be cyano.
  • R V can be hydrogen. In other embodiments, R V can be C 1-4 alkyl.
  • R V can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl.
  • Y and Z can each be C and X can be N. In other embodiments, Y and Z can each be C and X can be CH.
  • R a can be hydrogen;
  • R b can be -(C 1-4 alkyl)-R c ;
  • R a can be hydrogen;
  • R b can be -(CH 2 -CH 2 )-R c ;
  • R K can be selected from the group consisting of: unsubstituted benzothiohenyl and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one substituent Q, wherein Q can be selected from the group consisting of: C 1-4 alkyl, halo, and cyano;
  • R f can be selected from the group consisting of hydrogen, phenyl, and fluorophenyl;
  • Y and Z each can be C;
  • the compound of Formula (I-B) can be 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- yl)oxy)propanamide.
  • R J O
  • G can be N substituted with R G ; joining G and J can be a single bond;
  • R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S;
  • U can N;
  • V can be NR v ;
  • R v can be C 1 -C 4 alkyl;
  • R f can be hydrogen; J can be C;
  • X can be N;
  • the compound of Formula (I-B) can be 3- (2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; U can be CR u ; R u can be cyano; V can be NR v ; R v can be C 1 -C 4 alkyl; R f can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-B) can be 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3- d]pyrimidine-5-carbonitrile.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be unsubstituted C 1-6 alkyl; U can be CR u ; R u can be hydrogen; V can be S; R f can be phenyl; J can be C; X can be N; Y can be C; Z can be C.
  • the compound of Formula (I-B) can be N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4- amine.
  • R J when R J can be –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be hydrogen; U can be CR u ; R u can be hydrogen; V can be S; R f can be fluorophenyl; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-B) can be N-(2-(1H-indol-3-yl)ethyl)-6- (4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine.
  • the compound of Formula (I-B), or a pharmaceutically acceptable salt thereof can selected from the group consisting of: 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-yl)oxy)propanamide; 3-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide; 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3- d]pyrimidine-5-carbonitrile; N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4-amine; and N-(2-(1H-indol-3-yl)eth
  • the compound of Formula (I) can have the structure of Formula (I- ncluding pharmaceutically acceptable salts thereof, wherein: R J can be –NR a R b ; R a can be hydrogen or C 1 -C 4 alkyl; R b can be R c or - (C 1 -C 4 alkyl)-R c ; R c can be selected from the group consisting of: unsubstituted C 6-10 aryl; substituted C 6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R c moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting
  • R K can be -NH(C 1-4 alkyl).
  • R K can be -NH(CH 3 ), -NH(CH 2 CH 3 ), -NH(isopropyl), or -NH(sec-butyl).
  • R K can be unsubstituted benzothiophenyl.
  • R K can be substituted pyridinyl.
  • R K can be methylpyridinyl, ethylpyridinyl, cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl.
  • A can be N and B can be N. In other embodiments, A can be N and B can be CH. In still other embodiments, A can be CH and B can be N. In yet still other embodiments, A can be CH and B can be CH. [00337] In some embodiments, R g can be hydrogen. In other embodiments, R g can be - N(C 1-4 alkyl)2. In certain embodiments, R g can be -N(CH 3 ) 2 .
  • R a can be hydrogen;
  • R b can be -(C 1 -C 4 alkyl)-R c ;
  • R c can be selected from the group consisting of: unsubstituted C 6-10 aryl; substituted C 6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R c moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, -O(C 1 -C 4 alkyl), and -O(C 1 -C 4 haloalkyl);
  • R K can be selected from the
  • R a can be hydrogen;
  • R b can be -(C 1 -C 4 alkyl)-R c ;
  • R c can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, -O(C 1 -C 4 alkyl), and -O(C 1 -C 4 haloalkyl);
  • R K can be selected from the group consisting of: -NH(C 1-4 alkyl); unsubstituted benzothiophenyl; and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -
  • R a can be hydrogen;
  • R b can be -(CH 2 CH 2 )-R c ;
  • R c can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E can be -OH;
  • R K can be selected from the group consisting of: -NH(sec-butyl); unsubstituted benzothiohenyl, and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: C 1-4 alkyl, halo, and cyano; and
  • R g can be hydrogen or -N(CH 3 )2.
  • R J when A is C and B is C, R J can be –NR a R b ; G can be N; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; or unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R g can be hydrogen; J can be C; X can be N; Y can be C; and Z is C.
  • R J when R J is –NR a R b ; G can be N; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K is unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; A can be N; B can be N; R g can be -N(C 1-4 alkyl)2; J can be C; X can be N; Y can be C; and Z is C.
  • the compound of Formula (I-C) can be 4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4- yl)amino)ethyl)phenol.
  • R J when R J is –NR a R b ; G can be N; R a can be hydrogen R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R K moiety indicated as substituted is substituted with one or more Q, wherein Q can be halo; A can be CH; B can be CH; R g can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-C) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5- fluoropyridin-3-yl)quinazolin-4-amine.
  • R a can be hydrogen
  • R b can be –CH 2 CH 2 -R c
  • R c can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S
  • R K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S
  • a R K moiety indicated as substituted is substituted with one or more Q, wherein Q can be cyano
  • A can be CH
  • B can be CH
  • R g can be hydrogen
  • J can be cyano
  • A can be CH
  • B can be CH
  • R g can be hydrogen
  • J can be cyano
  • the compound of Formula (I-C) can be 5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R K can be -NH(C 1-4 alkyl); A can be CH; B can be CH; R g can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C.
  • the compound of Formula (I-C) can be N 4 - (2-(1H-indol-3-yl)ethyl)-N 2 -(sec-butyl)quinazoline-2,4-diamine.
  • the compound of Formula (I-C), or a pharmaceutically acceptable salt thereof can selected from the group consisting of: 4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4- yl)amino)ethyl)phenol; N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine; 5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile; and N 4 -(2-(1H-indol-3-yl)ethyl)-N 2 -(sec-butyl)quinazoline-2,4-diamine.
  • the compound of Formula (I) can have the structure of Formula (I ncluding pharmaceutically acceptable salts thereof, wherein: R J can be –NR a R b ; R a can be hydrogen or C 1 -C 4 alkyl; R b can be R c or -(C 1-4 alkyl)-R c ; R c can be selected from the group consisting of: unsubstituted C 6-10 aryl; substituted C 6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R c moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of:
  • R h can be hydrogen. In other embodiments, R h can be C 1-4 alkyl. For example, R h can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl.
  • D can be N. In other embodiments, D can be CH.
  • Y when D is N, Y can be N, Z can be C, and X can be N. In other embodiments, when D is N, Y can be N, Z can be C, and X can be CH.
  • R a can be hydrogen; R b can be -(C 1-4 alkyl)-R c ; R c can be selected from the group consisting of: unsubstituted C 6-10 aryl; substituted C 6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a R c moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C 1 -C 4 alkyl
  • R a can be hydrogen;
  • R b can be -(C 1 -C 4 alkyl)-R c ;
  • R c can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, -O(C 1 -C 4 alkyl), and -O(C 1 -C 4 haloalkyl);
  • R K can be unsubstituted benzothiophenyl; and
  • R h can be hydrogen or C 1-4 alkyl.
  • R a can be hydrogen;
  • R b can be -(CH 2 -CH 2 )-R c ;
  • R c can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E can be -OH;
  • R K can be unsubstituted benzothiophenyl; and
  • R h can be hydrogen or C 1-4 alkyl.
  • R J when D is N; R J is –NR a R b ; G can be N; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; or substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R h can be C 1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms.
  • R J when R J is –NR a R b ; G can be N; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S or substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; D can be N; R h can be C 1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms.
  • the compound of Formula (I-D) can be N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine.
  • R J when R J is –NR a R b ; G can be N; joining G and J can be a double bond; R a can be hydrogen; R b can be –CH 2 CH 2 -R c ; R c can be substituted C 6-10 aryl, substituted with one or more E, wherein E is –OH; R K can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; D can be N; R h can be C 1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms.
  • the compound of Formula (I-D) can be 4-(2-((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5- a]pyrazin-8-yl)amino)ethyl)phenol.
  • the compound of Formula (I-D), or a pharmaceutically acceptable salt thereof can selected from the group consisting of: N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine; and 4-(2-((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-yl)amino)ethyl)phenol.
  • the compounds provided herein may be enantiomerically pure, such as a single enantiomer or a single diastereomer, or be stereoisomeric mixtures, such as a mixture of enantiomers, e.g., a racemic mixture of two enantiomers; or a mixture of two or more diastereomers.
  • a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.
  • NK cells can be isolated or enriched, for example, by staining cells, in one embodiment, with antibodies to CD56 and CD3, and selecting for CD56 + CD3 – cells.
  • the NK cells are enriched for CD56 + CD3 – cells in comparison with total cells produced using the three-stage method, described herein.
  • NK cells e.g., cells produced using the three-stage method, described herein, can be isolated using a commercially available kit, for example, the NK Cell Isolation Kit (Miltenyi Biotec).
  • NK cells e.g., cells produced using the three-stage method, described herein
  • NK cells e.g., cells produced using the three-stage method, described herein
  • Negative isolation can be carried out using a commercially available kit, e.g., the NK Cell Negative Isolation Kit (Dynal Biotech).
  • Cells isolated by these methods may be additionally sorted, e.g., to separate CD11a+ and CD11a- cells, and/or CD117+ and CD117- cells, and/or CD16 + and CD16 – cells, and/or CD94 + and CD94 – .
  • cells e.g., cells produced by the three-step methods described herein, are sorted to separate CD11a+ and CD11a- cells.
  • CD11a+ cells are isolated.
  • the cells are enriched for CD11a + cells in comparison with total cells produced using the three-stage method, described herein.
  • CD11a- cells are isolated.
  • the cells are enriched for CD11a- cells in comparison with total cells produced using the three-stage method, described herein.
  • cells are sorted to separate CD117+ and CD117- cells.
  • CD117+ cells are isolated.
  • the cells are enriched for CD117 + cells in comparison with total cells produced using the three-stage method, described herein.
  • CD117- cells are isolated.
  • the cells are enriched for CD117- cells in comparison with total cells produced using the three-stage method, described herein.
  • cells are sorted to separate CD16 + and CD16 – cells.
  • CD16 + cells are isolated.
  • the cells are enriched for CD16 + cells in comparison with total cells produced using the three-stage method, described herein.
  • CD16 – cells are isolated.
  • the cells are enriched for CD16- cells in comparison with total cells produced using the three-stage method, described herein.
  • cells are sorted to separate CD94 + and CD94 – cells.
  • CD94 + cells are isolated.
  • the cells are enriched for CD94 + cells in comparison with total cells produced using the three-stage method, described herein.
  • CD94 – cells are isolated.
  • the cells are enriched for CD94- cells in comparison with total cells produced using the three-stage method, described herein.
  • isolation is performed using magnetic separation.
  • isolation is performed using flow cytometry.
  • Methods of isolating ILC3 cells are known in the art and can be used to isolate the ILC3 cells, e.g., ILC3 cells produced using the three-stage method, described herein.
  • ILC3 cells can be isolated or enriched, for example, by staining cells, in one embodiment, with antibodies to CD56, CD3, and CD11a, and selecting for CD56 + CD3 – CD11a – cells.
  • ILC3 cells e.g., cells produced using the three-stage method, described herein
  • ILC3 cells e.g., cells produced using the three-stage method, described herein
  • NK cells isolated by these methods may be additionally sorted, e.g., to separate CD117 + and CD117 – cells.
  • NK cells can be isolated or enriched, for example, by staining cells, in one embodiment, with antibodies to CD56, CD3, CD94, and CD11a, and selecting for CD56 + CD3 – CD94 + CD11a + cells.
  • NK cells e.g., cells produced using the three-stage method, described herein, can also be isolated or enriched by removal of cells other than NK cells in a population of cells that comprise the NK cells, e.g., cells produced using the three-stage method, described herein.
  • the NK cells are enriched for CD56 + CD3 – CD94 + CD11a + cells in comparison with total cells produced using the three-stage method, described herein.
  • ILC3 cells are isolated or enriched by selecting for CD56 + CD3 – CD11a – cells. In certain embodiments, the ILC3 cells are enriched for CD56 + CD3 – CD11a – cells in comparison with total cells produced using the three-stage method, described herein. In one embodiment, ILC3 cells are isolated or enriched by selecting for CD56 + CD3 – CD11a – CD117+ cells.
  • the ILC3 cells are enriched for CD56 + CD3 – CD11a – CD117+ cells in comparison with total cells produced using the three-stage method, described herein. In one embodiment, ILC3 cells are isolated or enriched by selecting for CD56 + CD3 – CD11a – CD117 + CDIL1R1 + cells. In certain embodiments, the ILC3 cells are enriched for CD56 + CD3 – CD11a – CD117 + CDIL1R1 + cells in comparison with total cells produced using the three-stage method, described herein. [00362] In one embodiment, NK cells are isolated or enriched by selecting for CD56 + CD3 – CD94 + CD11a + cells.
  • the NK cells are enriched for CD56 + CD3 – CD94 + CD11a + cells in comparison with total cells produced using the three-stage method, described herein.
  • NK cells are isolated or enriched by selecting for CD56 + CD3 – CD94 + CD11a + CD117 – cells.
  • the NK cells are enriched for CD56 + CD3 – CD94 + CD11a + CD117 – cells in comparison with total cells produced using the three- stage method, described herein.
  • Cell separation can be accomplished by, e.g., flow cytometry, fluorescence- activated cell sorting (FACS), or, in one embodiment, magnetic cell sorting using microbeads conjugated with specific antibodies.
  • FACS fluorescence- activated cell sorting
  • the cells may be isolated, e.g., using a magnetic activated cell sorting (MACS) technique, a method for separating particles based on their ability to bind magnetic beads (e.g., about 0.5-100 ⁇ m diameter) that comprise one or more specific antibodies, e.g., anti-CD56 antibodies.
  • Magnetic cell separation can be performed and automated using, e.g., an AUTOMACSTM Separator (Miltenyi).
  • a variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically recognizes a particular cell surface molecule or hapten.
  • the beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker.
  • these cells can then isolated and re-mixed with magnetic beads coupled to an antibody against additional cell surface markers.
  • the cells are again passed through a magnetic field, isolating cells that bound both the antibodies.
  • Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation. 5.5.
  • NK cells and/or ILC3 cells e.g., NK cell and/or ILC3 cell populations produced according to the three-stage method described herein may be produced from hematopoietic cells, e.g., hematopoietic stem or progenitors from any source, e.g., placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, or the like.
  • the hematopoietic stem cells are combined hematopoietic stem cells from placental perfusate and from cord blood from the same placenta used to generate the placental perfusate.
  • Placental perfusate comprising placental perfusate cells that can be obtained, for example, by the methods disclosed in U.S. Patent Nos.7,045,148 and 7,468,276 and U.S. Patent Application Publication No.2009/0104164, the disclosures of which are hereby incorporated in their entireties. 5.5.1.
  • the placental perfusate and perfusate cells, from which hematopoietic stem or progenitors may be isolated, or useful in tumor suppression or the treatment of an individual having tumor cells, cancer or a viral infection, e.g., in combination with the NK cells and/or ILC3 cells, e.g., NK cell and/or ILC3 cell populations produced according to the three-stage method provided herein, can be collected by perfusion of a mammalian, e.g., human post-partum placenta using a placental cell collection composition.
  • Perfusate can be collected from the placenta by perfusion of the placenta with any physiologically-acceptable solution, e.g., a saline solution, culture medium, or a more complex cell collection composition.
  • a physiologically-acceptable solution e.g., a saline solution, culture medium, or a more complex cell collection composition.
  • a cell collection composition suitable for perfusing a placenta, and for the collection and preservation of perfusate cells is described in detail in related U.S. Application Publication No.2007/0190042, which is incorporated herein by reference in its entirety.
  • the cell collection composition can comprise any physiologically-acceptable solution suitable for the collection and/or culture of stem cells, for example, a saline solution (e.g., phosphate-buffered saline, Kreb’s solution, modified Kreb’s solution, Eagle’s solution, 0.9% NaCl. etc.), a culture medium (e.g., DMEM, H.DMEM, etc.), and the like.
  • a saline solution e.g., phosphate-buffered saline, Kreb’s solution, modified Kreb’s solution, Eagle’s solution, 0.9% NaCl. etc.
  • a culture medium e.g., DMEM, H.DMEM, etc.
  • the cell collection composition can comprise one or more components that tend to preserve placental cells, that is, prevent the placental cells from dying, or delay the death of the placental cells, reduce the number of placental cells in a population of cells that die, or the like, from the time of collection to the time of culturing.
  • Such components can be, e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesium sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin- releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.); a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); a TNF- ⁇ inhibitor; and/or an oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bro
  • the cell collection composition can comprise one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, a hyaluronidase, an RNase, or a DNase, or the like.
  • tissue-degrading enzymes include, but are not limited to, collagenases (e.g., collagenase I, II, III or IV, a collagenase from Clostridium histolyticum, etc.); dispase, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.
  • the cell collection composition can comprise a bacteriocidally or bacteriostatically effective amount of an antibiotic.
  • the antibiotic is a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc.
  • a macrolide e.g., tobramycin
  • a cephalosporin e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil
  • a clarithromycin an erythromycin
  • the antibiotic is active against Gram(+) and/or Gram(–) bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus, and the like.
  • the cell collection composition can also comprise one or more of the following compounds: adenosine (about 1 mM to about 50 mM); D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50 mM); a macromolecule of molecular weight greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain endothelial integrity and cellular viability (e.g., a synthetic or naturally occurring colloid, a polysaccharide such as dextran or a polyethylene glycol present at about 25 g/l to about 100 g/l, or about 40 g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, buty
  • a human placenta is recovered shortly after its expulsion after birth.
  • the placenta is recovered from a patient after informed consent and after a complete medical history of the patient is taken and is associated with the placenta. In one embodiment, the medical history continues after delivery.
  • the umbilical cord blood and placental blood Prior to recovery of perfusate, the umbilical cord blood and placental blood are removed. In certain embodiments, after delivery, the cord blood in the placenta is recovered. The placenta can be subjected to a conventional cord blood recovery process.
  • a needle or cannula is used, with the aid of gravity, to exsanguinate the placenta (see, e.g., Anderson, U.S. Patent No.5,372,581; Hessel et 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 aid in draining cord blood from the placenta.
  • cord blood recovery may be performed commercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell.
  • the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.
  • a placenta is transported from the delivery or birthing room to another location, e.g., a laboratory, for recovery of cord blood and collection of perfusate.
  • the placenta can be transported in a sterile, thermally insulated transport device (maintaining the temperature of the placenta between 20-28 °C), for example, by placing the placenta, with clamped proximal umbilical cord, in a sterile zip-lock plastic bag, which is then placed in an insulated container.
  • the placenta is transported in a cord blood collection kit substantially as described in U.S. Patent No.7,147,626.
  • the placenta is delivered to the laboratory four to twenty-four hours following delivery.
  • the proximal umbilical cord is clamped, for example within 4-5 cm (centimeter) of the insertion into the placental disc prior to cord blood recovery.
  • the proximal umbilical cord is clamped after cord blood recovery but prior to further processing of the placenta.
  • the placenta, prior to collection of the perfusate can be stored under sterile conditions and at either room temperature or at a temperature of 5 to 25 °C (centigrade).
  • the placenta may be stored for a period of longer than forty eight hours, or for a period of four to twenty-four hours prior to perfusing the placenta to remove any residual cord blood.
  • the placenta can be stored in an anticoagulant solution at a temperature of 5 °C to 25 °C (centigrade). Suitable anticoagulant solutions are well known in the art. For example, a solution of heparin or warfarin sodium can be used.
  • the anticoagulant solution comprises a solution of heparin (e.g., 1% w/w in 1:1000 solution).
  • the exsanguinated placenta is stored for no more than 36 hours before placental perfusate is collected. 5.5.3.
  • Placental Perfusion Methods of perfusing mammalian placentae and obtaining placental perfusate are disclosed, e.g., in Hariri, U.S. Patent Nos.7,045,148 and 7,255,879, and in U.S. Application Publication Nos.2009/0104164, 2007/0190042 and 20070275362, issued as U.S. Pat No. 8,057,788, the disclosures of which are hereby incorporated by reference herein in their entireties.
  • Perfusate can be obtained by passage of perfusion solution, e.g., saline solution, culture medium or cell collection compositions described above, through the placental vasculature.
  • a mammalian placenta is perfused by passage of perfusion solution through either or both of the umbilical artery and umbilical vein.
  • the flow of perfusion solution through the placenta may be accomplished using, e.g., gravity flow into the placenta.
  • the perfusion solution is forced through the placenta using a pump, e.g., a peristaltic pump.
  • the umbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula, that is connected to a sterile connection apparatus, such as sterile tubing.
  • the sterile connection apparatus is connected to a perfusion manifold.
  • the placenta can be oriented in such a manner that the umbilical artery and umbilical vein are located at the highest point of the placenta.
  • the placenta can be perfused by passage of a perfusion solution through the placental vasculature, or through the placental vasculature and surrounding tissue.
  • the umbilical artery and the umbilical vein are connected simultaneously to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution.
  • the perfusion solution is passed into the umbilical vein and artery.
  • the perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation.
  • the perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the maternal uterine wall.
  • the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins, that is, is passed through only the placental vasculature (fetal tissue).
  • the umbilical artery and the umbilical vein are connected simultaneously, e.g., to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution.
  • the perfusion solution is passed into the umbilical vein and artery.
  • the perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation.
  • the perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the maternal uterine wall.
  • Placental cells that are collected by this method are typically a mixture of fetal and maternal cells.
  • the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins.
  • Placental cells collected by this method which can be referred to as a “closed circuit” method, are typically almost exclusively fetal.
  • the closed circuit perfusion method can, in one embodiment, be performed as follows. A post-partum placenta is obtained within about 48 hours after birth. The umbilical cord is clamped and cut above the clamp.
  • the umbilical cord can be discarded, or can processed to recover, e.g., umbilical cord stem cells, and/or to process the umbilical cord membrane for the production of a biomaterial.
  • the amniotic membrane can be retained during perfusion, or can be separated from the chorion, e.g., using blunt dissection with the fingers. If the amniotic membrane is separated from the chorion prior to perfusion, it can be, e.g., discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce, e.g., an amniotic membrane biomaterial, e.g., the biomaterial described in U.S. Application Publication No.2004/0048796.
  • the umbilical cord vessels are exposed, e.g., by partially cutting the umbilical cord membrane to expose a cross-section of the cord.
  • the vessels are identified, and opened, e.g., by advancing a closed alligator clamp through the cut end of each vessel.
  • the apparatus e.g., plastic tubing connected to a perfusion device or peristaltic pump, is then inserted into each of the placental arteries.
  • the pump can be any pump suitable for the purpose, e.g., a peristaltic pump.
  • Plastic tubing connected to a sterile collection reservoir, e.g., a blood bag such as a 250 mL collection bag, is then inserted into the placental vein.
  • a sterile collection reservoir e.g., a blood bag such as a 250 mL collection bag
  • the tubing connected to the pump is inserted into the placental vein, and tubes to a collection reservoir(s) are inserted into one or both of the placental arteries.
  • the placenta is then perfused with a volume of perfusion solution, e.g., about 750 ml of perfusion solution. Cells in the perfusate are then collected, e.g., by centrifugation.
  • the proximal umbilical cord is clamped during perfusion, and, more specifically, can be clamped within 4-5 cm (centimeter) of the cord’s insertion into the placental disc.
  • the first collection of perfusion fluid from a mammalian placenta during the exsanguination process is generally colored with residual red blood cells of the cord blood and/or placental blood. The perfusion fluid becomes more colorless as perfusion proceeds and the residual cord blood cells are washed out of the placenta. Generally from 30 to 100 mL of perfusion fluid is adequate to initially flush blood from the placenta, but more or less perfusion fluid may be used depending on the observed results.
  • cord blood is removed from the placenta prior to perfusion (e.g., by gravity drainage), but the placenta is not flushed (e.g., perfused) with solution to remove residual blood.
  • cord blood is removed from the placenta prior to perfusion (e.g., by gravity drainage), and the placenta is flushed (e.g., perfused) with solution to remove residual blood.
  • the volume of perfusion liquid used to perfuse the placenta may vary depending upon the number of placental cells to be collected, the size of the placenta, the number of collections to be made from a single placenta, etc.
  • the volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL.
  • the placenta is perfused with 700-800 mL of perfusion liquid following exsanguination. [00385]
  • the placenta can be perfused a plurality of times over the course of several hours or several days.
  • the placenta may be maintained or cultured under aseptic conditions in a container or other suitable vessel, and perfused with a cell collection composition, or a standard perfusion solution (e.g., a normal saline solution such as phosphate buffered saline (“PBS”) with or without an anticoagulant (e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g., ⁇ - mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 ⁇ g/ml), penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 0.5 ⁇ g/ml).
  • PBS phosphate buffered saline
  • an anticoagulant e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin
  • an antimicrobial agent e.g.,
  • an isolated placenta is maintained or cultured for a period of time without collecting the perfusate, such that the placenta is maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days before perfusion and collection of perfusate.
  • the perfused placenta can be maintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a second time with, e.g., 700-800 mL perfusion fluid.
  • the placenta can be perfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours.
  • perfusion of the placenta and collection of perfusion solution e.g., placental cell collection composition, is repeated until the number of recovered nucleated cells falls below 100 cells/ml.
  • the perfusates at different time points can be further processed individually to recover time-dependent populations of cells, e.g., total nucleated cells. Perfusates from different time points can also be pooled. 5.5.4.
  • placental perfusate from a single placental perfusion comprises about 100 million to about 500 million nucleated cells, including hematopoietic cells from which NK cells and/or ILC3 cells, e.g., NK cells and/or ILC3 cells produced according to the three-stage method described herein, may be produced by the method disclosed herein.
  • the placental perfusate or perfusate cells comprise CD34 + cells, e.g., hematopoietic stem or progenitor cells.
  • Such cells can, in a more specific embodiment, comprise CD34 + CD45 – stem or progenitor cells, CD34 + CD45 + stem or progenitor cells, or the like.
  • the perfusate or perfusate cells are cryopreserved prior to isolation of hematopoietic cells therefrom.
  • the placental perfusate comprises, or the perfusate cells comprise, only fetal cells, or a combination of fetal cells and maternal cells.
  • an isolated NK cell population produced by a three-stage method described herein wherein said NK cell population comprises a greater percentage of CD3–CD56+ cells than an NK progenitor cell population produced by a three-stage method described herein, e.g., an NK progenitor cell population produced by the same three-stage method with the exception that the third culture step used to produce the NK progenitor cell population was of shorter duration than the third culture step used to produce the NK cell population.
  • said NK cell population comprises about 70% or more, in some embodiments, 75%, 80%, 85%, 90%, 95%, 98%, or 99% CD3–CD56+ cells.
  • said NK cell population comprises no less than 80%, 85%, 90%, 95%, 98%, or 99% CD3–CD56+ cells. In another specific embodiment, said NK cell population comprises between 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-99% CD3–CD56+ cells. [00389] In certain embodiments, said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally NKp46 + . In certain embodiments, said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally CD16-.
  • said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally CD16+. In certain embodiments, said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally CD94-. In certain embodiments, said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally CD94+. In certain embodiments, said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally CD11a + . In certain embodiments, said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally NKp30 + .
  • said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally CD161 + . In certain embodiments, said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally DNAM-1 + . In certain embodiments, said CD3 – CD56 + cells in said NK cell population comprises CD3 – CD56 + cells that are additionally T-bet + . [00390] In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are CD117+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are NKG2D+.
  • an NK cell population produced by a three-stage method described herein comprises cells which are NKp44+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are CD244+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which express perforin. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which express EOMES. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which express granzyme B. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which secrete IFN ⁇ , GM-CSF and/or TNF ⁇ . 5.7.
  • ILC3 Cells 5.7.1. ILC3 Cells Produced by Three-Stage Method [00391] In another embodiment, provided herein is an isolated ILC3 cell population, wherein said ILC3 cells are produced according to the three-stage method described above. [00392] In one embodiment, provided herein is an isolated ILC3 cell population produced by a three-stage method described herein, wherein said ILC3 cell population comprises a greater percentage of CD3–CD56+ cells than an ILC3 progenitor cell population produced by a three- stage method described herein, e.g., an ILC3 progenitor cell population produced by the same three-stage method with the exception that the third culture step used to produce the ILC3 progenitor cell population was of shorter duration than the third culture step used to produce the ILC3 cell population.
  • said ILC3 cell population comprises about 70% or more, in some embodiments, 75%, 80%, 85%, 90%, 95%, 98%, or 99% CD3–CD56+ cells. In another specific embodiment, said ILC3 cell population comprises no less than 80%, 85%, 90%, 95%, 98%, or 99% CD3–CD56+ cells. In another specific embodiment, said ILC3 cell population comprises between 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%- 99% CD3–CD56+ cells. [00393] In certain embodiments, said CD3 – CD56 + cells in said ILC3 cell population comprises CD3 – CD56 + cells that are additionally NKp46 – .
  • said CD3 – CD56 + cells in said ILC3 cell population comprises CD3 – CD56 + cells that are additionally CD16-. In certain embodiments, said CD3 – CD56 + cells in said ILC3 cell population comprises CD3 – CD56 + cells that are additionally IL1R1+. In certain embodiments, said CD3 – CD56 + cells in said ILC3 cell population comprises CD3 – CD56 + cells that are additionally CD94-. In certain embodiments, said CD3 – CD56 + cells in said ILC3 cell population comprises CD3 – CD56 + cells that are additionally ROR ⁇ t+.
  • said CD3 – CD56 + cells in said ILC3 cell population comprises CD3 – CD56 + cells that are additionally CD11a – . In certain embodiments, said CD3 – CD56 + cells in said ILC3 cell population comprises CD3 – CD56 + cells that are additionally T-bet+.
  • an ILC3 cell population produced by a three-stage method described herein comprises cells which are CD117+. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are NKG2D – . In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are NKp30 – .
  • an ILC3 cell population produced by a three-stage method described herein comprises cells which are CD244+. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are DNAM-1+. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which express AHR. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which do not express perforin. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which do not express EOMES. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which do not express granzyme B.
  • an ILC3 cell population produced by a three-stage method described herein comprises cells which secrete IL-22 and/or IL-8.
  • cell populations produced by the three-stage method described herein comprise CD11a+ cells and CD11a– cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50.
  • a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 50:1.
  • a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 20:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 10:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 5:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:5. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:10.
  • a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:20. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:50. [00396] In certain aspects, cell populations described herein are produced by combining the CD11a+ cells with the CD11a– cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50 to produce a combined population of cells.
  • a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 50:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 20:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 10:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 5:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:1.
  • a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:5. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:10. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:20. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:50.
  • cell populations produced by the three-stage method described herein comprise NK cells and ILC3 cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50.
  • a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 50:1.
  • a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 20:1.
  • a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 10:1.
  • a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 5:1. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:1. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:5. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:10. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:20. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:50.
  • cell populations described herein are produced by combining the NK cells with the ILC3 cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50 to produce a combined population of cells.
  • a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 50:1.
  • a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 20:1.
  • a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 10:1.
  • a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 5:1. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:1. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:5. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:10. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:20. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:50. 5.8.
  • compositions Comprising NK Cells and/or ILC3 Cells 5.8.1. NK Cells and/or ILC3 Cells Produced Using The Three-Stage Method [00399]
  • a composition e.g., a pharmaceutical composition, comprising an isolated NK cell and/or ILC3 cell population produced using the three-stage method described herein.
  • said isolated NK cell and/or ILC3 cell population is produced from hematopoietic cells, e.g., hematopoietic stem or progenitor cells isolated from placental perfusate, umbilical cord blood, and/or peripheral blood.
  • said isolated NK cell and/or ILC3 cell population comprises at least 50% of cells in the composition.
  • said isolated NK cell and/or ILC3 cell population e.g., CD3 – CD56 + cells, comprises at least 80%, 85%, 90%.95%, 98% or 99% of cells in the composition.
  • no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the cells in said isolated NK cell and/or ILC3 cell population are CD3 – CD56 + cells.
  • said CD3 – CD56 + cells are CD16-.
  • NK cell and/or ILC3 cell populations produced using the three-stage method described herein can be formulated into pharmaceutical compositions for use in vivo.
  • Such pharmaceutical compositions comprise a population of NK cells and/or ILC3 cells in a pharmaceutically-acceptable carrier, e.g., a saline solution or other accepted physiologically- acceptable solution for in vivo administration.
  • Pharmaceutical compositions of the invention can comprise any of the NK cell and/or ILC3 cell populations described elsewhere herein.
  • the pharmaceutical compositions of the invention comprise populations of cells that comprise 50% viable cells or more (that is, at least 50% of the cells in the population are functional or living). Preferably, at least 60% of the cells in the population are viable.
  • the pharmaceutical compositions of the invention can comprise one or more compounds that, e.g., facilitate engraftment; stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, and the like.
  • the pharmaceutical composition of the invention comprises about 1.25% HSA and about 2.5% dextran.
  • Other injectable formulations, suitable for the administration of cellular products, may be used.
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for systemic or local administration.
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for parenteral administration.
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for injection, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor administration.
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for administration via a device, a matrix, or a scaffold.
  • the compositions, e.g., pharmaceutical compositions provided herein are suitable for injection.
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for administration via a catheter.
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for local injection.
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for local injection directly into a solid tumor (e.g., a sarcoma).
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for injection by syringe.
  • the compositions, e.g., pharmaceutical compositions, provided herein are suitable for administration via guided delivery.
  • compositions e.g., pharmaceutical compositions, provided herein are suitable for injection aided by laparoscopy, endoscopy, ultrasound, computed tomography, magnetic resonance, or radiology.
  • compositions e.g., pharmaceutical compositions provided herein, comprising NK cells and/or ILC3 cells produced using the methods described herein, are provided as pharmaceutical grade administrable units.
  • Such units can be provided in discrete volumes, e.g., 15 mL, 20 mL, 25 mL, 30 nL.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 so as to contain a specified number of cells, e.g., NK cells and/or ILC3 cells, e.g., 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 or more cells per milliliter, or 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , 1 x 10 11 or more cells per unit.
  • NK cells and/or ILC3 cells e.g., 1 x 10 4 , 5 x 10
  • the units can comprise about, at least about, or at most about 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 or more NK cells and/or ILC3 cells per milliliter, or 1 x 10 4 , 5 x 10 4 , 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , 1 x 10 11 or more cells per unit.
  • Such units can be provided to contain specified numbers of NK cells and/or ILC3 cells or NK cell and/or ILC3 cell populations and/or any of the other cells.
  • the NK cells and ILC3 cells are present in ratios provided herein.
  • said isolated NK cells and/or ILC3 cells in said composition are from a single individual.
  • said isolated NK cells and/or ILC3 cells comprise NK cells and/or ILC3 cells from at least two different individuals.
  • said isolated NK cells and/or ILC3 cells in said composition are from a different individual than the individual for whom treatment with the NK cells and/or ILC3 cells is intended.
  • said NK cells have been contacted or brought into proximity with an immunomodulatory compound or thalidomide in an amount and for a time sufficient for said NK cells to express detectably more granzyme B or perforin than an equivalent number of natural killer cells, i.e. NK cells not contacted or brought into proximity with said immunomodulatory compound or thalidomide.
  • said composition additionally comprises an immunomodulatory compound or thalidomide.
  • the immunomodulatory compound is a compound described below. See, e.g., U.S. Patent No.7,498,171, the disclosure of which is hereby incorporated by reference in its entirety.
  • the immunomodulatory compound is an amino-substituted isoindoline.
  • the immunomodulatory compound is 3-(4-amino-1-oxo-1,3-dihydroisoindol- 2-yl)-piperidine-2,6-dione; 3-(4'aminoisolindoline-1'-one)-1-piperidine-2,6-dione; 4-(amino)-2- (2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; or 4-Amino-2-(2,6-dioxopiperidin-3-yl)isoindole- 1,3-dione.
  • the immunomodulatory compound is pomalidomide, or lenalidomide.
  • R 1 is H, (C 1 –C 8 )alkyl, (C3–C7)cycloalkyl, (C 2 –C 8 )alkenyl, (C 2 –C 8 )alkynyl, benzyl, aryl, (C 0 -C 4 )alkyl–(C 1 -C 6 )heterocycloalkyl, (C 0 -C 4 )alkyl–(C 2 -C 5 )heteroaryl, C(O)R 3 , C(S)R 3 , C(O)OR 4 , (C 1 –C 8 )alkyl–N(R 6 )2, (C 1 –C 8 )alkyl–OR 5 , (C 1 –C 8 )alkyl–C(O)OR 5 , C(O)
  • the composition additionally comprises one or more anticancer compounds, e.g., one or more of the anticancer compounds described below.
  • the composition comprises NK cells and/or ILC3 cells from another source, or made by another method.
  • said other source is placental blood and/or umbilical cord blood.
  • said other source is peripheral blood.
  • the NK cell and/or ILC3 cell population in said composition is combined with NK cells and/or ILC3 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
  • the composition comprises an NK cell and/or ILC3 cell population produced using the three-stage method described herein and either isolated placental perfusate or isolated placental perfusate cells.
  • said placental perfusate is from the same individual as said NK cell and/or ILC3 cell population.
  • said placental perfusate comprises placental perfusate from a different individual than said NK cell and/or ILC3 cell population.
  • all, or substantially all (e.g., greater than 90%, 95%, 98% or 99%) of cells in said placental perfusate are fetal cells.
  • the placental perfusate or placental perfusate cells comprise fetal and maternal cells.
  • the fetal cells in said placental perfusate comprise less than about 90%, 80%, 70%, 60% or 50% of the cells in said perfusate.
  • said perfusate is obtained by passage of a 0.9% NaCl solution through the placental vasculature.
  • said perfusate comprises a culture medium.
  • said perfusate has been treated to remove erythrocytes.
  • said composition comprises an immunomodulatory compound, e.g., an immunomodulatory compound described below, e.g., an amino-substituted isoindoline compound.
  • the composition additionally comprises one or more anticancer compounds, e.g., one or more of the anticancer compounds described below.
  • the composition comprises an NK cell and/or ILC3 cell population and placental perfusate cells.
  • said placental perfusate cells are from the same individual as said NK cell and/or ILC3 cell population.
  • said placental perfusate cells are from a different individual than said NK cell and/or ILC3 cell population.
  • the composition comprises isolated placental perfusate and isolated placental perfusate cells, wherein said isolated perfusate and said isolated placental perfusate cells are from different individuals.
  • said placental perfusate comprises placental perfusate from at least two individuals.
  • said isolated placental perfusate cells are from at least two individuals.
  • said composition comprises an immunomodulatory compound.
  • the composition additionally comprises one or more anticancer compounds, e.g., one or more of the anticancer compounds described below. [00411] 6.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the compositions described herein, e.g., a composition comprising NK cells and/or ILC3 cells produced by a method described herein, e.g., NK cell and/or ILC3 cell populations produced using the three-stage method described herein.
  • a composition comprising NK cells and/or ILC3 cells produced by a method described herein, e.g., NK cell and/or ILC3 cell populations produced using the three-stage method described herein.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • kits encompassed herein can be used in accordance with the methods described herein, e.g., methods of suppressing the growth of tumor cells and/or methods of treating cancer, e.g., hematologic cancer, and/or methods of treating viral infection.
  • a kit comprises NK cells and/or ILC3 cells produced by a method described herein or a composition thereof, in one or more containers.
  • a kit comprising an NK cell and/or ILC3 cell population produced by a three-stage method described herein, or a composition thereof. 7. EXAMPLES 7.1.
  • Example 1 Three-stage method of producing natural killer cells from hematopoietic stem or progenitor cells [00414] CD34 + cells are cultured in the following medium formulations for the indicated number of days, and aliquots of cells are taken for assessment of cell count, cell viability, characterization of natural killer cell differentiation and functional evaluation.
  • Stage 1 medium 90% Stem Cell Growth Medium (SCGM) (CellGro®), 10% Human Serum-AB, supplemented with 25 ng/mL or 250 ng/mL recombinant human thrombopoietin (TPO), 25 ng/mL recombinant human Flt3L, 27 ng/mL recombinant human stem cell factor (SCF), 25 ng/mL recombinant human IL-7, 0.05 ng/mL or 0.025 ng/mL recombinant human IL-6, 0.25 ng/mL or 0.125 ng/mL recombinant human granulocyte colony-stimulating factor (G-CSF), 0.01 ng/mL or 0.025 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), 0.10% gentamicin, and 1 to 10 ⁇ m StemRegenin-1 (SR-
  • Stage 2 medium 90% SCGM, 10% Human Serum-AB, supplemented with 25 ng/mL recombinant human Flt3L, 27 ng/mL recombinant human SCF, 25 ng/mL recombinant human IL-7, 20 ng/mL recombinant human IL-15, 0.05 ng/mL or 0.025 ng/mL recombinant human IL-6, 0.25 ng/mL or 0.125 ng/mL recombinant human granulocyte colony-stimulating factor (G-CSF), 0.01 ng/mL or 0.025 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), 0.10% gentamicin, and 1 to 10 ⁇ m SR1 or other stem cell mobilizing agent.
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-
  • Stage 3 medium 90% STEMMACS TM , 10% Human Serum-AB, 0.025 mM 2- mercaptoethanol (55 mM), supplemented with 22 ng/mL recombinant human SCF, 1000 U/mL recombinant human IL-2, 20 ng/mL recombinant human IL-7, 20 ng/mL recombinant human IL- 15, 0.05 ng/mL or 0.025 ng/mL recombinant human IL-6, 0.25 ng/mL or 0.125 ng/mL recombinant human granulocyte colony-stimulating factor (G-CSF), 0.01 ng/mL or 0.025 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), and 0.10% gentamicin.
  • G-CSF granulocyte colony-stimulating factor
  • G-CSF granulocyte colony-stimulating
  • Cells are seeded at Day 0 at 3 ⁇ 10 4 cells/mL in Stage 1 media, and cells are tested for purity by a CD34+ and CD45+ count and viability by 7AAD staining.
  • At Day 5 cells are counted and seeded to a concentration of 1 ⁇ 10 5 cells/mL with Stage 1 medium.
  • At Day 7 cells are counted and seeded to a concentration of 1 ⁇ 10 5 cells/mL with Stage 1 medium.
  • cells are counted and seeded to a concentration of 1 ⁇ 10 5 cells/mL in Stage 2 medium.
  • cells are counted and seeded to a concentration of 3 ⁇ 10 5 cells/mL in Stage 2 medium.
  • cells are counted and seeded in Stage 3 medium. Cells are maintained in Stage 3 media until day 35. [00420] Alternatively, the following protocol is used through Day 14: Cells seeded at Day 0 at 7.5 ⁇ 10 3 cells/mL in Stage 1 media, and cells are tested for purity by a CD34+ and CD45+ count and viability by 7AAD staining. At Day 7 cells are counted and seeded to a concentration of 3 ⁇ 10 5 cells/mL with Stage 1 medium. At Day 9 cells are counted and seeded to a concentration of 3 ⁇ 10 5 cells/mL with Stage 2 medium. At Day 12, cells are counted and seeded to a concentration of 3 ⁇ 10 5 cells/mL in Stage 2 medium.
  • cells are counted and seeded to a concentration of 3 ⁇ 10 5 cells/mL in Stage 2 medium.
  • Seeding of cells into at passage is performed either by dilution of the culture with fresh media or by centrifugation of cells and resuspension / addition of fresh media.
  • cells are spun at 400 ⁇ g for seven minutes, followed by suspension of the pellet in an equal volume of Plasmalyte A. The suspension is spun at 400 ⁇ g for seven minutes, and the resulting pellet is suspended in 10% HSA (w/v), 60% Plasmalyte A (v/v) at the target cell concentration.
  • Example 2 Selection of stem cell mobilizing agents for the expansion of NK cells
  • the following compounds were investigated for their ability to promote the expansion of NK cell populations in vitro: 4-(2-((2-(benzo[b]thiophen-3-yl)-6-(isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol) (“CRL1”) 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol)) (“CRL2”)
  • Example 3 Characterization of three-stage NK cells .
  • UCB CD34+ cells were cultivated in presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 for 35 days to produce three-stage NK cells, as described in Example 1. Multi-color flow cytometry was used to determine the phenotypic characteristics of three-stage NK cells.
  • the compounds were provided to culture to evaluate their effects on NK cell expansion and differentiation. Specifically, donors of CD34+ cells (StemCell Technology) were thawed and expanded in vitro following NK culture protocol.
  • each CRL compounds was dissolved in DMSO and added to the culture at 10 ⁇ M concentration.
  • SR1 (at 10 ⁇ M) served as a positive control compound, while DMSO alone without any compound served as a negative control.
  • NK natural killer
  • cytotoxicity of the cells against K562 tumor cell line were characterized. Due to the large number of the compounds, the testing was performed in two experiments, CRL1-11 and CRL 12-22. The same donors were used for each experiment. Positive and negative controls were also included in both experiments. Results [00426] Cell expansion data showed that 20 out of the 22 compounds supported NK expansion at 10 ⁇ M concentration.
  • PBMC Peripheral blood derived NKs
  • PB-NK Peripheral blood derived NKs
  • CYNK cells were generated from umbilical cord blood-derived CD34+ stem cells (Ref: Zhang et al. J Immunother Cancer.2015).
  • the CD34+ cells were cultivated in the presence of cytokines including thromobopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 for 35 days.
  • PBNK and CYNK cells were cryopreserved until analysis.
  • Magnetic-activated cell sorting PNK cells were stained with PE Mouse Anti- Human CD11a (BD) and CD11a+ PNK cells concentrated using anti-PE MicroBeads according to manufacturer’s instructions (Miltenyi Biotec).
  • Single cell RNA sequencing CYNK cells were combined with PB-NK at 1:1 ratio and gene expression analyzed on single cell level using 10X Genomics Chromium platform and Illumina sequencing.
  • Bioinformatics analysis utilized 10X Genomics Cell Ranger analysis pipeline.
  • Flow Cytometry Cryopreserved cells were rapidly thawed in a 37°C water bath and washed once in RPMI1640 + 10% hiFBS (heat inactivated Fetal Bovine Serum, Gibco), followed by LIVE/DEADTM Fixable Aqua Stain in PBS. Cells were washed with FACS buffer (PBS + 2% FBS) followed by incubation in blocking solution (Brilliant Stain buffer, Mouse IgG2a isotype k control and Human BD Fc Block (all from BD)).
  • CYNK cells efficiently kill various tumor cell lines in vitro, however, the mechanisms CYNK cells use to induce cell death remains poorly understood (ref).
  • scRNAseq single-cell RNA sequencing
  • PB-NK peripheral blood NK cells
  • FIG.6A Unbiased transcriptional clustering revealed two distinct signatures differentiating between CYNK and PB-NK cells (FIG.6B).
  • Tables 1 and 2 list top 50 upregulated genes per cluster in PB-NK and CYNK cells, respectively.
  • the gene set expressed higher in PB- NK cells included genes associated with NK cell functional roles, including FGFBP2, granzymes (GZMH, GZMM), CXCR4, KLRF1, KLF2, IFNG (Table 1).
  • FGFBP2 encoding fibroblast growth factor-binding protein, is known to be secreted by cytotoxic lymphocytes.
  • ⁇ Granzymes are a group of serine proteases which are stored in the cytotoxic granules of NK cells and cytotoxic T lymphocytes (ref). While GzmA and GzmB induce target cell death upon release to their cytoplasm and have been extensively studied, less is known about the functional role of GzmH, GzmK and GzmM.
  • ⁇ CXCR4 regulates NK cell homing to bone marrow.
  • ⁇ KLRF1 encodes NKp80, an activating C-type lectin-like immunoreceptor that is activated upon binding to activation-induced C-type lectin (AICL), inducing NK cell cytotoxicity and cytokine secretion.
  • AICL activation-induced C-type lectin
  • ⁇ Transcription factor KLF2 that regulates both NK cell proliferation and survival.
  • ⁇ NK cell-derived IFN- ⁇ a key immunoregulatory factor secreted from activated NK cells that promotes adaptive immune response by modulating dendritic cell and T cell responses.
  • Top differentially expressed genes in CYNK cluster that are encode factors associated with NK cell functional role include surface receptors and co-receptors (CD96, NCR3, CD59, KLRC1), TNFSF10, immune checkpoint genes (TNFRSF18, TNFRSF4, HAVCR2), NK cell receptor adaptor molecule genes (FCER1G and LAT2) (Table 2). Table 2. Top 50 upregulated genes per CYNK cluster.
  • qRT-PCR demonstrated high expression of CD69, KLRK1 and KLRB1 relative to the housekeeping gene GAPDH in both CYNK and PB- NK cells, whereas, KLRK1 and KLRB1, encoding for NKG2D and CD161/KLRB1, respectively, were significantly higher expressed in PB-NK cells.
  • KLRD1 was higher expressed on PB-NK compared to CYNK cells.
  • KLRB1, KLRD1, KLRF1 The two C-type lectin receptor genes KLRC1 and KLRC2, encoding the inhibitory NKG2A and the activating NKG2C, were higher expressed in CYNK cells.
  • NCR2 cytotoxicity receptor 2 (encoding NKp44) was differentially expressed with high expression in CYNK cells and almost no expression in PB-NK cells.
  • CD244 Two co-activating NK cell receptor genes CD244 (2B4) and CD226 (DNAM-1) were slightly higher expressed in PB-NK compared to CYNK cells.
  • FCGR3A encoding an Fc receptor CD16 that is required for antibody-dependent cell-mediated cytotoxicity.
  • NK cells express high level of the NK cell marker CD56 and lack the expression of T cell, B cell and myeloid cell markers CD3, CD19 and CD14, respectively (FIG.8). Whereas a majority of PB-NK cells express CD56 at a low level, a small subset of PB-NK cells express CD56 at a level seen in CYNK cells (FIG.9). NCR analysis demonstrated a high expression of NKp44 in CYNK cells, whereas, NKp44 was expressed at a low level in PB-NK, corresponding well to our transcriptional analysis (FIG.7).
  • NKp80 was expressed on PB-NK cell and little on CYNK, also confirming the transcriptional data of KLRF1 expression (Table 1 and FIG.7).
  • CD16 was virtually not expressed on CYNK cells, whereas the majority of PB-NK cells expressed CD16 at a high level. CD16 protein expression, therefore, also corresponds well to transcriptional analysis (Table 1 and FIG.7).
  • the expression of killer cell lectin-like receptors was comparable between CYNK and PB-NK cells, with CYNK cells demonstrating higher mean fluorescence intensity compared to PB-NK cells for NKG2D, NKG2C, CD94 (NKG2C) and NKG2A.
  • GITR a checkpoint inhibitor molecule, encoded by TNFRSF18, was not expressed on PB-NK cells but highly on all CYNK cells, correlating well to qRT-PCR data.
  • FIG.8 and FIG.9 We used the flow cytometry dataset (FIG.8 and FIG.9) to perform an unbiased analysis of the surface marker expression on CYNK and PB-NK cell populations (FIG.10). Antibody-stained CYNK and PBMC cells were mixed for acquisition and analyzed by flow cytometry. It is evident from the tSNE plots that CYNK and PB-NK cells cluster separately from each other and other peripheral blood cells when looking at the localization of CD56- and CD3/CD14/CD19-positive cells on the plot.
  • NKp44 CD336
  • GITR GITR
  • PB-NK cells highly express CD16 and NKp80 that are not expressed on CYNK cells.
  • MFC multiparameter flow cytometry
  • PFS progression-free survival
  • OS duration of morphologic CR
  • TTP time to progression
  • OS overall survival
  • CR Complete Remission
  • CRi Complete Remission with incomplete hematologic recovery
  • MLFS Morphologic leukemic-free state
  • Additional secondary endpoints for subjects with relapsed or refractory AML include Duration of Response (DoR), and Overall Survival (OS).
  • Exploratory objectives include monitoring immune reconstitution following CYNK-001 dosing, in vivo persistence and expansion of CYNK-001 cells during treatment and up to 60 days following the first CYNK-001 infusion, characterization of immune cell populations in the bone marrow and peripheral blood, serum analysis of immune correlates, alloreactivity characterization, anti-HLA antibody analysis, and transcriptome analysis of bone marrow immune microenvironment.
  • Depth of MRD Response for both MRD positive population and R/R population who achieve CR, CRi, or MLFS
  • R/R AML population changes in blast count will be assessed. Aggregated data will be used to determine biomarker correlations to MRD Response.
  • the proposed study will enroll and treat up to approximately 94 subjects, which includes the option of further dose escalation based on the DMC recommendation.
  • the study is divided into 3 study periods: Treatment Eligibility Period, Treatment Period, and Follow-up Period.
  • Subjects will have a BMA Collection to determine AML disease status either following signing the Pre-Screening BMA Collection ICF prior to the potential subject signing the main Study ICF, or after signing the main Study ICF as part of the collective Treatment Eligibility screening activities. Each period has associated evaluations and procedures that must be performed at specific timepoints.
  • Subject participation is dependent on slot availability based on time of entry into the study. Error! Reference source not found.
  • rhIL-2 will be administered in combination with CYNK-001 cells.
  • the decision to administer rhIL-2 in Cohorts 6a, 6b, 7a and 7b will be based on review of safety, efficacy, and translational data in Cohorts 5a and 5b, respectively, after thorough review and recommendation from the DMC and based on Sponsor decision.
  • Pre-Screening BMA Collection (not required): Clinical sites have the option to collect and screen BMA samples prior to subjects signing the main Study ICF, approximately 2 weeks prior to the start of the treatment eligibility screening period. Subjects must consent to the Pre-Screening BMA Collection by signing the Pre-Screening BMA Collection ICF. Thereafter, pre-screening BMA will be collected and sent to the MRD central laboratory for flow cytometric analysis. A local morphological assessment should be performed as well.
  • Treatment Eligibility Screening Period If the pre-screening BMA collection was not previously collected as part of pre-screening BMA Collection, this BMA collection will be done as part of main Treatment Eligibility Screening after signing the main Study ICF.
  • MRD positive subjects The Treatment Eligibility Screening Period is defined as the period from Day -28 to Day -7 (or Day -6 for Cohorts 1, 2, and 3 only) in which subjects will be confirmed to be in morphological CR (or CRi, MLFS) with MRD positivity as measured on BMA by MFC and assessed for inclusion/exclusion criteria to confirm eligibility to participate in the study.
  • MRD positivity is defined as greater than or equal to 0.1% blasts detected by MFC on BMA by the Sponsor-selected Central MRD analysis laboratory, where assay sensitivity allows for a Lower Limit of Detection (LOD) of 1 x 10 -4 (i.e., 0.01%) or lower.
  • LOD Lower Limit of Detection
  • R/R subjects The Treatment Eligibility Screening Period is defined as the period from Day -28 to Day -7 in which subjects will have a confirmed diagnosis of R/R AML based on local disease assessment. [00450] During this period, a 14-day washout of prior chemotherapy will occur before each subject receives the Lymphodepletion Regimen (Cy-Flu). Consultation with the Medical Monitor is encouraged when determining appropriate washout period. Treatment Eligibility Criteria are provided. A Treatment Eligibility Checklist will be provided to aide in confirmation of eligibility. [00451] Only AEs associated with study-related procedures which are not considered standard of care will be reported in this study period.
  • Subjects may be rescreened for Treatment Eligibility up to two times for a total of 3 screens at a maximum. Subjects who are found to have relapsed upon central MRD analysis would be eligible to screen for R/R cohorts dependent on slot availability. [00453] Central BMA analysis for R/R subjects is not required for eligibility, however, a sample will be obtained for analyses. R/R subjects who have had local BMA assessment confirming R/R disease within 1 month prior to signing the main ICF are eligible.
  • Treatment Period The Treatment Period will consist of 2 parts: [00455] Part 1: Lymphodepletion Regimen (Cy-Flu) starting on Study Day -6 or -5 through Study Day -3, followed by two days with no treatment on Study Days -2 and -1. Mesna shall be administered, for Cohorts 4 and above, on days of Lymphodepletion for the inhibition of hemorrhagic cystitis induced by cyclophosphamide. Route of administration, dosage, and frequency of Mesna should be based on institutional standards.
  • Cyclophosphamide dose is 300 mg/m 2 to be administered on Study Days -5, -4, and -3 Fludarabine dose is 25 mg/m 2 to be administered on Study Days -5, -4, and -3 o Cy 900 Flu 30 Regimen (all other cohorts): Cyclophosphamide dose is 900 mg/m 2 to be administered on Study Days -6, -5, -4, and -3 Fludarabine dose is 30 mg/m 2 to be administered on Study Days -6, -5, -4, and -3 [00456] Subjects enrolled to the expansion portion of a treatment arm may receive Cy 900 Flu 30 for 3 days only (-5, -4 and -3) per Sponsor decision based on analysis of safety, efficacy and translational data.
  • o CYNK-001 Treatment period begins on Study Day 0 with the first of up to 4 CYNK-001 IV infusions. Three CYNK-001 infusions occur on Study Days 0, 7, and 14, followed by 2 weeks of no treatment for Cohorts 1 through 5 and Study Days 1, 7, 14, and 21 for Cohorts 6a, 6b, 7a and 7b followed by one week of no treatment through the end of the 28-day DLT period o rhIL-2 Treatment: Subjects treated in Cohorts 5a and 5b will receive rhIL-2 injections SC at least 1 to 3 hours prior CYNK-001 infusion.
  • Treatment with 6M IU rhIL-2 begins on the day of the first CYNK-001 infusion with the first of 7 total rhIL-2 injections. rhIL-2 injections should occur on each CYNK-001 infusion day and every other day in between CYNK-001 infusions (Days 0, 2, 4, 7, 9, 11, and 14). On non-CYNK-001 days, rhIL-2 may be delayed or skipped due to logistical constraints or adverse events at the PI’s discretion. rhIL-2 must not be administered on 2 consecutive days. Consultation with the Medical Monitor is encouraged when determining the rhIL-2 dosing schedule.
  • rhIL-2 in Cohorts 6a, 6b, 7a and 7b will be determined based on careful review of safety, efficacy, and translational data from subjects treated in Cohorts 5a and 5b. Those subjects will receive injections on the same days as Cohorts 5a and 5b and have one additional rhIL-2 injection on Day 21. The decision to include rhIL-2 will be based on DMC recommendation and Sponsor decision.
  • Pre- and post-medication of acetaminophen 650 mg orally (PO) and diphenhydramine 25 mg (PO/IV) is to be administered at the following schedule: ⁇ Cohorts 1, 2, 3 only: approximately 1 hour prior to each CYNK-001 infusion and approximately 4 hours after each infusion. ⁇ All other cohorts on rhIL-2 plus CYNK-001 infusion days: approximately 1 hour prior to rhIL-2 injection and approximately 2 hours after CYNK-001 infusion. ⁇ All other cohorts on rhIL-2 only days: approximately 1 hour prior to rhIL-2 injection and approximately 3 hours after rhIL-2 injection. ⁇ Meperidine may also be administered to control rigors, if clinically indicated.
  • Subjects will undergo optional BMA collection during the first 28 days on study and a protocol-mandated BMA collection on Study Day 28, both including MRD analysis by MFC by the Sponsor-selected Central MRD analysis laboratory (for MRD positive subjects only) and local Response Assessment (for MRD positive subjects and R/R subjects).
  • MRD analysis by MFC by the Sponsor-selected Central MRD analysis laboratory (for MRD positive subjects only) and local Response Assessment (for MRD positive subjects and R/R subjects).
  • MRD analysis by MFC by the Sponsor-selected Central MRD analysis laboratory (for MRD positive subjects only) and local Response Assessment (for MRD positive subjects and R/R subjects).
  • MRD positive subjects will be treated with CYNK-001 at 1.2 x 10 9 cells which will be administered on Study Days 0, 7, and 14.
  • Subjects will be followed for a 28- day DLT period.
  • Subjects in each cohort may be treated concurrently. Thereafter, dose escalation or cohort expansion will follow the rules outlined below.
  • Bone Marrow assessments should occur approximately 90 to 100 days after aSCT and every other month through Month 12.
  • Day 61 to Month 12 or Early Termination [00465]
  • MRD subjects Morphologic CR and MRD positive disease at study entry
  • Subjects may opt for this treatment, at the discretion of the treating physician.
  • Subjects may receive aSCT no sooner than 60 days after the first dose of CYNK-001 on Study Day 0, unless clinically indicated.
  • aSCT must occur after completion of the 28-day DLT period, provided that the Day 28 BMA has been collected for MRD Central Laboratory assessment.
  • BMA collection a Bone Marrow assessment
  • BMA collection including MRD analysis by MFC will occur at approximately 90 to 100 days after the aSCT and every other month thereafter through Month 12. Medications for the prevention of GVHD following aSCT are permitted.
  • MRD positive subjects After Study Day subjects may receive medication with the intent to maintain AML disease status (i.e., maintenance therapy) after Study Day 28.
  • Early Termination for PD Subjects who experience PD should have Early Termination visit completed (if possible) and will be followed for survival, every three months from early termination up to and including Month 12, loss to follow-up, death, or withdrawal from study, whichever occurs first. Survival follow-up may be done by telephone calls every three months.
  • Number of Subjects The study will enroll up to approximately 94 subjects, including a total of 10 subjects treated at the selected MTD/MPD for each population (MRD positive and R/R). The Sponsor may open the enrollment to 10 additional subjects for a total of 20 per expansion cohort based on review of safety and efficacy data.
  • Dose Limiting Toxicity Adverse events occurring up to Study Day 28 will be included in the dose-limiting toxicity (DLT) determination. Known toxicities associated with cyclophosphamide and fludarabine will be carefully considered and differentiated from CYNK- 001 in order to identify CYNK-001-related toxicities.
  • DLT Dose Limiting Toxicity Definition: A DLT is defined as the development of any new (not pre-existing) event that is deemed related to CYNK-001 and meets one of the following criteria: ⁇ Grade 4 or 5 event in any organ system with the following exceptions: o hematologic and infectious events o disease progression/relapse ⁇ Grade 3 AE for > 24 hour duration in the following organ systems: cardiac (excluding hypertension), pulmonary, hepatic, renal, central nervous system (CNS). ⁇ Grade ⁇ 3 allergic reaction that is suspected to be related to CYNK-001. ⁇ Grade ⁇ 3 hypertension event for > 48 hour duration. ⁇ Grade ⁇ 3 GVHD event occurring within the first 28 days following CYNK-001 infusion.
  • Treatment Assignment Upon confirmation of eligibility during the Treatment Eligibility Screening Period, eligible subjects may be sequentially assigned to one of the following dose level cohorts based on time of eligibility and treatment slot availability. [00477] There will be dose escalation of CYNK-001 cells in two patient populations with CYNK-001 in escalating doses alone or with CYNK-001 and rhIL-2. Doses and schedules are as listed in Table 5 and Table 6 for MRD Positive subjects and Table 7 and Table 8 for R/R subjects.
  • the study will utilize a 3 + 3 dose escalation design with 3 to 6 subjects enrolled into each dose cohort.
  • the final dose level cohort (either MTD or MPD) will enroll an additional 4 subjects to bring the total to 10 subjects treated at MTD/MPD.
  • the Sponsor may open the enrollment to 10 additional subjects for a total of 20 per expansion cohort based on review of safety and efficacy data. Dose de-escalations are shown in Table 6 and Table 6.
  • the use of rhIL-2 in MRD cohorts 6a, and 7a will be determined based on careful review of safety, efficacy, and translational data from subjects treated in Cohorts 5a. The decision to include rhIL-2 will be based on DMC recommendation and Sponsor decision.
  • Subjects enrolled to the expansion portion of a treatment arm may receive Cy 900 Flu 30 for 3 days only (-5, -4 and -3) per Sponsor decision based on analysis of safety, efficacy and translational data.
  • a 4a de-escalation The 4a cohort is intended to determine safety at 1.8x 10 9 cells at an increased lymphodepletion dose and schedule than in Cohort 3. If this 4a cohort is not tolerated, the de-escalation plan is to determine if the increased lymphodepletion regimen will be tolerated at a 1.2 x 10 9 CYNK-001 cell dose to determine if increased lymphodepletion would be tolerable in this population.
  • b 5a de-escalation The 5a cohort is intended to determine safety at 4a dose plus inclusion of rhIL-2.
  • the de-escalation plan is to determine if rhIL-2 will be tolerated at a 1.2 x 10 9 CYNK-001 cell dose before concluding that the rh-IL2 is tolerable in this population.
  • Subjects enrolled to the expansion portion of a treatment arm may receive Cy 900 Flu 30 for 3 days only (-5, -4 and -3) per Sponsor decision based on analysis of safety, efficacy and translational data.
  • Table 8 R/R Subjects with Possible De-escalation a 4b de-escalation: The 4b cohort is intended to determine safety at 1.8x 10 9 cells at with an increased lymphodepletion dose and schedule than in Cohort 3.
  • the de-escalation plan is to determine if the increased lymphodepletion regimen will be tolerated at a 1.2 x 10 9 CYNK-001 cell dose to determine if increased lymphodepletion would be tolerable in this population.
  • b 5b de-escalation The 5b cohort is intended to determine safety at 4b dose plus inclusion of rhIL-2. If this 5b cohort is not tolerated, the de-escalation plan is to determine if rhIL-2 will be tolerated at a 1.2 x 10 9 CYNK-001 cell dose before concluding that the rh-IL2 is tolerable in this population.
  • CYNK-001 cells are administered to all subjects on Study Days 0, 7, and 14 for Cohorts 1 through 5 and Study Days 0, 7, 14 and 21 for Cohorts 6a, 6b, 7a and 7b at the dose amount indicated by the appropriate cohort (listed in Table 5, Table 6, Table 7, and Table 8).
  • Subjects treated in Cohort 5a and Cohort 5b will receive rhIL-2 injections SC at least 1 to 3 hours prior CYNK-001 infusion. Treatment with 6M IU rhIL-2 begins on the day of the first CYNK-001 infusion with the first of 7 total rhIL-2 injections.
  • rhIL-2 injections should occur on each CYNK-001 infusion day and every other day in between CYNK-001 infusions (Days 0, 2, 4, 7, 9, 11, and 14). On non-CYNK-001 days, rhIL-2 may be delayed or skipped due to logistical constraints or clinically significant adverse events at the PI’s discretion. rhIL-2 must not be administered on 2 consecutive days. Consultation with the Medical Monitor is encouraged when determining the rhIL-2 dosing schedule. The use of rhIL-2 in R/R cohorts 6a, 6b, 7a and 7b will be determined based on careful review of safety, efficacy, and translational data from subjects treated in Cohorts 5a and 5b.
  • ⁇ Dose reductions are not permitted in this study. ⁇ Should dose delays for CYNK-001 be required: o Day 0 dose may not be delayed for longer than 48 hours o Day 7 dose may not be started early or delayed for longer than 48 hours ⁇ If delayed longer than 48 hours, the dose will be skipped. ⁇ A subject that skips Day 7 dosing may receive Day 14 dosing. o Day 14 dose may not be started early or delayed for longer than 48 hours ⁇ If delayed longer than 48 hours, the dose will be skipped. o If applicable, Day 21 dose may not be started early or delayed for longer than 48 hours ⁇ If delayed longer than 48 hours, the dose will be skipped.
  • rhIL-2 Dose Adjustments may occur if the subject weighs ⁇ 45 kgs. The dose of rhIL-2 should be reduced to 3M IU rhIL-2. rhIL-2 injections should not be given on 2 consecutive days or on the day prior to planned CYNK-001 infusion. On non-CYNK-001 days, rhIL-2 may be delayed or skipped due to logistical constraints or clinically significant adverse events at the PI’s discretion. Consultation with the Medical Monitor is encouraged when determining the rhIL-2 dosing schedule.
  • MRD definitions are based on the consensus document from the European LeukemiaNet MRD Working Party [Schuurhuis, 2018 50 ] and in accordance with the assay specifications from the Sponsor-selected Central MRD analysis laboratory, as follows: ⁇ MRD positive greater than or equal to 0.1%: MRD positivity is defined as greater than or equal to 0.1% blasts detected by MFC on BMA by the Sponsor-selected central MRD analysis laboratory, where assay sensitivity allows for a Lower Limit of Detection (LOD) of 1 x 10 -4 (0.01%) or lower.
  • LOD Lower Limit of Detection
  • ⁇ MRD positive less than 0.1% MRD positivity less than the protocol-defined threshold for eligibility, detected by MFC on BMA by the Sponsor-selected central MRD analysis laboratory, where assay sensitivity allows for a Lower Limit of Detection (LOD) of 1x10 -4 (0.01%) or lower.
  • LOD Lower Limit of Detection
  • ⁇ MRD Indeterminate major phenotype shift or mild change from normal (regardless of quantity).
  • ⁇ MRD negative No MFC MRD identified (i.e., 0% blasts).
  • ⁇ MRD MFC not possible sample could not be analyzed (e.g., insufficient sample volume or total cell number to determine MRD status, compromised sample quality, etc.).
  • MRD Response defined as conversion of MRD status from positive ( ⁇ 0.1 blasts) to either negative (no MRD identified; i.e., 0% blasts), MRD positive less than 0.1%, or MRD Indeterminate as measured by MFC on BMA with assay lower limit of detection at 1:10 4 or lower.
  • Time to MRD Response defined as time from first CYNK-001 infusion to MRD Response.
  • Duration of MRD Response defined as date of first MRD response after CYNK-001 infusion to date of MRD positivity ( ⁇ 0.1% blasts).
  • Duration of Morphologic CR for MRD positive cohorts, defined as duration from first morphologic CR (or CRi, MLFS) observation measured during front-line setting therapy to the time of disease progression per AML Response Criteria (Error! Reference source not found.), with deaths from causes other than progression censored. (The rationale for using first observation of morphologic CR is to allow for comparison to clinical response in AML as described in literature.
  • morphologic CR is defined as bone marrow blasts ⁇ 5% with other associated criteria outlined in ELN response criteria guidelines based on type of response (CR, CRi, MLFS) [Dohner, 2017 16 ] and may include subjects with MRD positivity, negativity, or unknown MRD status) [00493]
  • Time to Progression defined as the time from the date of the first CYNK-001 infusion to the date of disease progression per AML Response Criteria (Error! Reference source not found.), with deaths from causes other than progression censored.
  • OS Overall Survival
  • Duration of Response, DoR applicable for R/R cohorts, defined only for subjects who experience an objective response, as the time from the first CR (MLFS or CRi) after CYNK-001 to relapse or death due to disease progression.
  • Time to Response for R/R cohorts, defined as the length of time from first CYNK-001 treatment to the time of response of CR, CRi, or MLFS per AML Response Criteria.
  • Overall Response Rate ORR: defined as the percentage of subjects whose AML disease have a response of CR, CRi, or MLFS per AML Response Criteria.
  • HBcAB Core Antibody
  • Hepatitis B T Hepatitis B T
  • Mesna shall be administered, for rrhagic cystitis induced by ds.
  • Subjects enrolled to the expansionnalysis of safety, efficacy and translational nt (including during Treatment Eligibilityurs of collection; 3 mL send to infusion; BMA samples must be collected practice allow, a separate puncture is to-Chex tube for Immune phenotyping and um heparin tubes at each designated d will be collected in marbled red top urs (optional), and 24 hours after 2, 4, 9, and 11) a pre-infusion blood and egrees Celsius until shipment of all m separation tubes for serum analysis at eachd samples at the 24-hour timepoint.
  • CYNK-001 ndicated during the treatment period if e does not need to be repeated. usions for a total of 7 doses. An ECG should ormed at each follow-up visit. Dose y -3 during the Lymphodepletion Regimen, ct’s disease progresses, survival status should be collected. Survival follow-up can be done by visit or telephone call every three months until ubject withdraws or is lost to follow up.
  • a, 6b, 7a and 7b Subjects will receive a total of 4 CYNK-001 infusions on Study Days 0, 7, 14, and 21.
  • Treatment Eligibility Screening Period Subject Inclusion Criteria (all subjects): Subjects must satisfy the following criteria to be enrolled in the study: 1. Subject has eligible disease status: MRD positive population: Primary or Secondary AML subjects in first or second Morphological Complete Remission (CR), Morphological Complete Remission with incomplete hematologic recovery (CRi), or Morphologic Leukemia-free State (MLFS) as defined by the European LeukemiaNet (ELN) recommendations for AML Response Criteria [Dohner, 2017 16 ].
  • MRD positive population Primary or Secondary AML subjects in first or second Morphological Complete Remission (CR), Morphological Complete Remission with incomplete hematologic recovery (CRi), or Morphologic Leukemia-free State (MLFS) as defined by the European LeukemiaNet (ELN) recommendations for AML Response Criteria [Dohner, 2017 16 ].
  • CR Morphological Complete Remission
  • CRi Morphological Complete Remission with incomplete hematologic recovery
  • MLFS Morphologic Leukemia-free State
  • R/R population R/R diagnosis based on confirmed diagnosis with local pathology report following any re- induction/salvage therapy ELN guidelines.
  • ⁇ Relapsed AML are defined as having relapsed after achieving ⁇ 1 CR, including relapse after allogeneic stem cell transplantation ( ⁇ 2 months after transplant).
  • ⁇ Refractory AML defined as not achieving CR, CRi, or MLFS after 2 or more cycles of induction therapy (primary refractory) or not achieving CR after treatment for relapsed AML.
  • Secondary AML (MDS transformation) Secondary AML subjects are eligible to participate if they have received a minimum of one prior line of treatment for AML.
  • Treatment-related AML Treatment-related AML subjects are eligible to participate if they have received a minimum of one prior line of treatment for AML. 2. Subjects with prior central nervous system involvement by malignancy are eligible provided that it has been treated and cerebral spinal fluid sampled at least two weeks prior to the start of the lymphodepletion regimen was negative for AML by both cytology and flow cytometry. 3. MRD positive population only: Subject is MRD positive, as assessed on BMA by Multiparameter Flow Cytometry (MFC) at time of Treatment Eligibility assessment. a.
  • MFC Multiparameter Flow Cytometry
  • MRD positivity is defined as greater than or equal to 0.1% blasts detected by MFC on BMA by the Sponsor-selected Central analysis laboratory, where assay sensitivity allows for a Lower Limit of Detection (LOD) of 1 x 10 -4 (0.01%) or lower.
  • LOD Lower Limit of Detection
  • Subject is ⁇ 18 and ⁇ 80 years of age at the time of signing the Study informed consent form (ICF). 5.
  • Subject understands and voluntarily signs the Study ICF prior to any study-related assessments/procedures are conducted. 6.
  • Subject is willing and able to adhere to the study schedule and other protocol requirements. 7. Performance status of Eastern Cooperative Oncology Group (ECOG) ⁇ 2. 8.
  • FCBP is a female who: 1) has achieved menarche at some point, 2) has not undergone a hysterectomy or bilateral oophorectomy and 3) has not been naturally postmenopausal (amenorrhea following cancer therapy does not rule out childbearing potential) for at least 24 consecutive months (i.e., has had menses at any time in the preceding 24 consecutive months). 10. Male subject must agree to use a condom during sexual contact for at least 28 days following the last infusion of CYNK-001, even if he has undergone a successful vasectomy. [00504] Treatment Eligibility Screening Period Subject Exclusion Criteria (all subjects): The presence of any of the following will exclude the subject from enrollment: 1.
  • Subject has any significant medical condition, laboratory abnormality, or psychiatric illness that would prevent the subject from participating in the study. 2. Subject has any condition including the presence of laboratory abnormalities which places the subject at unacceptable risk if he or she were to participate in the study. 3. Subject has any condition that confounds the ability to interpret data from the study. 4. Subject has bi-phenotypic acute leukemia. 5. Subject has acute promyelocytic leukemia (APL). 6. Exclusion criterion 6 was removed with Protocol Amendment 3.0. 7. Subject has inadequate organ function as defined below at time of Treatment Eligibility Period: a.
  • APL acute promyelocytic leukemia
  • Subject has aspartate aminotransferase (AST), alanine aminotransferase (ALT), or alkaline phosphatase ⁇ 2.5 x the upper limit of normal (ULN).
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • UPN upper limit of normal
  • b Creatinine clearance less than 40 mL/minute.
  • Subject has a bilirubin level > 2 mg/dL (unless subject has known Gilbert’s disease). 8.
  • Subject has had prior treatment with biologic antineoplastic agents less than 7 days or 5 half-lives before the first CYNK-001 infusion, whichever is longer. (Exception will be granted for monoclonal antibodies that are known to have long half-lives, in which case a minimum of 2 weeks from last dose will be required).
  • Subject is pregnant or breastfeeding. 10.
  • Subject has new or progressive pulmonary infiltrates or pleural effusion large enough to be detected by chest x-ray or CT scan within 2 weeks of first CYNK-001 infusion.
  • Subject has active autoimmune disease other than controlled connective tissue disorder or those who are not on active therapy.
  • Subject has had a ASCT ⁇ 60 days prior to Treatment Eligibility Screening Visit or plans to have transplant within the 28 day period following first CYNK-001 infusion. Consultation with the Medical Monitor is encouraged when considering enrollment of subjects with prior ASCT. 13.
  • Subject has a history of malignancy other than AML or other underlying hematologic conditions such as MDS or MPN, unless the subject has been in remission or free of disease for greater than 1 year prior to CYNK-001 infusion. Other exceptions will include the following malignancies: a. Basal cell carcinoma of the skin b. Squamous cell carcinoma of the skin c. Carcinoma in situ of the cervix d. Carcinoma in situ of the breast e. Incidental biological finding of prostate cancer (TNM stage of T1a or T1b) f. Superficial Bladder Cancer g.
  • Subjects with therapy-related AML the underlying malignancy which led to secondary AML must have no evidence of the underlying malignant disease as of the last surveillance and the subject must not be planned for further treatment of the underlying malignant disease. 14.
  • Subject has a history of severe asthma for which the subject is presently on chronic medications or has a history of other symptomatic pulmonary disease. 15.
  • Subjects with the following prior history of GVHD will be excluded: ⁇ Acute GVHD: Subjects with prior history of acute GVHD where signs and/or symptoms did not completely resolve (no clinical signs/symptoms and not on more than 7.5 mg of prednisone per day) within 90 days of ongoing immunosuppression.
  • Chronic GVHD Subjects with prior history of chronic GVHD where signs and/or symptoms did not completely resolve (no clinical signs/symptoms and not on more than 7.5 mg of prednisone per day) within 90 days of ongoing immunosuppression. 16. Subject has an untreated chronic infection or has received treatment of any uncontrolled or progressive infection with systemic antibiotics within 2 weeks prior to first CYNK-001 infusion. Prophylactic antibiotic, antiviral, and antifungal medication are permissible (and required prior to or at start of lymphodepletion regimen). 17. Subject has any other organ dysfunction (CTCAE Version 5.0 Grade 3 or greater) that will interfere with the administration of the therapy according to this protocol. 18.
  • Subject has a resting left ventricular ejection fraction (LVEF) of ⁇ 40% obtained by echocardiography or multi-gated acquisition scan (MUGA). 19.
  • Subject was treated with an investigational product within 28 days of first CYNK-001 infusion. Subject must no longer be a participant in the previous interventional study at the time of CYNK-001 infusion. (Subjects who are under survival follow-up or observation associated with a study are permitted, and if treatment information is collected for this period, “Investigational Study” must be used to capture the study treatment.).
  • Treatment of Subjects [00506] Description of Study Drug: For full description of CYNK-001, refer to the Investigator’s Brochure (IB). Celularity will supply CYNK-001 for IV administration.
  • Subjects will receive CYNK-001 according to the protocol-specified treatment plan.
  • [00507] Commercially available acetaminophen and diphenhydramine will be used for pre-and post- medication. Subjects enrolled should obtain commercially available product through the local hospital pharmacy or licensed distributor.
  • [00508] Commercially available cyclophosphamide and fludarabine will be used as Lymphodepletion Regimen as outlined in this protocol.
  • rhIL-2 will be used as outlined in this protocol.
  • Lymphodepletion Regimen Each subject will undergo a Lymphodepletion Regimen beginning on Study Day -5 and ending on Study Day -3 followed by two days with no treatment on Study Days -2 and -1 (where Study Day 0 is the day of the first CYNK-001 infusion).
  • Cy 300 Flu 25 Regimen (Cohorts 1, 2, 3 only): Cyclophosphamide dose is 300 mg/m2 to be administered on Study Days -5, -4, and -3. Fludarabine dose is 25 mg/m2 to be administered on Study Days -5, -4, and -3.
  • Cyclophosphamide dose is 900 mg/m2 to be administered on Study Days -6, -5, -4, and -3.
  • Fludarabine dose is 30 mg/m2 to be administered on Study Days -6, -5, -4, and -3.
  • Dose calculations for cyclophosphamide and fludarabine may be based on actual or adjusted body weight based at the treating physician’s discretion and per institutional practices; doses may be rounded to the nearest 5% per institutional practices.
  • Mesna shall be administered, for Cohorts 4 and above, at the start of Lymphodepletion (e.g.
  • CYNK-001 is an allogeneic off the shelf cell therapy enriched for CD56+/CD3- NK cells culture-expanded from human placental CD34+ cells.
  • Culture- expanded cells are harvested, washed in Plasma-Lyte A and then packaged at 30 x 10 6 cells/mL in a total volume of 20 mL of cryopreservation solution containing 10% (w/v) HSA, 5.5% (w/v) Dextran 40, 0.21% NaCl (w/v), 32% (v/v) Plasma-Lyte A, and 5% (v/v) DMSO. It is filled into the container closure, frozen using a controlled rate freezer, and cryopreserved. Prior to releasing to the site, all release and characterization testing will be complete.
  • CYNK-001 When required by site, CYNK-001 is shipped in vapor phase LN2 to the designated clinical site where it will be processed for dose preparation in a standardized manner just prior to IV administration. [00517] On Study Days 0, 7, and 14 (and Day 21 for subjects treated in Cohorts 6a, 6b, 7a and 7b), subjects will receive acetaminophen 650 mg PO and diphenhydramine 25 mg as outlined. [00518] CYNK-001 will be administered at either 6 x 10 8 cells per dose, 1.2 x 10 9 cells per dose, 1.8 x 10 9 cells per dose, or 3.0 x 10 9 cells per dose (depending on Dose Cohort assignment), with or without rhIL-2.
  • CYNK-001 is administered IV, using a gravity IV administration set with a 16- to 22-gauge (or equivalent) needle or catheter with no filters.
  • a central line may be used to infuse CYNK-001 after confirming that the catheter diameter is 16- to 22-gauge (or equivalent) needle. For substantial deviation from this catheter diameter, consultation with the medical monitor is required.
  • the recommended infusion rate is approximately 240 mL per hour. No other medications or blood products should be in the IV line at the time of CYNK-001 infusion. Vital signs should be taken during CYNK-001 infusion if clinically indicated and any abnormal clinically significant findings should be documented. Immediately following the infusion, the infusion line will be flushed with 30 to 60 mL of normal saline.
  • Overdose As defined for this protocol, refers to CYNK-001, cyclophosphamide, fludarabine, and rhIL-2. On a per dose basis, an overdose is defined as the following amount over the protocol-specified dose of CYNK-001 assigned to a given subject, regardless of any associated AEs or sequalae: [00520] CYNK-001: 30% over the assigned protocol-specified dose of 6 x 10 8 cells, 1.2 x 10 9 cells, 1.8 x 10 9 cells, or 3.0 x 10 9 cells. [00521] Refer to cyclophosphamide, fludarabine, and rhIL-2 package inserts for overdose information.
  • rhIL-2 to Facilitate CYNK-001 Cell Survival and Expansion: Subjects treated in Cohort 5a, Cohort 5b, Cohort 5a De-escalation, Cohort 5b De-escalation, Cohort 6a*, Cohort 6b*, Cohort 7a*, and Cohort 7b* will receive rhIL-2 SC injections which will be administered according to the following instructions: ⁇ On CYNK-001 infusion days (Days 0, 7, 14 [and 21 for Cohorts 6a, 7b, 7a and 7b]), start subcutaneous rhIL-2 injections 1 to 3 hours prior to CYNK-001 cell infusion in the absence of Grade 4 infusion-related toxicity.
  • ⁇ *Cohorts 6a, 6b, 7a and 7b may receive rhIL-2 after thorough review of all safety, efficacy, and translational data in Cohorts 5a and 5b, respectively.
  • the decision to administer or hold rhIL-2 will be made by the Sponsor after review and DMC recommendation.
  • the location of the injection site is at the discretion of the treating physician following local institutional practices. Suggested locations for subcutaneous rhIL- 2 injections include upper arm, upper thigh, or abdomen. The location of each injection is to be collected in the electronic case report form (eCRF). ⁇ If subject is experiencing a Grade 4 AE on days where rhIL-2 will be given, then administration of rhIL-2 can be skipped, according to PI discretion.
  • rhIL-2 injections should not be given on 2 consecutive days or on the day prior to planned CYNK-001 infusion. If rhIL-2 dose delays occur where rhIL-2 is to be administered on the day prior to planned CYNK-001 infusion, that rhIL-2 dose should be skipped and the next rhIL-2 dose would be given on the same day of CYNK-001 infusion, 1 to 3 hours prior to CYNK-001 administration. ⁇ If there is an immediate clinically significant adverse reaction to rhIL-2 administration, CYNK-001 infusion can be delayed until recovered, according to PI discretion.
  • rhIL-2 will be given at a dose of 6M approximately every other day (e.g., 0, 2, 4, 7, 9, 11, and 14 [and Cohorts 6a, 6b, 7a and 7b: additional rhIL-2 dose on Day 21]) for a total of up to 8 doses ⁇
  • the rhIL-2 will be given at rhIL-2 3M IU on planned rhIL-2 dosing days.
  • ECG should be administered prior to each CYNK-001 infusion and after the third rhIL-2 injection. ECGs should continue to be performed at each follow-up visit.
  • Pulmonary function tests should be performed if clinically indicated during the treatment period if there is a history of or ongoing pulmonary condition.
  • Meperidine may be administered to control rigors if clinically indicated.
  • Pre- and post- IL-2 medications for each rhIL-2 injection are outlined.
  • Treatment Compliance CYNK-001 is to be administered IV at the clinical study site. Study personnel will review the dosing treatment allocation and ensure treatment is administered according to the subject’s treatment plan. Treatment compliance will be noted on the appropriate CRFs and source records based on administration records.
  • CYNK-001 Dose reductions are not permitted in this study. ⁇ Should dose delays for CYNK-001 be required o Day 0 dose may not be delayed for longer than 48 hours o Day 7 dose may not be started early or delayed for longer than 48 hours ⁇ If delayed longer than 48 hours, the dose will be skipped. ⁇ A subject that skips Day 7 dosing may receive Day 14 dosing. o Day 14 dose may not be started early or delayed for longer than 48 hours ⁇ If delayed longer than 48 hours, the dose will be skipped. o Cohorts 6a, 6b, 7a and 7b: Day 21 dose may not be started early or delayed for longer than 48 hours ⁇ If delayed longer than 48 hours, the dose will be skipped.
  • Subjects assigned to treatment in Cohort 5a, Cohort 5b, Cohort 6a*, Cohort 6b*, Cohort 7a*, and Cohort 7b* are to receive rhIL-2, approximately 1 to 3 hours prior to CYNK-001 infusion.
  • rhIL-2 Dose Adjustments to 3M IU may occur if the subject weighs ⁇ 45 kgs. *Note: decision to include rhIL-2 in cohorts 6a, 6b, 7a and 7b are based on DMC recommendation and Sponsor decision.
  • MRD positive population MRD Response (as defined herein) as assessed centrally by MFC at defined time points after CYNK-001 infusion. Additionally, time to MRD Response, duration of MRD Response, PFS, duration of morphologic CR, TTP, and OS will be evaluated.
  • R/R population Overall Response Rate (ORR) defined as achievement of Complete Remission (CR), Complete Remission with incomplete (CRi) hematologic recovery, or Morphologic leukemic-free state (MLFS), Duration of Response (DoR), and Overall (OS).
  • ORR Overall Response Rate
  • Safety Parameters Subject safety will be assessed in all subjects who receive any amount of CYNK-001 and will include AEs, vital signs, body weight measurements, physical examination findings, clinical laboratory test results, infusion site assessments, x-ray, magnetic resonance imaging (MRI) or computerized tomography (CT) scan results, electrocardiogram (ECG) interpretations, electroencephalography (EEG) if clinically indicated, pregnancy testing for FCBP, and concomitant medications and procedures will be tabulated and summarized by cohort. Timing of evaluations will be assessed as outlined in the Table of Events.
  • AEs will be reported and recorded in the electronic case report form (eCRF).
  • eCRF electronic case report form
  • SAEs serious adverse events
  • the rate of AEs, SAEs, abnormal laboratory AEs and vital signs (graded according to the NCI CTCAE Version 5.0) will be measured while the subject is on study.
  • the ASTCT Consensus Grading for CRS and Neurologic Toxicity Associated with Immune Effector Cells will be used for the purposes of grading of CRS considered associated with CYNK-001 by the Investigator. CRS at any grade is an expected event and immediately reportable.
  • IL-2- based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial. Bone Marrow Transplant 2003;32(2):177-86. 6. Caligiuri MA, Murray C, Robertson MJ, Wang E, Cochran K, Cameron C. Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin 2. J Clin Invest 1993;91:123-32. 7. Chen X, Xie H, Wood B, Walter R, Pagel J et al. Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia.
  • Trinchieri G Matsumoto-Kobayashi M, Clark SC, Seehra J, London L, Perussia B. Response of resting human peripheral blood natural killer cells to interleukin 2. J Exp Med 1984 Apr;160:1147-69.
  • Trinchieri G Natural killer cells wear different hats: effector cells of innate resistance and regulatory cells of adaptive immunity and of hematopoiesis. Semin Immunol 1995;7(2):83-8. Equivalents: [00535] The present invention is not to be limited in scope by the specific embodiments described herein.

Abstract

Provided herein are methods of treating acute myeloid leukemia (AML) in a human subject comprising administering to the subject an effective amount of CYNK cells to the subject so as thereby to provide an effective treatment of the cancer in the subject. The CYNK cells can be placental-derived natural killer (NK) cells or placental CD34+ cell-derived natural killer (NK) cells. The AML to be treated includes AML in remission with MRD and relapsed/refractory AML. The present invention also provides compositions comprising CYNK cells for the treatment of AML.

Description

HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS IN ACUTE MYELOID LEUKEMIA (AML) REMISSION WITH MINIMAL RESIDUAL DISEASE (MRD) OR RELAPSED/REFRACTORY AML [0001] This application claims priority to U.S. Provisional Patent Application No. 63,216,128, filed June 29, 2021, the disclosure of which is incorporated herein by reference in its entirety. 1. FIELD [0002] Provided herein are methods of producing populations of natural killer (NK) cells and/or ILC3 cells from a population of hematopoietic stem or progenitor cells in media comprising stem cell mobilizing factors, e.g., three-stage methods of producing NK cells and/or ILC3 cells in media comprising stem cell mobilizing factors starting with hematopoietic stem or progenitor cells from cells of the placenta, for example, from placental perfusate (e.g., human placental perfusate) or other tissues, for example, umbilical cord blood or peripheral blood. Further provided herein are methods of using the placental perfusate, the NK cells and/or ILC3 cells and/or NK progenitor cells described herein, to, e.g., suppress the proliferation of tumor cells, including multiple myeloma and acute myeloid leukemia cells. 2. BACKGROUND [0003] Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy of the myeloid precursor cell line, characterized by the clonal expansion of abnormal cells, which accumulate in the bone marrow, peripheral blood and/or other tissues, and interfere with the production of normal blood cells. [National Comprehensive Cancer Network [NCCN], 201939]. According to the Surveillance, Epidemiology, and End Results (SEER) database, approximately 19,520 individuals were diagnosed with AML and approximately 10,670 deaths due to AML occurred in the United States in 2018. The median age at diagnosis is 68 years [SEER, 201950]. AML is generally classified as primary or secondary, secondary referring to either exposure to prior cytotoxic chemotherapy or by transformation from an underlying hematologic condition such as myelodysplastic syndromes (MDS) or myeloproliferative neoplasm (MPN). Advances in mutational profiling and gene sequencing have allowed for enhanced risk stratification and prognosis [Dohner, 201716]. [0004] The risk factors associated with poor outcomes include older age (i.e., ≥ 60 years old), adverse cytogenetics and transformation of existing myelodysplasia, etc. [Dohner, 201015]. Of patients who are fit enough to receive standard induction therapy, accumulated data demonstrate that about 60% to 80% of younger adults and 40% to 50% of older adults achieve complete remission (CR), leaving a substantial population of surviving patients who are refractory to initial induction therapy. Patients whose disease does not respond to the first cycle of induction chemotherapy are sometimes categorized as refractory. [0005] A widely used remission-induction chemotherapy is the combination of cytarabine and anthracycline, consisting of cytarabine 100 to 200 mg/m2/day for 7 days and daunorubicin 45 to 90 mg/m2/day for 3 days, [Löwenberg, 199935; Tallman, 200552] often referred to as the “7 + 3 protocol.” A retrospective analysis of six Eastern Cooperative Oncology Group studies which included both younger and older adults demonstrated that 26% of patients treated with anthracycline and cytarabine-based induction therapy required a second cycle of identical induction therapy to achieve CR [Mangan, 201136]. If morphological CR is achieved, a consolidation regimen is typically employed, which may consist of additional chemotherapy cycles or stem cell transplant, typically allogeneic hematopoietic stem cell transplantation (aSCT). Alternatively, treatment options for subjects who choose not to receive emission-induction standard chemotherapy or are considered ineligible to receive remission-induction chemotherapy include low-dose cytarabine, azacitidine or decitabine [Deschler, 200614]. [0006] Relapsed and/or Refractory Acute Myeloid Leukemia (AML): Additionally, there is a large population of patients whose disease relapses after achieving first CR. A 3-year study of 1,069 patients who did not undergo aSCT, conducted at MD Anderson Cancer Center, showed that the probability of relapse-free survival at 3 years was 29%. The patients had a median age of 55 years, included 22% with favorable cytogenetics, 64% with intermediate risk cytogenetics, and 14% with adverse cytogenetics. Younger age and more favorable karyotype were associated with significantly increased rates of relapse-free survival at 1 year [Mangan, 201136]. The prognosis of relapsed or refractory AML is poor and the median survival is approximately 6 months [Ferrara, 200420; Giles, 200521; Ritchie, 201346; Craddock, 201410; Pleyer, 201444]. Among the few attempts to compare salvage therapies in AML, none demonstrated clear evidence of superiority [Feldman, 200519; Roboz, 201447]. [0007] For some patients with relapsed or refractory disease treated with chemotherapy alone, there is the potential for long-term disease-free survival (DFS), however this is most likely due to hematopoietic stem cell therapy (HSCT). Hematopoietic SCT in first relapse may be successful if a suitable donor can be identified, and the patient can proceed to transplant in a timely fashion. However, when treating patients with relapsed or refractory disease, challenges include accurately assessing the following: prognosis of disease, whether remission can be achieved, and the ability of patients to tolerate aggressive salvage therapies, choosing a successful salvage therapy, and ultimately identifying suitable patients for HSCT. The prognosis in relapsed AML patients is generally poor but depends largely on the timing of relapse (early versus late) and the possibility of allogeneic hematopoietic stem cell transplantation (HSCT). For patients potentially eligible for HSCT relapse post-SCT is frequent, and in spite of salvage attempts, less than 20% of these patients are alive after 5 years [Thol, F., Ganser, A., 2020]. For those patients that are not eligible for SCT, hypomethylating agents (HMA), low-dose AraC (LDAC and increasingly combination therapy of Venetoclax with demethylating agents achieves encouraging response rates. [0008] MRD Detection in AML: Mounting evidence has shown that risk of relapse in AML following chemotherapy has also been correlated to the detection of MRD. MRD is defined as leukemic cells at levels below morphologic detection [Ravandi, 201845; Ossenkoppele, 201341]. The presence of residual leukemia blasts in AML, known as MRD, can be determined by multiparameter flow cytometry (MFC) with reported detection limits of 1:104 to 1:106 white blood cells, compared to 1:20 as detected in morphological-based CR [Schuurhuis, 201849]. [0009] Recent advancements in the measurement of MRD in AML have indicated that the presence of MRD is a strong independent prognostic marker of increased risk of relapse and shorter survival in patients with AML [Grimwade, 201424; San Miguel, 200148; Buccisano, 20064; Jongen-Lavrencic 201827; Chen, 20157]. Immunophenotyping by MFC has emerged as a well-established strategy in MRD detection in AML. A retrospective analysis from the Southwest Oncology Group S0106 study showed that MRD detected by MFC after completion of induction chemotherapy could be used to stratify younger patients by risk of AML recurrence and that MRD status was the single most important predictor of overall survival and progression-free survival in individual patients [Othus, 201642; Schuurhuis, 201849]. [0010] Natural Killer Cells in the Treatment of AML: Evidence suggests chemotherapy alone does not result in a durable remission in AML patients due to a non-actively cycling subpopulation of leukemic cells, called leukemia stem cells [Jordan, 200728]. However, these leukemia stem cells are capable of entering into cell cycle and regenerating leukemia cells associated with relapse. There are several reasons why NK cell infusions may induce and/or prolong remission and ultimately survival in high-risk AML patients. Natural killer cells have demonstrated the ability to kill leukemia stem cells [Langenkamp, 200931], which may explain earlier studies demonstrating longer times to relapse in patients given cytoreductive therapy followed by the adoptive transfer of NK cells. In particular, one study demonstrated that adoptively transferred NK cells could expand in vivo, and that induction of remission in 5 of 19 poor-prognosis AML patients was associated with NK cell expansion and killer cell immunoglobulin-like receptor (KIR) ligand-mismatch donors [Miller, 200537; Bachanova, 20143]. More recently, infusion of haploidentical NK cells as post-CR consolidation in elderly AML patients was associated with prolonged disease-free survival [Curti, 201111; Curti, 201612]. [0011] In a phase I first-in-human study of PNK-007 (NCT02781467), 10 relapsed/refractory subjects with a median age of 66 years were treated with a single PNK-007 infusion followed by 5 to 6 recombinant human interleukin-2 (rhIL-2) injections. These subjects received a median of 3 prior lines of AML therapy and included 5 subjects with a history of MDS and 5 subjects who had received prior aSCT. [0012] One subject treated with 10 x 106 cells/kg PNK-007 developed Cytokine Release Syndrome (CRS) 14 days after infusion and was effectively managed with tocilizumab. This CRS event was deemed a dose-limiting toxicity. The other 9 subjects did not experience CRS symptoms and PNK-007 was well tolerated with no infusion reactions or graft-versus-host disease (GVHD). No deaths were attributed to PNK-007. [0013] Due to supply chain constraints, logistics constraints and a need to transition to an alternative manufacturing site capable of later stage and commercial manufacturing, several changes have been implemented to the manufacturing processes for PNK-007. The results of testing based on identity, purity, viability, fold expansion during manufacturing and performance of the Drug Products using a qualified cytotoxicity assay demonstrated comparability between PNK 007 and CYNK-001. [0014] CYNK-001 in the Treatment of AML: CYNK-001 is an allogeneic off the shelf cell therapy enriched for CD56+/CD3- NK cells expanded from human placental CD34+ cells. CYNK-001 is manufactured in a cryopreserved formulation that is thawed and diluted at the clinical site prior to dose preparation and direct infusion. CYNK-001 is packaged at 30 x 106 cells/mL in a total volume of 20 mL cryopreservation solution containing 10% (w/v) human serum albumin (HSA), 5.5% (w/v) Dextran 40, 0.21% sodium chloride (NaCl) (w/v), 32% (v/v) Plasma-Lyte A, and 5% (v/v) dimethyl sulfoxide (DMSO). It is filled into the container closure, frozen using a controlled rate freezer, and cryopreserved. Prior to releasing to the site, all release and characterization testing will be complete. When required by site, CYNK 001 is shipped in vapor phase LN2 to the designated clinical site where it will be processed for dose preparation in a standardized manner just prior to IV administration. [0015] This study is the first study to evaluate the safety and potential efficacy of CYNK- 001 in subjects with primary or secondary AML in morphological CR and MRD positivity and in subjects with R/R AML. The use of a 3 + 3 dose escalating tolerability algorithm with strict dose- limiting toxicity (DLT) criteria will allow detection of serious toxicity associated with the use of CYNK 001 in study subjects. [0016] The study will be comprised of Treatment Eligibility Period, Treatment Period and Follow-up Period. The Treatment Period will include a Lymphodepletion Regimen that will be used to help prevent rejection of donor cells and to maintain and augment CYNK 001 cells in study subjects. [0017] IL-15 is a cytokine critical for NK cell survival and proliferation. In a seminal AML study of haploidentical NK cell adoptive transfer, high-dose Cy/Flu preparative regimen (Cy: 60mg/kg x 2 days, Flu: 25mg/m2 x 5 days) was associated with elevated serum IL-15 levels and expansion of the transferred NK cells. Patients who received lower intensity regimens did not demonstrate in vivo expansion of donor cells and showed low to modest changes in serum IL-15 (Miller et al, Blood 2005). Preclinical data also support the need for exposure to IL-15 in vivo for maintaining CYNK-001 persistence. Whereas clinical study NCT02781467 evaluating PNK-007 using high-dose Cy/Flu (Cy: 8400mg total, Flu: 230mg total) conditioning confirmed elevated IL- 15 in AML patients, data generated for cohorts 1-3 following low dose Cy/Flu (Cy: 1656mg total, Flu: 230mg total) in the current study show negligible change to low baseline levels of serum IL- 15. An enhanced dose of Cy/Flu tested in cohort 4 (Cy: 6624mg total, Flu: 221mg total) [Dolstra, 201717] achieved the desired effect of raising serum IL-15 to support NK cell persistence. Evaluation of CYNK-001 persistence in cohorts 1-4 indicate limited persistence up to 28 days in the bone marrow, but not in follow up timepoints beyond 28 days (data not shown). [0018] In addition to raising circulating IL-15, enhanced lymphodepletion reduced the number of CD4+ regulatory T cell (Treg) in circulation over the treatment period compared to low dose Cy/Flu. This is hypothesized to be advantageous in cohort 5 which evaluates the inclusion of rhIL-2 to support CYNK-001 persistence. Treg proliferate in response to IL-2 and functionally antagonize immune effector responses, potentially limiting NK cell mediated tumor immunity. Administering CYNK-001 cells at days 0, 7, 14, and 21 using enhanced lymphodepletion takes advantage of the IL-15 and Treg changes during the treatment period to maximize the persistence of dosed CYNK-001 cells and their functional impact on eliminating AML blasts [0019] HLA matching and KIR mismatching will not be used in the selection of CYNK- 001 for an individual subject. However, these data will be collected for retrospective analysis. [0020] rhIL-2 Background: The use of rhIL-2 therapy has shown regression of tumors, has known adverse toxic effects, and has been in clinical trials for cancer therapy [Siegel, 199151 ]. One mechanism that induces tumor regression is due to the ability of rhIL-2 to stimulate the proliferation and cytotoxicity of NK cells in vitro and in vivo [Henney, 198126; Trinchieri, 198454; Caligiuri, 19936]. Moreover, rhIL-2 has been shown to enhance the expression of maturation markers on the surface of NK cells, making them more potent killers in vitro and in vivo [Trinchieri, 199555]. Adoptive transfer of haploidentical NK cells has shown a cytokine- dependent expansion phase after infusion, and a reduction after cytokine withdrawal in both the nonclinical setting [Miller, 201438] and clinical trial setting [Geller, 201122]. Similarly, cytokine-dependent cell expansion followed by reduction upon cytokine withdrawal was observed in the pre-clinical model of adoptively transferred PNK 007 cells into non-obese diabetic (NOD scid gamma [NSG]) immunodeficient mice supplemented with human (h)IL-15. Specifically, the rhIL-15 dosing regimen was designed to mimic the human clinical response to chemotherapy [Miller, 200537]. [0021] Clinical trials have assessed the effects of low-dose rhIL-2 administration on activation of NK cells in subjects with cancer. Miller et al demonstrated the safety and feasibility of daily subcutaneous rhIL-2 injections following high-dose chemotherapy and autologous hematopoietic cell transplantation (HCT). Whereas rhIL-2 significantly expanded the number of circulating NK cells in vivo, these NK cells were not maximally cytotoxic as determined by in vitro assays [Miller, 199737]. Subsequent studies tested an infusion of rhIL-2–activated NK-cell– enriched populations or intravenous rhIL-2 infusions combined with subcutaneous (SC) rhIL-2. Although these approaches augmented in vivo NK-cell function, no consistent efficacy of autologous NK cell therapy could be detected in cancer patients when compared with cohorts of matched controls [Burns, 20035]. [0022] The dose of rhIL-2 once every other day (QOD) for 6 doses has been described in other settings, including in patients with B-cell non-Hodgkin’s lymphoma in which rhIL-2 was given with rituximab in a Phase I/II study and demonstrated that doses of rhIL-2 given three times a week up to 6 doses resulted in NK-cell expansion that correlated with response [Gluck, 200423]. Miller et al showed that following rhIL-2 administered QOD after lymphodepletion and haploidentical NK cell infusions in patients with relapsed, primary refractory AML or secondary AML, 5 of 14 patients achieved morphologic CR. There were mild symptoms attributed to rhIL-2, but IL-2 doses of 10 million (M) international units (IU) QOD was well tolerated with no evidence of GVHD [Miller, 200537]. NK-cells persistence was detected 7+ days in patients treated with cyclophosphamide and fludarabine prior to haploidentical NK cell infusion and rhIL- 2. [0023] Published pharmacokinetic data for IL-2 administered by subcutaneous (SC) injection demonstrate measurable levels within one hour of administration and maximal bioavailability at 3-4 hours post injection with clearance from the body by 24 hours [Eton, 200218; Piscitelli, 199643; Kirchner,199830]. [0024] Also noted was an elevation of IL-15 during periods of lymphopenia [Miller, 200537]. In a study described the pretreatment with IL-2 diphteria toxin fusion protein (IL-2DT) which was used to remove endogenous IL-2R expressing T-cells, resulted in improved NK-cell expansion in patients who were treated with rhIL-29 M IU QOD x 6 doses compared to those who did not receive IL-2DT pre-treatment, showing the importance of optimizing conditions in which potential inhibitory cells, such as Treg cells, could impact NK cell expansion [Bachanova, 20143]. [0025] Similar to clinical studies of NK cell therapies [Geller, 201122], rhIL-2 was administered to facilitate NK cell survival and expansion in the PNK-007-AML-001 study (NCT02781467). IL-2 was administered at a dose of 6 M IU/dose SC every other day (QOD) for up to 6 doses after lymphodepleting chemotherapy with cyclophosphamide and fludarabine followed by a single infusion of PNK-007 cells. Ten patients received PNK-007 after lymphodepleting chemotherapy, with 10 evaluable for safety and 8 for efficacy. The efficacy population included one patient (12.5%) with complete remission with incomplete platelet recovery (CRp) assessed on day 21 and one patient (12.5%) with morphologic leukemia-free state (MLFS) assessed on day 28. Overall, rhIL-2 was well tolerated in 25 subjects treated within both CCT-PNK-007-AML-001 and a similar study in multiple myeloma patients, CCT-PNK-007-MM- 001, studies. There were no serious adverse events that were suspected to be related to rhIL-2 in both studies. In the CCT-PNK-007-AML-001, treatment-emergent adverse events leading to rhIL-2 discontinuation were reported in 2 subjects. One subject experienced rhIL-2 related Grade 2 acute kidney injury, pyrexia and chills and the other subject experienced pyrexia. Dose was reduced in one subject due to rhIL-2 related events of Grade 1 pyrexia and Grade 3 hypotension. [0026] In the CCT-PNK-007-MM-001 study, Treatment-emergent adverse events leading to rhIL-2 discontinuation were reported in 2 subjects. One subject experienced rhIL-2 injection‑related reactions (Grade 1 dyspnea, Grade 2 Pyrexia and Grade 3 maculo‑papular rash) and the other subject experienced rhIL‑2 injection‑related reaction (Grade 2 pyrexia and Grade 3 maculo-papular rash). [0027] There were no reported cases of graft-vs-host disease. One patient experienced manageable cytokine release syndrome diagnosed 15 days following administration of PNK-007. PNK-007 persisted 7-28 days following a single IV infusion. This study provides proof-of- concept of the antileukemic activity of PNK-007 following lymphodepleting chemotherapy in R/R AML. [Cooley, 20199]. We are incorporating rhIL-2 into several of the cohorts in this amended protocol, using the dosing and treatment schedule that was used in the previously described studies with PNK-007 in order to determine if this is safe and tolerable in combination with CYNK-001 and can affect enhanced CYNK-001 activity and persistence in this study. [0028] Human Placental Hematopoietic Stem Cell Derived Natural Killer Cell Dosing: PNK-007, which has been previously used in an AML study (CCT PNK 007 AML 001, NCT02781467), was produced with a cryopreserved Drug Substance, which was subsequently thawed, cultured, washed, filtered, and reformulated as a fresh Drug Product in Plasma-Lyte®-A solution containing 10% (weight/volume) HSA. The cells were formulated at concentrations of 0.5 x 106 cells/mL, 1.5 x 106 cells/mL, 5 x 106 cells/mL or 15 x 106 cells/mL, which allowed a range of clinical doses with similar infusion volumes. PNK-007 is dosed based on subject weight (e.g., 106 cells/kg) so the volume of the infusion scales with the subject weight (approximately 2 mL/kg). Each unit of PNK-007 was custom filled based on the subject weight, so that a full unit delivered the allocated cell dose. [0029] A total of 10 subjects were treated with a single infusion of PNK-007 (range 1 x 106 cells/kg to 10 x 106 cells/kg) followed by 5 or 6 total rhIL-2 injections QOD starting on day of PNK-007 infusion to facilitate PNK-007 expansion. Four subjects were treated in the highest dose administered in the PNK-007-AML-001 study, 10 x106 cells/kg PNK-007, with an actual dose infused ranging from 5.86 x 108 to 8.49 x 108 total cells associated with subject weight ranges from 59.3 kg to 83.1 kg. [0030] The CCT-PNK-007-AML-001 study was terminated prior to completion due to a business decision. Therefore, the maximum tolerated dose (MTD) was not identified. 3. SUMMARY [0031] The present invention provides methods of treating acute myeloid leukemia (AML) in a subject comprising administering to the subject an effective amount of CYNK cells to the subject so as thereby to provide an effective treatment of the AML in the subject. [0032] In some embodiments of the invention the CYNK cells are placental-derived natural killer (NK) cells. [0033] In some embodiments of the invention the CYNK cells are placental CD34+ cell- derived natural killer (NK) cells. [0034] In some embodiments of the invention the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells and / or expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 which is higher than expression of said markers in peripheral blood natural killer cells. [0035] In some embodiments of the invention the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells. [0036] In some embodiments of the invention expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS is lower than expression of said markers in peripheral blood natural killer cells. [0037] The method of any one of claims 1-6, wherein the CYNK cells are characterized by expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 which is higher than expression of said markers in peripheral blood natural killer cells. [0038] In some embodiments of the invention expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 is higher than expression of said markers in peripheral blood natural killer cells. [0039] In some embodiments of the invention the CYNK cells are prepared by the methods presented herein. [0040] In some embodiments of the invention the acute myeloid leukemia is primary acute myeloid leukemia or secondary acute myeloid leukemia. [0041] In some embodiments of the invention the acute myeloid leukemia is in remission with minimal residual disease. [0042] In some embodiments of the invention the acute myeloid leukemia is relapsed/refractory AML. [0043] In some embodiments of the invention providing an effective treatment comprises reducing the rate of minimal residual disease (MRD) relative to placebo. [0044] In some embodiments of the invention providing an effective treatment comprises converting the subject to MRD negative. [0045] In some embodiments of the invention providing an effective treatment comprises converting the subject to MRD positive less than 0.1%. [0046] In some embodiments of the invention the MRD is measured by flow cytometry. [0047] In some embodiments of the invention the MRD is measured by nucleic acid sequencing, preferably by next generation sequencing. [0048] In some embodiments of the invention providing an effective treatment comprises reducing the time to minimal residual disease (MRD) response relative to placebo. [0049] In some embodiments of the invention providing an effective treatment comprises increasing the duration of minimal residual disease (MRD) response relative to placebo. [0050] In some embodiments of the invention providing an effective treatment comprises reducing the incidence, severity, or duration of the disease as measured by one or more International Myeloma Working Group (IMWG) response criteria relative to placebo. [0051] In some embodiments of the invention providing an effective treatment comprises reducing the incidence, severity, or duration of the disease as measured by the Eastern Cooperative Oncology Group (ECOG) Performance Status relative to placebo. [0052] In some embodiments of the invention providing an effective treatment comprises increasing the duration of clinical response relative to placebo. [0053] In some embodiments of the invention providing an effective treatment comprises increasing the rate of progression free survival, the rate of front-line progression free survival, or the rate of survival relative to placebo. [0054] In some embodiments of the invention providing an effective treatment comprises increasing the time to progression, the front-line time to progression or the time to death relative to placebo. [0055] In some embodiments of the invention providing an effective treatment comprises increasing the overall survival or front-line overall survival relative to placebo. [0056] In some embodiments of the invention providing an effective treatment comprises increasing the patient reported outcome relative to placebo or relative to pretreatment. [0057] In some embodiments of the invention administering the cells to the subject is performed intravenously. [0058] In some embodiments of the invention from about 6 x 108 to about 3.0 x 109 cells are administered per administration. [0059] In some embodiments of the invention from about 9 x 108 to about 1.8 x 109 cells are administered per administration. [0060] In some embodiments of the invention about about 6 x 108 cells are administered per administration. [0061] In some embodiments of the invention about about 1.2 x 109 cells are administered per administration. [0062] In some embodiments of the invention about about 1.8 x 109 cells are administered per administration. [0063] In some embodiments of the invention about about 3.0 x 109 cells are administered per administration. [0064] In some embodiments of the invention the treatment comprises 1 to 5 administrations of cells. [0065] In some embodiments of the invention the treatment comprises 3 administrations of cells. [0066] In some embodiments of the invention the treatment comprises 4 administrations of cells. [0067] In some embodiments of the invention the administrations occur approximately 1 week apart. [0068] In some embodiments of the invention one administration of cells occurs at approximately day 0 of the treatment. [0069] In some embodiments of the invention one administration of cells occurs at approximately day 7 of the treatment. [0070] In some embodiments of the invention one administration of cells occurs at approximately day 14 of the treatment. [0071] In some embodiments of the invention one administration of cells occurs at approximately day 21 of the treatment. [0072] In some embodiments of the invention the treatment comprises about 3 administrations of cells occurring at about days 0, 7, and 14 of the treatment. [0073] In some embodiments of the invention the treatment comprises about 3 administrations of cells occurring at about days 0, 7, 14, and 21 of the treatment. [0074] In some embodiments of the invention the treatment further comprises a lymphodepletion regimen. [0075] In some embodiments of the invention the lymphodepletion regimen comprises administering cyclophosphamide and fludarabine to the subject. [0076] In some embodiments of the invention the lymphodepletion regimen comprises administering about 300 mg/m2/day cyclophosphamide and 25 mg/m2/day fludarabine to the subject. [0077] In some embodiments of the invention the lymphodepletion regimen comprises administering about 900 mg/m2/day cyclophosphamide and 30 mg/m2/day fludarabine to the subject. [0078] In some embodiments of the invention the cyclophosphamide and fludarabine are administered to the subject on days -5, -4, and -3. [0079] In some embodiments of the invention the cyclophosphamide and fludarabine are administered to the subject on days -6, -5, -4, and -3. [0080] In some embodiments of the invention the treatment further comprises administering recombinant human interleukin-2 (rhIL-2) to the subject. [0081] In some embodiments of the invention the administration of rhIL-2 occurrs on each day that CYNK cells are administered. [0082] In some embodiments of the invention the administration of rhIL-2 occurrs on days 0, 7, and 14. [0083] In some embodiments of the invention the administration of rhIL-2 occurrs on days 0, 7, 14, and 21. [0084] In some embodiments of the invention the rhIL-2 is additionally administered every other day between each administration of CYNK cells. [0085] In some embodiments of the invention about 4M IU to about 8MIU of rhIL-2 is administered to the subject for each administration. [0086] In some embodiments of the invention about 6M IU of rhIL-2 is administered to the subject for each administration. [0087] In some embodiments of the invention the treatment days are measured relative to administration fo the first dose of CYNK cells at day 0 of the treatment. [0088] The present invention provides compositions comprising human CYNK cells for use in the treatment of acute myeloid leukemia (AML) in a subject. [0089] The present invention provides uses of compositions comprising human CYNK cells for use in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML) in a subject. [0090] In some embodiments of the invention the cancer is multiple myeloma. [0091] In some embodiments of the invention the acute myeloid leukemia is primary acute myeloid leukemia or secondary acute myeloid leukemia. [0092] In some embodiments of the invention the acute myeloid leukemia is in remission with minimal residual disease. [0093] In some embodiments of the invention the acute myeloid leukemia is relapsed/refractory AML. [0094] In some embodiments of the invention the CYNK cells are placental-derived natural killer (NK) cells. [0095] The composition or use of any one of claims 59-64, wherein the CYNK cells are placental CD34+ cell-derived natural killer (NK) cells. [0096] In some embodiments of the invention the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells and / or expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 which is higher than expression of said markers in peripheral blood natural killer cells. [0097] In some embodiments of the invention the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells. [0098] In some embodiments of the invention expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS is lower than expression of said markers in peripheral blood natural killer cells. [0099] In some embodiments of the invention the CYNK cells are characterized by expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 which is higher than expression of said markers in peripheral blood natural killer cells. [00100] In some embodiments of the invention expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 is higher than expression of said markers in peripheral blood natural killer cells. [00101] In some embodiments of the invention the CYNK cells are prepared by the methods presented herein. Terminology [00102] As used herein, the term CYNK are CD34+ cell-derived NK cells produced by the methods described herein. In specific embodiments, CYNK cells are placental-deived NK cells. In other specific embodiments, CYNK-001 is a specific formulation of CYNK cells. [00103] As used herein, the terms “immunomodulatory compound” and “IMiDTM” do not encompass thalidomide. [00104] As used herein, “lenalidomide” means 3-(4'aminoisoindoline-1'-one)-1-piperidine- 2,6-dione (Chemical Abstracts Service name) or 2,6-Piperidinedione,3-(4-amino-1,3-dihydro-1- oxo-2H-isoindol-2-yl)- (International Union of Pure and Applied Chemistry (IUPAC) name). As used herein, “pomalidomide” means 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione. [00105] As used herein, “multipotent,” when referring to a cell, means that the cell has the capacity to differentiate into a cell of another cell type. In certain embodiments, “a multipotent cell” is a cell that has the capacity to grow into a subset of the mammalian body's approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all of the cell types. [00106] As used herein, “feeder cells” refers to cells of one type that are co-cultured with cells of a second type, to provide an environment in which the cells of the second type can be maintained, and perhaps proliferate. Without being bound by any theory, feeder cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors (e.g., bFGF), other factors (e.g., cytokines), and metabolic nutrients to target cells. In certain embodiments, feeder cells grow in a mono-layer. [00107] As used herein, the “natural killer cells” or “NK cells” produced using the methods described herein, without further modification, include natural killer cells from any tissue source. [00108] As used herein, the “ILC3 cells” produced using the methods described herein, without further modification, include ILC3 cells from any tissue source. [00109] As used herein, “placental perfusate” means perfusion solution that has been passed through at least part of a placenta, e.g., a human placenta, e.g., through the placental vasculature, and includes a plurality of cells collected by the perfusion solution during passage through the placenta. [00110] As used herein, “placental perfusate cells” means nucleated cells, e.g., total nucleated cells, isolated from, or isolatable from, placental perfusate. [00111] As used herein, “tumor cell suppression,” “suppression of tumor cell proliferation,” and the like, includes slowing the growth of a population of tumor cells, e.g., by killing one or more of the tumor cells in said population of tumor cells, for example, by contacting or bringing, e.g., NK cells or an NK cell population produced using a three-stage method described herein into proximity with the population of tumor cells, e.g., contacting the population of tumor cells with NK cells or an NK cell population produced using a three-stage method described herein. In certain embodiments, said contacting takes place in vitro or ex vivo. In other embodiments, said contacting takes place in vivo. [00112] As used herein, the term “hematopoietic cells” includes hematopoietic stem cells and hematopoietic progenitor cells. [00113] As used herein, the “undefined component” is a term of art in the culture medium field that refers to components whose constituents are not generally provided or quantified. Examples of an “undefined component” include, without limitation, serum, for example, human serum (e.g., human serum AB) and fetal serum (e.g., fetal bovine serum or fetal calf serum). [00114] As used herein, “+”, when used to indicate the presence of a particular cellular marker, means that the cellular marker is detectably present in fluorescence activated cell sorting over an isotype control; or is detectable above background in quantitative or semi-quantitative RT-PCR. [00115] As used herein, “–”, when used to indicate the presence of a particular cellular marker, means that the cellular marker is not detectably present in fluorescence activated cell sorting over an isotype control; or is not detectable above background in quantitative or semi- quantitative RT-PCR. 4. BRIEF DESCRIPTION OF THE FIGURES [00116] FIG.1 shows expansion of NK cells for compounds CRL1 – CRL11. [00117] FIG.2 shows expansion of NK cells for compounds CRL12 – CRL22. [00118] FIG.3 shows expansion of NK cells relative to SR1 positive control. [00119] FIG.4 shows expansion of CD34+ cells from which the NK cells were derived. [00120] FIG.5 shows cytotoxicity of the expanded NK cultures. [00121] FIG.6 shows that PNK cells highly express genes encoding the cytotoxic machinery. FIG.6A CYNK cells were combined with peripheral blood derived NK cells (PB- NK) at 1:1 ratio and gene expression analyzed on single cell level using 10X Genomics Chromium platform and Illumina sequencing. Bioinformatics analysis utilized 10X Genomics Cell Ranger analysis pipeline. Transcript analysis was restricted to Granzyme B (GZMB) expressing cells. FIG.6B A representative tSNE plot depicting PNK and PB-NK cells as distinct populations. FIG.6C tSNE plots of selected NK cell-associated genes. The data is representative of two donors. [00122] FIG.7 shows that PNK and PB-NK cells differentially express genes encoding NK cell receptors. The expression of selected NK cell receptor genes analyzed by real-time quantitative PCR in peripheral blood NK cells (PB-NK) and CD11a+-bead-purified PNK cells. An alternative name indicated above the histogram for selected markers. The data represents mean ± SD of three donors for CYNK and PBNK cells (n=3). * p<0.05, ** p<0.005, *** p<0.001. [00123] FIG.8 shows the gating strategy for PB-NK and CYNK cells. CYNK and PBMC cells were thawed and stained with fluorophore-coupled antibodies targeting NK cell receptors. The figure demonstrates representative dot plots and the gating strategy for the identification of CYNK and PB-NK cells. See FIG.9 for further characterization of the populations. [00124] FIG.9 shows differential expression of surface proteins on CYNK and PB-NK cells. CYNK and PB-NK cells were pre-gated as indicated in FIG.8. [00125] FIG.10 shows that CYNK cells form a distinct cell population from PB-NK cells based on surface protein expression. tSNE plots demonstrating differential clustering of CYNK and PB-NK cells based on their surface markers. tSNE plots were generated of flow cytometry data using FlowJo software. [00126] FIG.11 shows a schematic of the AML cohort dose escalation plan. 5. DETAILED DESCRIPTION [00127] Provided herein are novel methods of producing and expanding NK cells and/or ILC3 cells from hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells. Also provided herein are methods, e.g., three-stage methods, of producing NK cell populations and/or ILC3 cell populations from hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells. The hematopoietic cells (e.g., CD34+ hematopoietic stem cells) used to produce the NK cells and/or ILC3 cells, and NK cell populations and/or ILC3 cell populations, may be obtained from any source, for example, without limitation, placenta, umbilical cord blood, placental blood, peripheral blood, spleen or liver. In certain embodiments, the NK cells and/or ILC3 cells or NK cell populations and/or ILC3 cell populations are produced from expanded hematopoietic cells, e.g., hematopoietic stem cells and/or hematopoietic progenitor cells. In one embodiment, hematopoietic cells are collected from a source of such cells, e.g., placenta, for example from placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver (e.g., fetal liver) and/or bone marrow. [00128] The hematopoietic cells used to produce the NK cells and/or ILC3 cells, and NK cell populations and/or ILC3 cell populations, may be obtained from any animal species. In certain embodiments, the hematopoietic stem or progenitor cells are mammalian cells. In specific embodiments, said hematopoietic stem or progenitor cells are human cells. In specific embodiments, said hematopoietic stem or progenitor cells are primate cells. In specific embodiments, said hematopoietic stem or progenitor cells are canine cells. In specific embodiments, said hematopoietic stem or progenitor cells are rodent cells. 5.1. Hematopoietic Cells [00129] Hematopoietic cells useful in the methods disclosed herein can be any hematopoietic cells able to differentiate into NK cells and/or ILC3 cells, e.g., precursor cells, hematopoietic progenitor cells, hematopoietic stem cells, or the like. Hematopoietic cells can be obtained from tissue sources such as, e.g., bone marrow, cord blood, placental blood, peripheral blood, liver or the like, or combinations thereof. Hematopoietic cells can be obtained from placenta. In a specific embodiment, the hematopoietic cells are obtained from placental perfusate. In one embodiment, the hematopoietic cells are not obtained from umbilical cord blood. In one embodiment, the hematopoietic cells are not obtained from peripheral blood. Hematopoietic cells from placental perfusate can comprise a mixture of fetal and maternal hematopoietic cells, e.g., a mixture in which maternal cells comprise greater than 5% of the total number of hematopoietic cells. In certain embodiments, hematopoietic cells from placental perfusate comprise at least about 90%, 95%, 98%, 99% or 99.5% fetal cells. [00130] In another specific embodiment, the hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells, from which the NK cell populations and/or ILC3 cell populations produced using a three-stage method described herein are produced, are obtained from placental perfusate, umbilical cord blood, fetal liver, mobilized peripheral blood, or bone marrow. In another specific embodiment, the hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells, from which the NK cell populations and/or ILC3 cell populations produced using a three- stage method described herein are produced, are combined cells from placental perfusate and cord blood, e.g., cord blood from the same placenta as the perfusate. In another specific embodiment, said umbilical cord blood is isolated from a placenta other than the placenta from which said placental perfusate is obtained. In certain embodiments, the combined cells can be obtained by pooling or combining the cord blood and placental perfusate. In certain embodiments, the cord blood and placental perfusate are combined at 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 the like by volume to obtain the combined cells. In a specific embodiment, the cord blood and placental perfusate are combined at a ratio of from 10:1 to 1:10, from 5:1 to 1:5, or from 3:1 to 1:3. In another specific embodiment, the cord blood and placental perfusate are combined at a ratio of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5 or 1:10. In a more specific embodiment, the cord blood and placental perfusate are combined at a ratio of 8.5:1.5 (85%:15%). [00131] In certain embodiments, the cord blood and placental perfusate are combined at 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 the like by total nucleated cells (TNC) content to obtain the combined cells. In a specific embodiment, the cord blood and placental perfusate are combined at a ratio of from 10:1 to 10:1, from 5:1 to 1:5, or from 3:1 to 1: 3. In another specific embodiment, the cord blood and placental perfusate are combined at a ratio of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5 or 1:10. [00132] In another specific embodiment, the hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells from which said NK cell populations and/or ILC3 cell populations produced using a three-stage method described herein are produced, are from both umbilical cord blood and placental perfusate, but wherein said umbilical cord blood is isolated from a placenta other than the placenta from which said placental perfusate is obtained. [00133] In certain embodiments, the hematopoietic cells are CD34+ cells. In specific embodiments, the hematopoietic cells useful in the methods disclosed herein 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 of CD2–, CD3–, CD11b–, CD11c–, CD14–, CD16–, CD19–, CD24–, CD56–, CD66b– and/or glycophorin A–. In another specific embodiment, the hematopoietic 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–. In another more specific embodiment, the hematopoietic cells are CD34+CD38–CD33–CD117–CD235–CD36–. [00134] In another embodiment, the hematopoietic cells are CD45+. In another specific embodiment, the hematopoietic cells are CD34+CD45+. In another embodiment, the hematopoietic cell is Thy-1+. In a specific embodiment, the hematopoietic cell is CD34+Thy-1+. In another embodiment, the hematopoietic cells are CD133+. In specific embodiments, 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, the hematopoietic cells are positive for KDR (vascular growth factor receptor 2). In specific embodiments, the hematopoietic cells are CD34+KDR+, CD133+KDR+ or Thy-1+KDR+. In certain other embodiments, the hematopoietic cells are positive for aldehyde dehydrogenase (ALDH+), e.g., the cells are CD34+ALDH+. [00135] In certain other embodiments, the CD34+ cells are CD45–. In specific embodiments, the CD34+ cells, e.g., CD34+, CD45– cells express one or more, or all, of the miRNAs hsa-miR-380, hsa-miR-512, hsa-miR-517, hsa-miR-518c, hsa-miR-519b, hsa-miR-520a, hsa-miR-337, hsa-miR-422a, hsa-miR-549, and/or hsa-miR-618. [00136] In certain embodiments, the hematopoietic cells are CD34–. [00137] The hematopoietic cells can also lack certain markers that indicate lineage commitment, or a lack of developmental naiveté. 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, the hematopoietic cells are negative for one or more, or all, of lineage markers CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin A. [00138] Thus, hematopoietic cells can be selected for use in the methods disclosed herein on the basis of the presence of markers that indicate an undifferentiated state, or on the basis of the absence of lineage markers indicating that at least some lineage differentiation has taken place. Methods of isolating cells, including hematopoietic cells, on the basis of the presence or absence of specific markers is discussed in detail below. [00139] Hematopoietic cells used in the methods provided herein can be a substantially homogeneous population, e.g., a population comprising at least about 95%, at least about 98% or at least about 99% hematopoietic cells from a single tissue source, or a population comprising hematopoietic cells exhibiting the same hematopoietic cell-associated cellular markers. For example, in various embodiments, the hematopoietic cells can comprise at least about 95%, 98% or 99% hematopoietic cells from bone marrow, cord blood, placental blood, peripheral blood, or placenta, e.g., placenta perfusate. [00140] Hematopoietic cells used in the methods provided herein can be obtained from a single individual, e.g., from a single placenta, or from a plurality of individuals, e.g., can be pooled. Where the hematopoietic cells are obtained from a plurality of individuals and pooled, the hematopoietic cells may be obtained from the same tissue source. Thus, in various embodiments, the pooled hematopoietic cells are all from placenta, e.g., placental perfusate, all from placental blood, all from umbilical cord blood, all from peripheral blood, and the like. [00141] Hematopoietic cells used in the methods disclosed herein can, in certain embodiments, comprise hematopoietic cells from two or more tissue sources. For example, in certain embodiments, when hematopoietic cells from two or more sources are combined for use in the methods herein, a plurality of the hematopoietic cells used to produce natural killer cells using a three-stage method described herein comprise hematopoietic cells from placenta, e.g., placenta perfusate. In various embodiments, the hematopoietic cells used to produce NK cell populations and/or ILC3 cell populations produced using a three-stage method described herein, comprise hematopoietic cells from placenta and from cord blood; from placenta and peripheral blood; from placenta and placental blood, or placenta and bone marrow. In one embodiment, the hematopoietic cells comprise hematopoietic cells from placental perfusate in combination with hematopoietic cells from cord blood, wherein the cord blood and placenta are from the same individual, i.e., wherein the perfusate and cord blood are matched. In embodiments in which the hematopoietic cells comprise hematopoietic cells from two tissue sources, the hematopoietic cells from 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. 5.1.1. Placental Hematopoietic Stem Cells [00142] In certain embodiments, the hematopoietic cells used in the methods provided herein are placental hematopoietic cells. In one embodiment, placental hematopoietic cells are CD34+. In a specific embodiment, the placental hematopoietic cells are predominantly (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) CD34+CD38– cells. In another specific embodiment, the placental hematopoietic cells are predominantly (e.g., 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 post-partum mammalian (e.g., human) placenta by any means known to those of skill in the art, e.g., by perfusion. [00143] In another embodiment, the placental hematopoietic cell is CD45–. In a specific embodiment, the hematopoietic cell is CD34+CD45–. In another specific embodiment, the placental hematopoietic cells are CD34+CD45+. 5.2. Production of Natural Killer and/or ILC3 Cells and Natural Killer Cell and/or ILC3 Cell Populations [00144] Production of NK cells and/or ILC3 cells and NK cell and/or ILC3 cell populations by the present methods comprises expanding a population of hematopoietic cells. During cell expansion, a plurality of hematopoietic cells within the hematopoietic cell population differentiate into NK cells and/or ILC3 cells. In one aspect, provided herein is a method of producing NK cells comprising culturing hematopoietic stem cells or progenitor cells, e.g., CD34+ stem cells or progenitor cells, in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells, subsequently culturing said first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells, and subsequently culturing said second population of cells in a third medium comprising IL-2 and IL-15, and lacking a stem cell mobilizing agent and LMWH, to produce a third population of cells, wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and wherein at least 70%, for example at least 80%, of the natural killer cells are viable. In certain embodiments, such natural killer cells comprise natural killer cells that are CD16-. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94+. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94+ or CD16+. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94- or CD16-. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94+ and CD16+. In certain embodiments, such natural killer cells comprise natural killer cells that are CD94- and CD16-. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00145] In one aspect, provided herein is a method of producing NK cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and CD11a+. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP- 1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00146] In one aspect, provided herein is a method of producing NK cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of stem cell factor (SCF) and LMWH, to produce a third population of cells; wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and CD11a+. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00147] In one aspect, provided herein is a method of producing NK cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of SCF, a stem cell mobilizing agent, and LMWH, to produce a third population of cells; wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and CD11a+. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00148] In one aspect, provided herein is a method of producing NK cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of a stem cell mobilizing agent and LMWH, to produce a third population of cells; and (d) isolating CD11a+ cells from the third population of cells to produce a fourth population of cells; wherein the fourth population of cells comprises natural killer cells that are CD56+, CD3-, and CD11a+. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00149] In certain embodiments, of any of the above embodiments, said natural killer cells express perforin and EOMES. In certain embodiments, said natural killer cells do not express either RORγt or IL1R1. [00150] In one aspect, provided herein is a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00151] In one aspect, provided herein is a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising a stem cell mobilizing agent, IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00152] In one aspect, provided herein is a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising SCF, IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00153] In one aspect, provided herein is a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) culturing the second population of cells in a third medium comprising a stem cell mobilizing agent, SCF, IL-2 and IL-15, and lacking LMWH, to produce a third population of cells; wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00154] In one aspect, provided herein is a method of producing ILC3 cells comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of a stem cell mobilizing agent and LMWH, to produce a third population of cells; and (d) isolating CD11a- cells, or removing CD11a+ cells, from the third population of cells to produce a fourth population of cells; wherein the fourth population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP-1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. [00155] In certain embodiments, said ILC3 cells express RORγt and IL1R1. In certain embodiments, said ILC3 cells do not express either perforin or EOMES. 5.2.1. Production of NK Cell and/or ILC3 Cell Populations Using a Three- Stage Method [00156] In one embodiment, provided herein is a three-stage method of producing NK cell and/or ILC3 cell populations. In certain embodiments, the method of expansion and differentiation of the hematopoietic cells, as described herein, to produce NK cell and/or ILC3 cell populations according to a three-stage method described herein comprises maintaining the cell population comprising said hematopoietic cells at between about 2 x 104 and about 6 x 106 cells per milliliter. In certain aspects, said hematopoietic stem or progenitor cells are initially inoculated into said first medium from 1 x 104 to 1 x 105 cells/mL. In a specific aspect, said hematopoietic stem or progenitor cells are initially inoculated into said first medium at about 3 x 104 cells/mL. [00157] In certain aspects, said first population of cells are initially inoculated into said second medium from 5 x 104 to 5 x 105 cells/mL. In a specific aspect, said first population of cells is initially inoculated into said second medium at about 1 x 105 cells/mL. [00158] In certain aspects said second population of cells is initially inoculated into said third medium from 1 x 105 to 5 x 106 cells/mL. In certain aspects, said second population of cells is initially inoculated into said third medium from 1 x 105 to 1 x 106 cells/mL. In a specific aspect, said second population of cells is initially inoculated into said third medium at about 5 x 105 cells/mL. In a more specific aspect, said second population of cells is initially inoculated into said third medium at about 5 x 105 cells/mL in a spinner flask. In a specific aspect, said second population of cells is initially inoculated into said third medium at about 3 x 105 cells/mL. In a more specific aspect, said second population of cells is initially inoculated into said third medium at about 3 x 105 cells/mL in a static culture. [00159] In a certain embodiment, the three-stage method comprises a first stage (“stage 1”) comprising culturing hematopoietic stem cells or progenitor cells, e.g., CD34+ stem cells or progenitor cells, in a first medium for a specified time period, e.g., as described herein, to produce a first population of cells. In certain embodiments, the first medium comprises a stem cell mobilizing agent and thrombopoietin (Tpo). In certain embodiments, the first medium comprises in addition to a stem cell mobilizing agent and Tpo, one or more of LMWH, Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF. In a specific embodiment, the first medium comprises in addition to a stem cell mobilizing agent and Tpo, each of LMWH, Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM- CSF. In a specific embodiment, the first medium lacks added LMWH. In a specific embodiment, the first medium lacks added desulphated glycosaminoglycans. In a specific embodiment, the first medium lacks LMWH. In a specific embodiment, the first medium lacks desulphated glycosaminoglycans. In a specific embodiment, in addition to a stem cell mobilizing agent and Tpo, each of Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF. In specific embodiments, the first medium lacks leukemia inhibiting factor (LIF), macrophage inhibitory protein-1alpha (MIP-1α) or both. [00160] In certain embodiments, subsequently, in “stage 2” said cells are cultured in a second medium for a specified time period, e.g., as described herein, to produce a second population of cells. In certain embodiments, the second medium comprises a stem cell mobilizing agent and interleukin-15 (IL-15) and lacks Tpo. In certain embodiments, the second medium comprises, in addition to a stem cell mobilizing agent and IL-15, one or more of LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF. In certain embodiments, the second medium comprises, in addition to a stem cell mobilizing agent and IL-15, each of LMWH, Flt-3, SCF, IL-6, IL-7, G- CSF, and GM-CSF. In a specific embodiment, the second medium lacks added LMWH. In a specific embodiment, the second medium lacks added desulphated glycosaminoglycans. In a specific embodiment, the second medium lacks heparin, e.g., LMWH. In a specific embodiment, the second medium lacks desulphated glycosaminoglycans. In certain embodiments, the second medium comprises, in addition to a stem cell mobilizing agent and IL-15, each of Flt-3, SCF, IL- 6, IL-7, G-CSF, and GM-CSF. In specific embodiments, the second medium lacks leukemia inhibiting factor (LIF), macrophage inhibitory protein-1alpha (MIP-1α) or both. [00161] In certain embodiments, subsequently, in “stage 3” said cells are cultured in a third medium for a specified time period, e.g., as described herein, to produce a third population of cell, e.g., natural killer cells. In certain embodiments, the third medium comprises IL-2 and IL-15, and lacks a stem cell mobilizing agent and LMWH. In certain embodiments, the third medium comprises in addition to IL-2 and IL-15, one or more of SCF, IL-6, IL-7, G-CSF, and GM-CSF. In certain embodiments, the third medium comprises, in addition to IL-2 and IL-15, each of SCF, IL-6, IL-7, G-CSF, and GM-CSF. In specific embodiments, the first medium lacks one, two, or all three of LIF, MIP-1α, and Flt3L. In specific embodiments, the third medium lacks added desulphated glycosaminoglycans. In specific embodiments, the third medium lacks desulphated glycosaminoglycans. In specific embodiments, the third medium lacks heparin, e.g., LMWH. [00162] In a specific embodiment, the three-stage method is used to produce NK cell and/or ILC3 cell populations. In certain embodiments, the three-stage method is conducted in the absence of stromal feeder cell support. In certain embodiments, the three-stage method is conducted in the absence of exogenously added steroids (e.g., cortisone, hydrocortisone, or derivatives thereof). [00163] In certain aspects, said first medium used in the three-stage method comprises a stem cell mobilizing agent and thrombopoietin (Tpo). In certain aspects, the first medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and Tpo, one or more of Low Molecular Weight Heparin (LMWH), Flt-3 Ligand (Flt-3L), stem cell factor (SCF), IL-6, IL-7, granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage- stimulating factor (GM-CSF). In certain aspects, the first medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and Tpo, each of LMWH, Flt-3L, SCF, IL- 6, IL-7, G-CSF, and GM-CSF. In certain aspects, the first medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and Tpo, each of Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF. In a specific aspect, the first medium lacks added LMWH. In a specific aspect, the first medium lacks added desulphated glycosaminoglycans. In a specific aspect, the first medium lacks LMWH. In a specific aspect, the first medium lacks desulphated glycosaminoglycans. In certain aspects, said Tpo is present in the first medium at a concentration of from 1 ng/mL to 100 ng/mL, from 1 ng/mL to 50 ng/mL, from 20 ng/mL to 30 ng/mL, or about 25 ng/mL. In other aspects, said Tpo is present in the first medium at a concentration of from 100 ng/mL to 500 ng/mL, from 200 ng/mL to 300 ng/mL, or about 250 ng/mL. In certain aspects, when LMWH is present in the first medium, the LMWH is present at a concentration of from 1U/mL to 10U/mL; the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL; the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL. In certain aspects, in the first medium, the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL; the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL. In certain aspects, when LMWH is present in the first medium, the LMWH is present at a concentration of from 4U/mL to 5U/mL; the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, in the first medium, the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, when LMWH is present in the first medium, the LMWH is present at a concentration of about 4.5U/mL; the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 25 ng/mL; the G-CSF is present at a concentration of about .25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL. In certain aspects, in the first medium, the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 25 ng/mL; the G-CSF is present at a concentration of about .25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL. In certain embodiments, said first medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite; ascorbic acid; ethanolamine; and glutathione. In certain embodiments, the medium that provides the base for the first medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially available cell/tissue culture medium such as SCGMTM, STEMMACSTM, GBGM®, AIM-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, Myelocult™ H5100, IMDM, and/or RPMI-1640; or is a medium that comprises components generally included in known cell/tissue culture media, such as the components included in GBGM®, AIM-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, Myelocult™ H5100, IMDM, and/or RPMI-1640. In certain embodiments, said first medium is not GBGM®. In specific embodiments of any of the above embodiments, the first medium lacks LIF, MIP-1α, or both. [00164] In certain aspects, said second medium used in the three-stage method comprises a stem cell mobilizing agent and interleukin-15 (IL-15), and lacks Tpo. In certain aspects, the second medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and IL-15, one or more of LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF. In certain aspects, the second medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and IL-15, each of LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF. In certain aspects, the second medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and IL-15, each of Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF. In a specific aspect, the second medium lacks added LMWH. In a specific aspect, the second medium lacks added desulphated glycosaminoglycans. In a specific aspect, the second medium lacks LMWH. In a specific aspect, the second medium lacks desulphated glycosaminoglycans. In certain aspects, said IL-15 is present in said second medium at a concentration of from 1 ng/mL to 50 ng/mL, from 10 ng/mL to 30 ng/mL, or about 20 ng/mL. In certain aspects, when LMWH is present in said second medium, the LMWH is present at a concentration of from 1U/mL to 10U/mL; the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL; the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL. In certain aspects, in said second medium, the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL; the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL. In certain aspects, when LMWH is present in the second medium, the LMWH is present in the second medium at a concentration of from 4U/mL to 5U/mL; the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, in the second medium, the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, when LMWH is present in the second medium, the LMWH is present in the second medium at a concentration of from 4U/mL to 5U/mL; the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, in the second medium, the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, when LMWH is present in the second medium, the LMWH is present in the second medium at a concentration of about 4.5U/mL; the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 25 ng/mL; the G-CSF is present at a concentration of about 0.25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL. In certain aspects, in the second medium, the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 25 ng/mL; the G-CSF is present at a concentration of about 0.25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL. In certain embodiments, said second medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite; ascorbic acid; ethanolamine; and glutathione. In certain embodiments, the medium that provides the base for the second medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially available cell/tissue culture medium such as SCGMTM, STEMMACSTM, GBGM®, AIM-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, Myelocult™ H5100, IMDM, and/or RPMI-1640; or is a medium that comprises components generally included in known cell/tissue culture media, such as the components included in GBGM®, AIM-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, Myelocult™ H5100, IMDM, and/or RPMI-1640. In certain embodiments, said second medium is not GBGM®. In specific embodiments of any of the above embodiments, the first medium lacks LIF, MIP-1α, or both. [00165] In certain aspects, said third medium used in the three-stage method comprises IL- 2 and IL-15, and lacks a stem cell mobilizing agent and LMWH. In certain aspects, said third medium used in the three-stage method comprises IL-2 and IL-15, and lacks LMWH. In certain aspects, said third medium used in the three-stage method comprises IL-2 and IL-15, and lacks SCF and LMWH. In certain aspects, said third medium used in the three-stage method comprises IL-2 and IL-15, and lacks SCF, a stem cell mobilizing agent and LMWH. In certain aspects, said third medium used in the three-stage method comprises a stem cell mobilizing agent, IL-2 and IL- 15, and lacks LMWH. In certain aspects, said third medium used in the three-stage method comprises SCF, IL-2 and IL-15, and lacks LMWH. In certain aspects, said third medium used in the three-stage method comprises a stem cell mobilizing agent, SCF, IL-2 and IL-15, and lacks LMWH. In certain aspects, said third medium used in the three-stage method comprises IL-2 and IL-15, and lacks a stem cell mobilizing agent and LMWH. In certain aspects, the third medium used in the three-stage method comprises, in addition to IL-2 and IL-15, one or more of SCF, IL- 6, IL-7, G-CSF, or GM-CSF. In certain aspects, the third medium used in the three-stage method comprises, in addition to IL-2 and IL-15, each of SCF, IL-6, IL-7, G-CSF, and GM-CSF. In certain aspects, said IL-2 is present in said third medium at a concentration of from 10 U/mL to 10,000 U/mL and said IL-15 is present in said third medium at a concentration of from 1 ng/mL to 50 ng/mL. In certain aspects, said IL-2 is present in said third medium at a concentration of from 100 U/mL to 10,000 U/mL and said IL-15 is present in said third medium at a concentration of from 1 ng/mL to 50 ng/mL. In certain aspects, said IL-2 is present in said third medium at a concentration of from 300 U/mL to 3,000 U/mL and said IL-15 is present in said third medium at a concentration of from 10 ng/mL to 30 ng/mL. In certain aspects, said IL-2 is present in said third medium at a concentration of about 1,000 U/mL and said IL-15 is present in said third medium at a concentration of about 20 ng/mL. In certain aspects, in said third medium, the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL-7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G-CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM- CSF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL. In certain aspects, in said third medium, the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G-CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, in said third medium, the SCF is present at a concentration of about 22 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 20 ng/mL; the G-CSF is present at a concentration of about 0.25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL. In certain aspects, the third medium comprises 100 ng/mL IL-7, 1000 ng/mL IL-2, 20 ng/mL IL-15, and 10 stem cell mobilizing agent and lacks SCF. In certain aspects, the third medium comprises 20 ng/mL IL-7, 1000 ng/mL IL-2, 20 ng/mL IL-15, and stem cell mobilizing agent and lacks SCF. In certain aspects, the third medium comprises 20 ng/mL IL-7, 20 ng/mL IL-15, and stem cell mobilizing agent and lacks SCF. In certain aspects, the third medium comprises 100 ng/mL IL-7, 22 ng/mL SCF, 1000 ng/mL IL-2, and 20 ng/mL IL-15 and lacks stem cell mobilizing agent. In certain aspects, the third medium comprises 22 ng/mL SCF, 1000 ng/mL IL-2, and 20 ng/mL IL-15 and lacks stem cell mobilizing agent. In certain aspects, the third medium comprises 20 ng/mL IL-7, 22 ng/mL SCF, 1000 ng/mL IL-2, and 20 ng/mL IL-15 and lacks stem cell mobilizing agent. In certain aspects, the third medium comprises 20 ng/mL IL-7, 22 ng/mL SCF, and 1000 ng/mL IL-2 and lacks stem cell mobilizing agent. In specific embodiments of any of the above embodiments, the first medium lacks one, two, or all three of LIF, MIP-1α, Flt-3L. [00166] In certain embodiments, said third medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite; ascorbic acid; ethanolamine; and glutathione. In certain embodiments, the medium that provides the base for the third medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially available cell/tissue culture medium such as SCGMTM, STEMMACSTM, GBGM®, AIM-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, Myelocult™ H5100, IMDM, and/or RPMI-1640; or is a medium that comprises components generally included in known cell/tissue culture media, such as the components included in GBGM®, AIM-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, Myelocult™ H5100, IMDM, and/or RPMI-1640. In certain embodiments, said third medium is not GBGM®. [00167] Generally, the particularly recited medium components do not refer to possible constituents in an undefined component of said medium. For example, said Tpo, IL-2, and IL-15 are not comprised within an undefined component of the first medium, second medium or third medium, e.g., said Tpo, IL-2, and IL-15 are not comprised within serum. Further, said LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and/or GM-CSF are not comprised within an undefined component of the first medium, second medium or third medium, e.g., said LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and/or GM-CSF are not comprised within serum. [00168] In certain aspects, said first medium, second medium or third medium comprises human serum-AB. In certain aspects, any of said first medium, second medium or third medium comprises 1% to 20% human serum-AB, 5% to 15% human serum-AB, or about 2, 5, or 10% human serum-AB. [00169] In certain embodiments, in the three-stage methods described herein, said hematopoietic stem or progenitor cells are cultured in said first medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In certain embodiments, in the three-stage methods described herein, cells are cultured in said second medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In certain embodiments, in the three-stage methods described herein, cells are cultured in said third medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or for more than 30 days. [00170] In a specific embodiment, in the three-stage methods described herein, said hematopoietic stem or progenitor cells are cultured in said first medium for 7-13 days to produce a first population of cells, before said culturing in said second medium; said first population of cells are cultured in said second medium for 2-6 days to produce a second population of cells before said culturing in said third medium; and said second population of cells are cultured in said third medium for 10-30 days, i.e., the cells are cultured a total of 19-49 days. [00171] In a specific embodiment, in the three-stage methods described herein, in the three- stage methods described herein, said hematopoietic stem or progenitor cells are cultured in said first medium for 8-12 days to produce a first population of cells, before said culturing in said second medium; said first population of cells are cultured in said second medium for 3-5 days to produce a second population of cells before said culturing in said third medium; and said second population of cells are cultured in said third medium for 15-25 days, i.e., the cells are cultured a total of 26-42 days. [00172] In a specific embodiment, in the three-stage methods described herein, said hematopoietic stem or progenitor cells are cultured in said first medium for about 10 days to produce a first population of cells, before said culturing in said second medium; said first population of cells are cultured in said second medium for about 4 days to produce a second population of cells before said culturing in said third medium; and said second population of cells are cultured in said third medium for about 21 days, i.e., the cells are cultured a total of about 35 days. [00173] In certain aspects, the three-stage method disclosed herein produces at least 5000- fold more natural killer cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 10,000-fold more natural killer cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 50,000-fold more natural killer cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 75,000-fold more natural killer cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, the viability of said natural killer cells is determined by 7-aminoactinomycin D (7AAD) staining. In certain aspects, the viability of said natural killer cells is determined by annexin-V staining. In specific aspects, the viability of said natural killer cells is determined by both 7-AAD staining and annexin-V staining. In certain aspects, the viability of said natural killer cells is determined by trypan blue staining. [00174] In certain aspects, the three-stage method disclosed herein produces at least 5000- fold more ILC3 cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 10,000-fold more ILC3 cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 50,000-fold more ILC3 cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. In certain aspects, said three-stage method produces at least 75,000-fold more ILC3 cells as compared to the number of hematopoietic stem cells initially inoculated into said first medium. [00175] In certain aspects, the three-stage method produces natural killer cells that comprise at least 20% CD56+CD3– natural killer cells. In certain aspects, the three-stage method produces natural killer cells that comprise at least 40% CD56+CD3– natural killer cells. In certain aspects, the three-stage method produces natural killer cells that comprise at least 60% CD56+CD3– natural killer cells. In certain aspects, the three-stage method produces natural killer cells that comprise at least 70% CD56+CD3– natural killer cells. In certain aspects, the three- stage method produces natural killer cells that comprise at least 80% CD56+CD3– natural killer cells. [00176] In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 20% CD56+CD3–CD11a+ natural killer cells. In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 40% CD56+CD3– CD11a+ natural killer cells. In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 60% CD56+CD3– CD11a+ natural killer cells. In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 80% CD56+CD3– CD11a+ natural killer cells. [00177] In certain aspects, the three-stage method disclosed herein produces ILC3 cells that comprise at least 20% CD56+CD3– CD11a– ILC3 cells. In certain aspects, the three-stage method disclosed herein produces ILC3 cells that comprise at least 40% CD56+CD3– CD11a– ILC3 cells. In certain aspects, the three-stage method disclosed herein produces ILC3 cells that comprise at least 60% CD56+CD3– CD11a– ILC3 cells. In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 80% CD56+CD3– CD11a– ILC3 cells. [00178] In certain aspects, the three-stage method produces natural killer cells that exhibit at least 20% cytotoxicity against K562 cells when said natural killer cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces natural killer cells that exhibit at least 35% cytotoxicity against the K562 cells when said natural killer cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces natural killer cells that exhibit at least 45% cytotoxicity against the K562 cells when said natural killer cells and said K562 cells are co- cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces natural killer cells that exhibit at least 60% cytotoxicity against the K562 cells when said natural killer cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces natural killer cells that exhibit at least 75% cytotoxicity against the K562 cells when said natural killer cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. [00179] In certain aspects, the three-stage method produces ILC3 cells that exhibit at least 20% cytotoxicity against K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces ILC3 cells that exhibit at least 35% cytotoxicity against the K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces ILC3 cells that exhibit at least 45% cytotoxicity against the K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces ILC3 cells that exhibit at least 60% cytotoxicity against the K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. In certain aspects, the three-stage method produces ILC3 cells that exhibit at least 75% cytotoxicity against the K562 cells when said ILC3 cells and said K562 cells are co-cultured in vitro or ex vivo at a ratio of 10:1. [00180] In certain aspects, after said third culturing step, said third population of cells, e.g., said population of natural killer cells and/or ILC3 cells, is cryopreserved. In certain aspects, after said fourth step, said fourth population of cells, e.g., said population of natural killer cells and/or ILC3 cells, is cryopreserved. [00181] In certain aspects, provided herein are populations of cells comprising natural killer cells, i.e., natural killers cells produced by a three-stage method described herein. Accordingly, provided herein is an isolated natural killer cell population produced by a three- stage method described herein. In a specific embodiment, said natural killer cell population comprises at least 20% CD56+CD3– natural killer cells. In a specific embodiment, said natural killer cell population comprises at least 40% CD56+CD3– natural killer cells. In a specific embodiment, said natural killer cell population comprises at least 60% CD56+CD3– natural killer cells. In a specific embodiment, said natural killer cell population comprises at least 80% CD56+CD3– natural killer cells. In a specific embodiment, said natural killer cell population comprises at least 60% CD16- cells. In a specific embodiment, said natural killer cell population comprises at least 80% CD16- cells. In a specific embodiment, said natural killer cell population comprises at least 20% CD94+ cells. In a specific embodiment, said natural killer cell population comprises at least 40% CD94+ cells. [00182] In certain aspects, provided herein is a population of natural killer cells that is CD56+CD3– CD117+CD11a+, wherein said natural killer cells express perforin and/or EOMES, and do not express one or more of RORγt, aryl hydrocarbon receptor (AHR), and IL1R1. In certain aspects, said natural killer cells express perforin and EOMES, and do not express any of RORγt, aryl hydrocarbon receptor, or IL1R1. In certain aspects, said natural killer cells additionally express T-bet, GZMB, NKp46, NKp30, and NKG2D. In certain aspects, said natural killer cells express CD94. In certain aspects, said natural killer cells do not express CD94. [00183] In certain aspects, provided herein is a population of ILC3 cells that is CD56+CD3– CD117+CD11a-, wherein said ILC3 cells express one or more of RORγt, aryl hydrocarbon receptor, and IL1R1, and do not express one or more of CD94, perforin, and EOMES. In certain aspects, said ILC3 cells express RORγt, aryl hydrocarbon receptor, and IL1R1, and do not express any of CD94, perforin, or EOMES. In certain aspects, said ILC3 cells additionally express CD226 and/or 2B4. In certain aspects, said ILC3 cells additionally express one or more of IL-22, TNFα, and DNAM-1. In certain aspects, said ILC3 cells express CD226, 2B4, IL-22, TNFα, and DNAM-1. [00184] In certain aspects, provided herein is a method of producing a cell population comprising natural killer cells and ILC3 cells, comprising (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; (c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking each of a stem cell mobilizing agent and LMWH, to produce a third population of cells; and (d) separating CD11a+ cells and CD11a– cells from the third population of cells; and (e) combining the CD11a+ cells with the CD11a– cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50 to produce a fourth population of cells. In certain embodiments, said first medium and/or said second medium lack leukemia inhibiting factor (LIF) and/or macrophage inflammatory protein-1 alpha (MIP-1α). In certain embodiments, said third medium lacks LIF, MIP-1α, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1α, and said third medium lacks LIF, MIP- 1α, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 50:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 20:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 10:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 5:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:1. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:5. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:10. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:20. In certain aspects, in the fourth population of cells, the CD11a+ cells and CD11a– cells are combined in a ratio of 1:50. 5.3. Stem Cell Mobilizing Factors 5.3.1. Chemistry definitions [00185] To facilitate understanding of the disclosure of stem cell mobilizing factors set forth herein, a number of terms are defined below. [00186] Generally, the nomenclature used herein and the laboratory procedures in biology, cellular biology, biochemistry, organic chemistry, medicinal chemistry, and pharmacology described herein are those well known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. [00187] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. [00188] As used herein, any "R" group(s) such as, without limitation, Ra, Rb, Rc, Rd, Re, Rf , Rg, Rh, Rm, RG, RJ, RK, RU, RV, RY, and RZ represent substituents that can be attached to the indicated atom. An R group may be substituted or unsubstituted. If two "R" groups are described as being "taken together" the R groups and the atoms they are attached to can form a cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocycle. For example, without limitation, if Ra and Rb of an NRa Rb group are indicated to be "taken together," it means that they are covalently bonded to one another to form a ring: [
Figure imgf000046_0001
[00190] In addition, if two “R” groups are described as being “taken together” with the atom(s) to which they are attached to form a ring as an alternative, the R groups are not limited to the variables or substituents defined previously. [00191] Whenever a group is described as being “optionally substituted” that group may be unsubstituted or substituted with one or more of the indicated substituents. Likewise, when a group is described as being “unsubstituted or substituted” if substituted, the substituent(s) may be selected from one or more the indicated substituents. If no substituents are indicated, it is meant that the indicated “optionally substituted” or “substituted” group may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, acylalkyl, hydroxy, alkoxy, alkoxyalkyl, aminoalkyl, amino acid, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxyalkyl, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, azido, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, an amino, a mono-substituted amino group and a di-substituted amino group. [00192] As used herein, “Ca to Cb” in which “a” and “b” are integers refer to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl, aryl, heteroaryl or heteroalicyclyl group. That is, the alkyl, alkenyl, alkynyl, ring(s) of the cycloalkyl, ring(s) of the cycloalkenyl, ring(s) of the aryl, ring(s) of the heteroaryl or ring(s) of the heteroalicyclyl can contain from “a” to “b”, inclusive, carbon atoms. Thus, for example, a “C1 to C4 alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that is, CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-. If no “a” and “b” are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl cycloalkenyl, aryl, heteroaryl or heteroalicyclyl group, the broadest range described in these definitions is to be assumed. [00193] As used herein, “alkyl” refers to a straight or branched hydrocarbon chain that comprises a fully saturated (no double or triple bonds) hydrocarbon group. The alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated). The alkyl group may also be a medium size alkyl having 1 to 10 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group of the compounds may be designated as “C1-C4 alkyl” or similar designations. By way of example only, “C1-C4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexyl. The alkyl group may be substituted or unsubstituted. [00194] As used herein, “alkenyl” refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more double bonds. Examples of alkenyl groups include allenyl, vinylmethyl and ethenyl. An alkenyl group may be unsubstituted or substituted. [00195] As used herein, “alkynyl” refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more triple bonds. Examples of alkynyls include ethynyl and propynyl. An alkynyl group may be unsubstituted or substituted. [00196] As used herein, “cycloalkyl” refers to a completely saturated (no double or triple bonds) mono- or multi- cyclic hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused fashion. Cycloalkyl groups can contain 3 to 10 atoms in the ring(s) or 3 to 8 atoms in the ring(s). A cycloalkyl group may be unsubstituted or substituted. Typical cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. [00197] As used herein, “cycloalkenyl” refers to a mono- or multi- cyclic hydrocarbon ring system that contains one or more double bonds in at least one ring; although, if there is more than one, the double bonds cannot form a fully delocalized pi-electron system throughout all the rings (otherwise the group would be “aryl,” as defined herein). Cycloalkenyl groups can contain 3 to 10 atoms in the ring(s) or 3 to 8 atoms in the ring(s). When composed of two or more rings, the rings may be connected together in a fused fashion. A cycloalkenyl group may be unsubstituted or substituted. [00198] As used herein, “aryl” refers to a carbocyclic (all carbon) monocyclic or multicyclic aromatic ring system (including fused ring systems where two carbocyclic rings share a chemical bond) that has a fully delocalized pi-electron system throughout all the rings. The number of carbon atoms in an aryl group can vary. For example, the aryl group can be a C6-C14 aryl group, a C6-C10 aryl group, or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. An aryl group may be substituted or unsubstituted. [00199] As used herein, “heteroaryl” refers to a monocyclic or multicyclic aromatic ring system (a ring system with fully delocalized pi-electron system) that contain(s) one, two, three or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. The number of atoms in the ring(s) of a heteroaryl group can vary. For example, the heteroaryl group can contain 4 to 14 atoms in the ring(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s). Furthermore, the term “heteroaryl” includes fused ring systems where two rings, such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, those described herein and the following: furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline and triazine. A heteroaryl group may be substituted or unsubstituted. [00200] As used herein, “heterocyclyl” or “heteroalicyclyl” refers to three-, four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-membered monocyclic, bicyclic, and tricyclic ring system wherein carbon atoms together with from 1 to 5 heteroatoms constitute said ring system. A heterocycle may optionally contain one or more unsaturated bonds situated in such a way, however, that a fully delocalized pi-electron system does not occur throughout all the rings. The heteroatom(s) is an element other than carbon including, but not limited to, oxygen, sulfur, and nitrogen. A heterocycle may further contain one or more carbonyl or thiocarbonyl functionalities, so as to make the definition include oxo-systems and thio-systems such as lactams, lactones, cyclic imides, cyclic thioimides and cyclic carbamates. When composed of two or more rings, the rings may be joined together in a fused fashion. Additionally, any nitrogens in a heterocyclyl may be quaternized. Heterocyclyl or heteroalicyclic groups may be unsubstituted or substituted. Examples of such “heterocyclyl” or “heteroalicyclyl” groups include, but are not limited to, those described herein and the following: 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3- dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole, 1,3- dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 1,3-thiazinane, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N- Oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, and their benzo-fused analogs (e.g., benzimidazolidinone, tetrahydroquinoline, and 3,4-methylenedioxyphenyl). [00201] As used herein, “aralkyl” and “aryl(alkyl)” refer to an aryl group connected, as a substituent, via a lower alkylene group. The lower alkylene and aryl group of an aralkyl may be substituted or unsubstituted. Examples include but are not limited to benzyl, 2-phenylalkyl, 3- phenylalkyl and naphthylalkyl. [00202] As used herein, “heteroaralkyl” and “heteroaryl(alkyl)” refer to a heteroaryl group connected, as a substituent, via a lower alkylene group. The lower alkylene and heteroaryl group of heteroaralkyl may be substituted or unsubstituted. Examples include but are not limited to 2- thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, imidazolylalkyl and their benzo-fused analogs. [00203] A “heteroalicyclyl(alkyl)” and “heterocyclyl(alkyl)” refer to a heterocyclic or a heteroalicyclylic group connected, as a substituent, via a lower alkylene group. The lower alkylene and heterocyclyl of a heteroalicyclyl(alkyl) may be substituted or unsubstituted. Examples include but are not limited tetrahydro-2H-pyran-4-yl(methyl), piperidin-4-yl(ethyl), piperidin-4-yl(propyl), tetrahydro-2H-thiopyran-4-yl(methyl), and 1,3-thiazinan-4-yl(methyl). [00204] “Lower alkylene groups” are straight-chained -CH2- tethering groups, forming bonds to connect molecular fragments via their terminal carbon atoms. Examples include but are not limited to methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and butylene (-CH2CH2CH2CH2-). A lower alkylene group can be substituted by replacing one or more hydrogen of the lower alkylene group with a substituent(s) listed under the definition of “substituted.” [00205] As used herein, “alkoxy” refers to the formula –OR wherein R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) is defined herein. A non-limiting list of alkoxys are methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, phenoxy and benzoxy. An alkoxy may be substituted or unsubstituted. [00206] As used herein, “acyl” refers to a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) connected, as substituents, via a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl and acryl. An acyl may be substituted or unsubstituted. [00207] As used herein, “acylalkyl” refers to an acyl connected, as a substituent, via a lower alkylene group. Examples include aryl-C(=O)-(CH2)n- and heteroaryl-C(=O)-(CH2)n-, where n is an integer in the range of 1 to 6. [00208] As used herein, “alkoxyalkyl” refers to an alkoxy group connected, as a substituent, via a lower alkylene group. Examples include C1-4 alkyl-O-(CH2)n- ,wherein n is an integer in the range of 1 to 6. [00209] As used herein, “aminoalkyl” refers to an optionally substituted amino group connected, as a substituent, via a lower alkylene group. Examples include H2N(CH2)n- ,wherein n is an integer in the range of 1 to 6. [00210] As used herein, “hydroxyalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a hydroxy group. Exemplary hydroxyalkyl groups include but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. A hydroxyalkyl may be substituted or unsubstituted. [00211] As used herein, “haloalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups include but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloro-fluoroalkyl, chloro-difluoroalkyl and 2-fluoroisobutyl. A haloalkyl may be substituted or unsubstituted. [00212] As used herein, “haloalkoxy” refers to an alkoxy group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkoxy, di- haloalkoxy and tri- haloalkoxy). Such groups include but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloro-fluoroalkyl, chloro-difluoroalkoxy and 2- fluoroisobutoxy. A haloalkoxy may be substituted or unsubstituted. [00213] A “sulfenyl” group refers to an “-SR” group in which R can be hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). A sulfenyl may be substituted or unsubstituted. [00214] A “sulfinyl” group refers to an “-S(=O)-R” group in which R can be the same as defined with respect to sulfenyl. A sulfinyl may be substituted or unsubstituted. [00215] A “sulfonyl” group refers to an “SO2R” group in which R can be the same as defined with respect to sulfenyl. A sulfonyl may be substituted or unsubstituted. [00216] An “O-carboxy” group refers to a “RC(=O)O-” group in which R can be hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein. An O- carboxy may be substituted or unsubstituted. [00217] The terms “ester” and “C-carboxy” refer to a “-C(=O)OR” group in which R can be the same as defined with respect to O-carboxy. An ester and C-carboxy may be substituted or unsubstituted. [00218] A “thiocarbonyl” group refers to a “-C(=S)R” group in which R can be the same as defined with respect to O-carboxy. A thiocarbonyl may be substituted or unsubstituted. [00219] A “trihalomethanesulfonyl” group refers to an “X3CSO2-” group wherein each X is a halogen. [00220] A “trihalomethanesulfonamido” group refers to an “X3CS(O)2N(RA)-” group wherein each X is a halogen, and RA hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). [00221] The term “amino” as used herein refers to a –NH2 group. [00222] As used herein, the term “hydroxy” refers to a –OH group. [00223] A “cyano” group refers to a “-CN” group. [00224] The term “azido” as used herein refers to a –N3 group. [00225] An “isocyanato” group refers to a “-NCO” group. [00226] A “thiocyanato” group refers to a “-CNS” group. [00227] An “isothiocyanato” group refers to an “ -NCS” group. [00228] A “carbonyl” group refers to a C=O group. [00229] An “S-sulfonamido” group refers to a “-SO2N(RARB)” group in which RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An S-sulfonamido may be substituted or unsubstituted. [00230] An “N-sulfonamido” group refers to a “RSO2N(RA)-” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An N-sulfonamido may be substituted or unsubstituted. [00231] An “O-carbamyl” group refers to a “-OC(=O)N(RARB)” group in which RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An O-carbamyl may be substituted or unsubstituted. [00232] An “N-carbamyl” group refers to an “ROC(=O)N(RA)-” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An N-carbamyl may be substituted or unsubstituted. [00233] An “O-thiocarbamyl” group refers to a “-OC(=S)-N(RARB)” group in which RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An O-thiocarbamyl may be substituted or unsubstituted. [00234] An “N-thiocarbamyl” group refers to an “ROC(=S)N(RA)-” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An N-thiocarbamyl may be substituted or unsubstituted. [00235] A “C-amido” group refers to a “-C(=O)N(RARB)” group in which RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). A C-amido may be substituted or unsubstituted. [00236] An “N-amido” group refers to a “RC(=O)N(RA)-” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An N-amido may be substituted or unsubstituted. [00237] A “urea” group refers to “N(R)-C(=O)-NRARB group in which R can be hydrogen or an alkyl, and RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). A urea may be substituted or unsubstituted. [00238] The term “halogen atom” or “halogen” as used herein, means any one of the radio- stable atoms of column 7 of the Periodic Table of the Elements, such as, fluorine, chlorine, bromine and iodine. [00239] As used herein, “--------” indicates a single or double bond, unless stated otherwise. [00240] Where the numbers of substituents is not specified (e.g. haloalkyl), there may be one or more substituents present. For example “haloalkyl” may include one or more of the same or different halogens. As another example, “C1-C3 alkoxyphenyl” may include one or more of the same or different alkoxy groups containing one, two or three atoms. [00241] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (See, Biochem. 11:942-944 (1972)). [00242] In certain embodiments, “optically active” and ”enantiomerically active” refer to a collection of molecules, which has an enantiomeric excess of no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, no less than about 91%, no less than about 92%, no less than about 93%, no less than about 94%, no less than about 95%, no less than about 96%, no less than about 97%, no less than about 98%, no less than about 99%, no less than about 99.5%, or no less than about 99.8%. In certain embodiments, the compound comprises about 95% or more of the desired enantiomer and about 5% or less of the less preferred enantiomer based on the total weight of the two enantiomers in question. [00243] In describing an optically active compound, the prefixes R and S are used to denote the absolute configuration of the optically active compound about its chiral center(s). The (+) and (-) are used to denote the optical rotation of an optically active compound, that is, the direction in which a plane of polarized light is rotated by the optically active compound. The (-) prefix indicates that an optically active compound is levorotatory, that is, the compound rotates the plane of polarized light to the left or counterclockwise. The (+) prefix indicates that an optically active compound is dextrorotatory, that is, the compound rotates the plane of polarized light to the right or clockwise. However, the sign of optical rotation, (+) and (-), is not related to the absolute configuration of a compound, R and S. [00244] The term “isotopic variant” refers to a compound that contains an unnatural proportion of an isotope at one or more of the atoms that constitute such a compound. In certain embodiments, an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen (1H), deuterium (2H), tritium (3H), carbon-11 (11C), carbon-12 (12C), carbon-13 (13C), carbon-14 (14C), nitrogen-13 (13N), nitrogen-14 (14N), nitrogen-15 (15N), oxygen-14 (14O), oxygen-15 (15O), oxygen-16 (16O), oxygen-17 (17O), oxygen- 18 (18O), fluorine-17 (17F), fluorine-18 (18F), phosphorus-31 (31P), phosphorus-32 (32P), phosphorus-33 (33P), sulfur-32 (32S), sulfur-33 (33S), sulfur-34 (34S), sulfur-35 (35S), sulfur-36 (36S), chlorine-35 (35Cl), chlorine-36 (36Cl), chlorine-37 (37Cl), bromine-79 (79Br), bromine-81 (81Br), iodine-123 (123I), iodine-125 (125I), iodine-127 (127I), iodine-129 (129I), and iodine-131 (131I). In certain embodiments, an “isotopic variant” of a compound is in a stable form, that is, non-radioactive. In certain embodiments, an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen (1H), deuterium (2H), carbon-12 (12C), carbon-13 (13C), nitrogen-14 (14N), nitrogen-15 (15N), oxygen-16 (16O), oxygen- 17 (17O), oxygen-18 (18O), fluorine-17 (17F), phosphorus-31 (31P), sulfur-32 (32S), sulfur-33 (33S), sulfur-34 (34S), sulfur-36 (36S), chlorine-35 (35Cl), chlorine-37 (37Cl), bromine-79 (79Br), bromine- 81 (81Br), and iodine-127 (127I). In certain embodiments, an “isotopic variant” of a compound is in an unstable form, that is, radioactive. In certain embodiments, an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, tritium (3H), carbon-11 (11C), carbon-14 (14C), nitrogen-13 (13N), oxygen-14 (14O), oxygen-15 (15O), fluorine-18 (18F), phosphorus-32 (32P), phosphorus-33 (33P), sulfur-35 (35S), chlorine-36 (36Cl), iodine-123 (123I), iodine-125 (125I), iodine-129 (129I), and iodine-131 (131I). It will be understood that, in a compound as provided herein, any hydrogen can be 2H, for example, or any carbon can be 13C, for example, or any nitrogen can be 15N, for example, or any oxygen can be 18O, for example, where feasible according to the judgment of one of skill. In certain embodiments, an “isotopic variant” of a compound contains unnatural proportions of deuterium (D). [00245] The term “solvate” refers to a complex or aggregate formed by one or more molecules of a solute, e.g., a compound provided herein, and one or more molecules of a solvent, which present in a stoichiometric or non-stoichiometric amount. Suitable solvents include, but are not limited to, water, methanol, ethanol, n-propanol, isopropanol, and acetic acid. In certain embodiments, the solvent is pharmaceutically acceptable. In one embodiment, the complex or aggregate is in a crystalline form. In another embodiment, the complex or aggregate is in a noncrystalline form. Where the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, a hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate. [00246] The phrase “an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof” has the same meaning as the phrase “(i) an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, or an isotopic variant of the compound referenced therein; (ii) a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of the compound referenced therein; or (iii) a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, or an isotopic variant of the compound referenced therein.” 5.3.2. Stem cell mobilizing compounds [00247] In certain aspects, the stem cell mobilizing factor is a compound having Formula (I), (I-A), (I-B), (I-C), or (I-D), as described below. Formula (I) [00248] Some embodiments disclosed herein relate to a compound of Formula (I), or a pharmaceutically acceptable salt thereof, having the structure:
Figure imgf000056_0001
wherein: each can independently represent a single bond or a double bond; RJ can be selected from the group consisting of –NRaRb, -ORb, and =O; wherein if RJ is =O, then joining G and J represents a single bond and G is N and the N is substituted with RG; otherwise joining G and J represents a double bond and G is N; Ra can be hydrogen or C1-C4 alkyl; Rb can be Rc or -(C1-C4 alkyl)-Rc; Rc can be selected from the group consisting of: -OH, -O(C1- C4 alkyl), -O(C1-C4 haloalkyl); -C(=O)NH2; unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted can be substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; substituted C1-6 alkyl; -NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted can be substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O- (C1-4 haloalkyl); RG can be selected from the group consisting of hydrogen, C1-4 alkyl, and -(C1-4 alkyl)-C(=O)NH2; RY and RZ can each independently be absent or be selected from the group consisting of: hydrogen, halo, C1-6 alkyl, -OH, -O-(C1-4 alkyl), -NH(C1-4 alkyl), and -N(C1-4 alkyl)2; or RY and RZ taken together with the atoms to which they are attached can joined together t
Figure imgf000057_0002
, , , be optionally substituted with one, two, or three groups independently selected from C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -OH, -O-(C1-4 alkyl), -N(C1-4 alkyl)2, unsubstituted C6-C10 aryl, C6-C10 aryl substituted with 1-5 halo atoms, and -O-(C1-4 haloalkyl); and wherein if RY and RZ taken together forms
Figure imgf000057_0001
, then RJ can be -ORb or =O; Rd can be hydrogen or C1-C4 alkyl; Rm can be selected from the group consisting of C1-4 alkyl, halo, and cyano; J can be C; and X, Y, and Z can each be independently N or C, wherein the valency of any carbon atom is filled as needed with hydrogen atoms. [00249] [0077] In some embodiments, can represent a single bond. In other embodiments, can represent a double bond. In some embodiments, joining Y and Z can represent a single bond. In other embodiments, joining Y and Z can represent a double bond. In some embodiments, when joining G and J representes a single bond, G can be N and the N is substituted with RG. In other embodiments, when joining G and J represents a double bond, G can be N. In some embodiments, when joining G and J representes a double bond, then joining J and RJ can be a single bond. In some embodiments, when joining G and J representes a double bond, then joining J and RJ can not be a double bond. In some embodiments, when joining J and RJ representes a double bond, then joining G and J can be a single bond. In some embodiments, when joining J and RJ representes a double bond, then joining G and J can not be a double bond. [00250] In some embodiments, RJ can be –NRaRb. In other embodiments, RJ can be - ORb. In still other embodiments, RJ can be =O. In some embodiments, when RJ is =O, then joining G and J represents a single bond and G is N and the N is substituted with RG. In some embodiments, RG is -CH2CH2-C(=O)NH2. [00251] In some embodiments, Ra can be hydrogen. In some embodiments, Ra can be C1- C4 alkyl. For example, Ra can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl. [00252] In some embodiments, Rb can be Rc. In some embodiments, Rb can be -(C1-C4 alkyl)-Rc. For example, Rb can be -CH2-Rc, -CH2CH2-Rc, -CH2CH2CH2-Rc, or -CH2CH2CH2CH2-Rc. In some embodiments, when Rb is -CH2CH2-Rc, Rc can be -O(C1-C4 alkyl). In other embodiments, when Rb is -CH2CH2-Rc, Rc can be -O(C1-C4 haloalkyl). In still other embodiments, when Rb is -CH2CH2- Rc, Rc can be -C(=O)NH2. [00253] In some embodiments, Rc can be –OH. In some embodiments, Rc can be -O(C1-C4 alkyl). In some embodiments, Rc can be -O(C1-C4 haloalkyl). In some embodiments, Rc can be -C(=O)NH2. In some embodiments, Rc can be unsubstituted C6-10 aryl. In some embodiments, Rc can be substituted C6-10 aryl. In some embodiments, Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, Rc can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, when a Rc moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents E. In some embodiments, E can be –OH. In some embodiments, E can be C1-C4 alkyl. In some embodiments, E can be C1-C4 haloalkyl. In some embodiments, E can be -O(C1-C4 alkyl). In some embodiments, E can be -O(C1-C4 haloalkyl). [00254] In some embodiments, when Rb is -CH2CH2-Rc, Rc can be unsubstituted C6-10 aryl. In other embodiments, when Rb is -CH2CH2-Rc, Rc can be substituted C6-10 aryl. In still other embodiments, when Rb is -CH2CH2-Rc, Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In yet still other embodiments, Rb can be -(C1-C4 alkyl)-Rc and Rc can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. When a Rc moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents E. In some embodiments, E can be –OH. In other embodiments, E can be C1-C4 alkyl. In still other embodiments, E can be C1-C4 haloalkyl. In still other embodiments, E can be -O(C1-C4 alkyl). In still other embodiments, E can be -O(C1-C4 haloalkyl). [00255] In some embodiments, when Rb is -CH2CH2-Rc, Rc can be phenyl. In other embodiments, when Rb is -CH2CH2-Rc, Rc can be naphthyl. In still other embodiments, when Rb is -CH2CH2-Rc, Rc can be hydroxyphenyl. In still other embodiments, when Rb is -CH2CH2-Rc, Rc can be indolyl. [00256] In some embodiments, RK can be hydrogen. In other embodiments, RK can be unsubstituted C1-6 alkyl. For example, in some embodiments, RK can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, pentyl (branched and straight-chained), or hexyl (branched and straight-chained). In other embodiments, RK can be substituted C1-6 alkyl. In other embodiments, RK can be -NH(C1-4 alkyl). For example, in some embodiments, RK can be - NH(CH3), -NH(CH2CH3), -NH(isopropyl), or -NH(sec-butyl). In other embodiments, RK can be - N(C1-4 alkyl)2. [00257] In some embodiments, RK can be unsubstituted C6-10 aryl. In other embodiments, RK can be substituted C6-10 aryl. In other embodiments, RK can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In other embodiments, RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. When a RK moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents substituents Q. In some embodiments, Q can be -OH. In other embodiments, Q can be C1-4 alkyl. In still other embodiments, Q can be C1-4 haloalkyl. In still other embodiments, Q can be halo. In still other embodiments, Q can be cyano. In still other embodiments, Q can be -O-(C1-4 alkyl). In still other embodiments, Q can be -O-(C1-4 haloalkyl). [00258] In some embodiments, RK can be phenyl or naphthyl. In other embodiments, RK can be benzothiophenyl. In other embodiments, RK can be benzothiophenyl. In other embodiments, RK can be benzothiophenyl. In still other embodiments, RK can be pyridinyl. In yet still other embodiments, RK can be pyridinyl substituted with one or more substituents Q. For example, RK can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl. [00259] In some embodiments, RG can be hydrogen. In some embodiments, RG can be C1- 4 alkyl. In some embodiments, RG can be -(C1-4 alkyl)-C(=O)NH2. [00260] In some embodiments, RY and RZ can independently be absent. In other embodiments, RY and RZ can independently be hydrogen. In other embodiments, RY and RZ can independently be halo. In other embodiments, RY and RZ can independently be C1-6 alkyl. In other embodiments, RY and RZ can independently be –OH. In still other embodiments, RY and RZ can independently be -O-(C1-4 alkyl). In other embodiments, RY and RZ can independently be -NH(C1-4 alkyl). For example, RY and RZ can independently be -NH(CH3), -NH(CH2CH3), -NH(isopropyl), or -NH(sec-butyl). In other embodiments, RY and RZ can independently be - N(C1-4 alkyl)2. [00261] In some embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form a ring. In some embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000060_0001
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000060_0002
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000060_0003
. In still other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000060_0004
. In yet still other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000060_0005
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000061_0001
. In yet other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000061_0002
. In yet still other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000061_0003
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000061_0004
. In still other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form and
Figure imgf000061_0005
. In some embodiments, when RY and RZ taken together with the atoms to which they are attached can be joined together to form a ring, the ring can be substituted with one, two, or three groups independently selected from C1-C4 alkyl, -N(C1-C4 alkyl)2, cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms. [00262] In some embodiments, when RY and RZ taken together forms
Figure imgf000061_0006
, then RJ can be -ORb or =O. [00263] In some embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000062_0001
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000062_0002
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000062_0003
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000062_0004
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000062_0005
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000062_0006
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000062_0007
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000063_0001
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000063_0002
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can be joined together to form
Figure imgf000063_0003
. In some embodiments, when RY and RZ taken together with the atoms to which they are attached can be joined together to form a ring, the ring can be substituted with one, two, or three groups independently selected from C1-C4 alkyl, -N(C1-C4 alkyl)2, cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms. In some embodiments, RY and RZ taken together with the atoms to which they are attached can be
Figure imgf000063_0004
. In other embodiments, RY and RZ taken together with the atoms to which they are attached can b
Figure imgf000063_0005
. till other embodiments, RY and RZ taken together with the atoms to which they are attached can be
Figure imgf000063_0006
. et still other embodiments, RY and RZ taken together with the atoms to which they are attached can be
Figure imgf000064_0001
In other embodiments, RY and RZ taken together with the atoms to which they are attached can be
Figure imgf000064_0002
. [00264] In some embodiments, Rd can be hydrogen. In other embodiments, Rd can be C1- C4 alkyl. For example Rd can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl. In still other embodiments, Rd can be halo. In other embodiments, Rd can be cyano. [00265] In some embodiments, Rm can be hydrogen. In other embodiments, Rm can be C1- C4 alkyl. For example Rm can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl. In still other embodiments, Rm can be halo. For example, Rm can be fluoro, chloro, bromo, or iodo. In other embodiments, Rm can be cyano. [00266] In some embodiments, X, Y, and Z can each be independently N or C, wherein the valency of any carbon atom is filled as needed with hydrogen atoms. In some embodiments, X can be N, Y can be N, and Z can be N. In other embodiments, X can be N, Y can be N, and Z can be CH. In some embodiments, X can be N, Y can be CH, and Z can be N. In still other embodiments, X can be CH, Y can be N, and Z can be N. In yet still other embodiments, X can be CH, Y can be CH, and Z can be N. In other embodiments, X can be CH, Y can be N, and Z can be CH. In yet other embodiments, X can be N, Y can be CH, and Z can be CH. In other embodiments, X can be CH, Y can be CH, and Z can be CH. [00267] In some embodiments, Ra can be hydrogen; Rb can be -(C1-C4 alkyl)-Rc; Rc can be selected from the group consisting of: -C(=O)NH2; unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; - NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); RG can be -(C1-4 alkyl)-C(=O)NH2; RY and RZ can each be independently absent or be selected from the group consisting of: hydrogen, C1-6 alkyl, and -NH(C1-4 alkyl); or RY and RZ taken together with the atoms to which they are attached can be joined together to form a ring selected from:
Figure imgf000065_0001
Figure imgf000065_0002
wherein said ring can be optionally substituted with one, two, or three groups independently selected from C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -OH, -O-(C1-4 alkyl), -N(C1-4 alkyl)2, unsubstituted C6-C10 aryl, C6-C10 aryl substituted with 1-5 halo atoms, and -O-(C1-4 haloalkyl); Rd can be C1-C4 alkyl; Rm can be cyano; and X, Y, and Z can each be independently N or C, wherein the valency of any carbon atom is filled as needed with hydrogen atoms. [00268] In some embodiments, Ra can be hydrogen; Rb can be -CH2CH2-Rc; Rc can be selected from the group consisting of: unsubstituted phenyl, substituted phenyl, indolyl, and - C(=O)NH2; RK can be selected from the group consisting of: hydrogen, methyl, substituted pyridinyl, unsubstituted benzothiophenyl, and -NH(C1-C4 alkyl); RG can be -CH2CH2-C(=O)NH2; RY can be -NH(C1-C4 alkyl); RZ can be absent or hydrogen; or RY and RZ taken together with the atoms to which they are attached can be joined together to form a ring selected from:
Figure imgf000065_0003
,
Figure imgf000066_0003
Figure imgf000066_0004
, ; wherein said ring can be optionally substituted with one, two, or three groups independently selected from C1-C4 alkyl, -N(C1-C4 alkyl)2, cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms; Rd can be C1-C4 alkyl; Rm can be cyano; and X can be N or CH. [00269] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; or Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; or RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; substituted with one or more Q, wherein Q can be selected from cyano, halo, or C1-C4 alkyl; RY and RZ taken together can b
Figure imgf000066_0001
Figure imgf000066_0002
[00270] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; or Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be hydrogen, C1-4 alkyl, or unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and RY and RZ taken together can b
Figure imgf000067_0001
Figure imgf000067_0002
[00271] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; or Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be hydrogen, C1-4 alkyl, or unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and RY and RZ taken together can b
Figure imgf000067_0003
Figure imgf000067_0004
. [00272] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond, Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be substituted C6-10 aryl; substituted with one or more E, wherein E can be –OH; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY can be -NH(C1-4 alkyl); RZ can be hydrogen; J can be C; X can be N; Y can be C; Z can be C; and joining Y and Z can be a double bond. In some embodiments, the compound of Formula (I) can be 4-(2-((2-(benzo[b]thiophen-3-yl)-6- (isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol. [00273] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc, Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E can be –OH; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together is
Figure imgf000068_0001
wherein the ring is substituted with C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be 4-(2-((2-(benzo[b]thiophen-3-yl)-7- isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol. [00274] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc, Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E can be –OH; RK can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together is
Figure imgf000068_0002
can be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be 4-(2-((2- (benzo[b]thiophen-3-yl)-7-isopropyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4- yl)amino)ethyl)phenol. [00275] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc, Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E can be –OH; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together is
Figure imgf000068_0003
can be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one. [00276] In some embodiments, when RJ is –ORb; G can be N; joining G and J can be a double bond; Rb can be –CH2CH2-Rc; Rc can be -C(=O)NH2; RK can unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together can b
Figure imgf000069_0001
an be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z is C. In some embodiments, the compound of Formula (I) can be 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- yl)oxy)propanamide. [00277] In some embodiments, when RJ is is –NRaRb; G can be N; joining G and J can be a double bond; Rb can be –CH2CH2-Rc; Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK is unsubstituted five- to ten-membered heteroaryl having 1- 4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together can be
Figure imgf000069_0002
wherein said ring is substituted with -N(C1-4 alkyl)2; J can be C; X can be N; Y can be C; and Z is C. In some embodiments, the compound of Formula (I) can be 4-(2-((2- (benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4-yl)amino)ethyl)phenol. [00278] In some embodiments, when RJ is is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; RY can be -NH(C1-4 alkyl); RZ can be absent; J can be C; X can be C; Y can be C; Z can be N; and joining Y and Z can be a double bond. In some embodiments, the compound of Formula (I) can be 5-(2-((2-(1H-indol-3-yl)ethyl)amino)-6-(sec- butylamino)pyrimidin-4-yl)nicotinonitrile. [00279] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be unsubstituted C1-6 alkyl; RY and RZ taken together can
Figure imgf000070_0001
herein the ring is substituted with unsubstituted C6-C10 aryl; J can be C; X can be N; Y can be C; Z can be C. . In some embodiments, the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6- phenylthieno[2,3-d]pyrimidin-4-amine [00280] In some embodiments, when RJ can be –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be hydrogen; RY and RZ taken together can b
Figure imgf000070_0002
wherein the ring is substituted with substituted C6-C10 aryl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine [00281] In some embodiments, when RJ is =O; G can be N substituted with RG; joining G and J can be a single bond; RG can be -(C1-4 alkyl)-C(=O)NH2; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together can b
Figure imgf000070_0003
can be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be 3-(2- (benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide. [00282] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond Ra can be hydrogen Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q can be halo; RY and RZ taken together can b
Figure imgf000071_0001
an be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine. [00283] In some embodiments, when RJ is –NRaRb; G is N; joining G and J can be a double bond; Ra can be hydrogen Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q can be cyano; RY and RZ taken together is
Figure imgf000071_0002
an be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be 5-(4- ((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile. [00284] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be -NH(C1-4 alkyl); RY and RZ taken together can b
Figure imgf000071_0003
an be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be N4-(2-(1H- indol-3-yl)ethyl)-N2-(sec-butyl)quinazoline-2,4-diamine. [00285] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together can b
Figure imgf000072_0001
wherein the ring is substituted with cyano; Rd can be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H- pyrrolo[2,3-d]pyrimidine-5-carbonitrile. [00286] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together can b
Figure imgf000072_0002
wherein the ring is substituted with C1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms. In some embodiments, the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3- isopropylimidazo[1,5-a]pyrazin-8-amine. [00287] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RY and RZ taken together can b
Figure imgf000072_0003
wherein the ring can be substituted with C1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms. In some embodiments, the compound of Formula (I) can be 4-(2- ((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-yl)amino)ethyl)phenol. [00288] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J represents a double bond; Ra can be hydrogen Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; RY and RZ taken together is
Figure imgf000073_0001
wherein the ring is substituted with C1-C4 alkyl;J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7- isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile. [00289] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J represents a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; RY and RZ taken together can be
Figure imgf000073_0002
wherein the ring is substituted with C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin- 3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine. [00290] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; RY and RZ taken together can b
Figure imgf000073_0003
an be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be N-(2- (1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)furo[3,2-d]pyrimidin-4-amine. [00291] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is C1-C4 alkyl; RY and RZ taken together can b
Figure imgf000074_0001
an be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine. [00292] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is C1-C4 alkyl; RY and RZ taken together can b
Figure imgf000074_0002
herein the ring is substituted with C1-C4 alkyl J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5- methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine. [00293] In some embodiments, when RJ is –NRaRb; G is N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; RY and RZ taken together can b
Figure imgf000075_0001
an be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I) can be 5-(4- ((2-(1H-indol-3-yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile. [00294] In some emdiments, provided herein is compound of Formula (I), wherein the compound can be selected from: 4-(2-((2-(benzo[b]thiophen-3-yl)-6-(isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol; 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol; 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4- yl)amino)ethyl)phenol; 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-5,7-dihydro-6H- pyrrolo[2,3-d]pyrimidin-6-one; 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-yl)oxy)propanamide; 4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4- yl)amino)ethyl)phenol; 5-(2-((2-(1H-indol-3-yl)ethyl)amino)-6-(sec-butylamino)pyrimidin-4-yl)nicotinonitrile; N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4-amine; N-(2-(1H-indol-3-yl)ethyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine; 3-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide; N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine; 5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile; N4-(2-(1H-indol-3-yl)ethyl)-N2-(sec-butyl)quinazoline-2,4-diamine; 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3- d]pyrimidine-5-carbonitrile; N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine; 4-(2-((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-yl)amino)ethyl)phenol; 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7-isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile; N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine; N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine; 5-(4-((2-(1H-indol-3-yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile; and pharmaceutically acceptable salts thereof. Formula (I-A) [00295] In some embodiments provided herein, the compound of Formula (I) can have the structure of Formula (I-
Figure imgf000076_0001
ncluding pharmaceutically acceptable salts thereof, wherein: RJ can be –NRaRb; Ra can be hydrogen or C1-C4 alkyl; Rb can be Rc or - (C1-C4 alkyl)-Rc; Rc can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and - O(C1-C4 haloalkyl); RK can be selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; -NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); Y and Z can each be C; X can be N or CH; W can be O or S; and Re can be hydrogen or C1-C4 alkyl. [00296] In some embodiments, Ra can be hydrogen. In other embodiments, Ra can be C1- C4 alkyl. [00297] In some embodiments, Rb can be -(C1-C4 alkyl)-Rc. For example, Rb can be -CH2- Rc, -CH2CH2-Rc, -CH2CH2CH2-Rc, or -CH2CH2CH2CH2-Rc. [00298] In some embodiments, Rc can be –OH. In some embodiments, Rc can be -O(C1-C4 alkyl). In some embodiments, Rc can be -O(C1-C4 haloalkyl). In some embodiments, Rc can be - C(=O)NH2. In some embodiments, Rc can be unsubstituted C6-10 aryl. In some embodiments, Rc can be substituted C6-10 aryl. In some embodiments, Rc can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, Rc can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, when a Rc moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents E. In some embodiments, E can be –OH. In some embodiments, E can be C1-C4 alkyl. In some embodiments, E can be C1-C4 haloalkyl. In some embodiments, E can be -O(C1-C4 alkyl). In some embodiments, E can be - O(C1-C4 haloalkyl). In some embodiments Rc can be phenyl. In other embodiments, Rc can be hydroxyphenyl. In still other embodiments, Rc can be indolyl. [00299] In some embodiments, RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein the substituted heteroaryl can substituted with one or more substituents Q, wherein each Q can independently selected from the group consisting of: -OH, C1- 4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl). In some embodiments, RK can be pyridinyl. In other embodiments, RK can be pyridinyl substituted with one or more substituents Q. For example, RK can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl. [00300] In some embodiments, Re can be hydrogen. In some embodiments, Re can be C1- C4 alkyl. For example, Re can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl. [00301] In some embodiments, Ra can be hydrogen; Rb can be -(C1-C4 alkyl)-Rc; Rc can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be selected from the group consisting of: unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein the substituted heteroaryl is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, - O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); and Re can be C1-C4 alkyl. [00302] In some embodiments, Ra can be hydrogen; Rb can be -(CH2-CH2)-Rc; Rc can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E can be -OH; RK can be selected from the group consisting of: unsubstituted benzothiophenyl and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one substituent Q, wherein Q can be selected from the group consisting of: C1-4 alkyl, halo, and cyano; and Re can be isopropyl. [00303] In some embodiments, when W is O, RJ can be –NRaRb; Ra can be hydrogen; Rb can be -CH2CH2-Rc; Rc can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, and -O(C1-C4 alkyl); RK can be selected from the group consisting of unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: - C1-4 alkyl, halo, cyano, and -O-(C1-4 alkyl); Y and Z can each be C; X can be N or CH; and Re can be hydrogen or C1-C4 alkyl. [00304] In some embodiments, when W is S, RJ can be –NRaRb; Ra can be hydrogen; Rb can be -CH2CH2-Rc; Rc can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, and -O(C1-C4 alkyl); RK can be selected from the group consisting of unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: - C1-4 alkyl, halo, cyano, and -O-(C1-4 alkyl); Y and Z can each be C; X can be N or CH; and Re can be hydrogen or C1-C4 alkyl. [00305] In some embodiments, when RJ is –NRaRb; G can be N; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is C1-C4 alkyl; W can be S; Re can be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-A) can be N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2- (5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine. [00306] In some embodiments, when RJ is –NRaRb; G can be N; Ra can be hydrogen Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; W can be S; Re can be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-A) can be 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7- isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile. [00307] In some embodiments, when RJ is –NRaRb; G can be N; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; W can be S; Re can be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-A) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5- fluoropyridin-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine. [00308] In some embodiments, when RJ is –NRaRb; G can be N; Ra can be hydrogen; Rb can be –CH2CH2-Rc, Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E can be –OH; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; W can be S; Re can be C1-C4 alkyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-A) can be 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol. [00309] In some embodiments, when RJ is –NRaRb; G can be N; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; W can be O; Re can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-A) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3- yl)furo[3,2-d]pyrimidin-4-amine. [00310] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is C1-C4 alkyl; W can be O; Re can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-A) can be N-(2- (1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine. [00311] In some embodiments, when RJ is –NRaRb; G is NRa can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; W can be O; Re can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-A) can be 5-(4-((2-(1H-indol-3- yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile. [00312] [0140] In some embodiments, the compound of Formula (I-A), or a pharmaceutically acceptable salt thereof, can selected from the group consisting of: N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine; 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7-isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile; N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine; 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol; N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; and 5-(4-((2-(1H-indol-3-yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile. Formula (I-B) [00313] In other embodiments provided herein, the compound of Formula (I) can have the structure of Formula (I-
Figure imgf000081_0001
including pharmaceutically acceptable salts thereof, wherein: Ra can be hydrogen or C1-C4 alkyl; Rb can be Rc or -(C1-4 alkyl)-Rc; Rc can be selected from the group consisting of: -OH, -O(C1-C4 alkyl), -O(C1-C4 haloalkyl); -C(=O)NH2; unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1- C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; substituted C1-6 alkyl; -NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); RG can be selected from the group consisting of hydrogen, C1-4 alkyl, and -(C1-4 alkyl)-C(=O)NH2; Rf can be selected from the group consisting of hydrogen, C1-4 alkyl, unsubstituted C6-C10 aryl, and C6-C10 aryl substituted with 1-5 halo atoms; U can be N or CRU; V can be S or NRV; RU can be selected from the group consisting of hydrogen, C1-4 alkyl, halo, and cyano; RV can be hydrogen or C1-C4 alkyl; wherein when U is CRU and V is NRV, RU is selected from the group consisting of C1-4 alkyl, halo, and cyano; Y and Z can each be C; and X can be N or CH. [00314] In some embodiments, Ra can be hydrogen. In other embodiments, Ra can be C1- C4 alkyl. [00315] In some embodiments, Rb can be -(C1-C4 alkyl)-Rc. For example, Rb can be -CH2- Rc, -CH2CH2-Rc, -CH2CH2CH2-Rc, or -CH2CH2CH2CH2-Rc. In certain embodiments, Rb can be - (CH2CH2)-Rc. In certain embodiments, Rb can be -(CH2CH2)-C(=O)NH2. In certain embodiments, Rb can be -(CH2CH2)-(indolyl). In certain embodiments, Rb can be -(CH2CH2)-(hydroxyphenyl). [00316] In some embodiments, Rc can be –OH. In some embodiments, Rc can be -O(C1-C4 alkyl). In some embodiments, Rc can be -O(C1-C4 haloalkyl). In some embodiments, Rc can be - C(=O)NH2. In some embodiments, Rc can be unsubstituted C6-10 aryl. In some embodiments, Rc can be substituted C6-10 aryl. In some embodiments, Rc can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, Rc can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, when a Rc moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents E. In some embodiments, E can be –OH. In some embodiments, E can be C1-C4 alkyl. In some embodiments, E can be C1-C4 haloalkyl. In some embodiments, E can be -O(C1-C4 alkyl). In some embodiments, E can be - O(C1-C4 haloalkyl). [00317] In some embodiments, RK can be hydrogen. In other embodiments, RK can be C1- C4 alkyl. For example, RK can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert- butyl. In some embodiments, RK can be selected from the group consisting of: unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein the substituted heteroaryl can substituted with one or more substituents Q, wherein each Q can independently selected from the group consisting of: -OH, C1- 4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl). In certain mbodiments, RK can be benzothiophenyl. In other embodiments, RK can be pyridinyl substituted with one or more substituents Q. For example, RK can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl. [00318] In some embodiments, RG can be selected from the group consisting of hydrogen, C1-4 alkyl, and -(C1-4 alkyl)-C(=O)NH2. In certain embodiments, RG can be -(CH2CH2)- C(=O)NH2. [00319] In some embodiments, Rf can be hydrogen. In other embodiments, Rf can be C1-4 alkyl. For example, Rf can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In some embodiments, Rf can be unsubstituted C6-C10 aryl. In other embodiments, Rf can be C6- C10 aryl substituted with 1-5 halo atoms. In certain embodiments, Rf can be phenyl substituted with 1-5 halo atoms. In certain embodiments, Rf can be fluorophenyl. [00320] In some embodiments, U can be N. In other embodiments, U can be CRU. [00321] In some embodiments, V can be S. In other embodiments, V can be NRV. [00322] In some embodiments, RU can be hydrogen. In some embodiments, RU can be C1-4 alkyl. In other embodiments RU can be halo. For example, RU can be fluoro, chloro, bromo, or iodo. In still other embodiments, RU can be cyano. [00323] In some embodiments, RV can be hydrogen. In other embodiments, RV can be C1-4 alkyl. For example, RV can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In some embodiments, Y and Z can each be C and X can be N. In other embodiments, Y and Z can each be C and X can be CH. [00324] In some embodiments, Ra can be hydrogen; Rb can be -(C1-4 alkyl)-Rc; Rc can be selected from the group consisting of: -C(=O)NH2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted can be substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be selected from the group consisting of: unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein the substituted heteroaryl is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); RG is C1-4 alkyl or -(C1-4 alkyl)-C(=O)NH2; Rf can be selected from the group consisting of hydrogen, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms; Y and Z each can be C; and X can be CH. [00325] In some embodiments, Ra can be hydrogen; Rb can be -(CH2-CH2)-Rc; Rc can be selected from the group consisting of: -C(=O)NH2, substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E can be -OH; RK can be selected from the group consisting of: unsubstituted benzothiohenyl and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one substituent Q, wherein Q can be selected from the group consisting of: C1-4 alkyl, halo, and cyano; RG can be -(CH2CH2)- C(=O)NH2; Rf can be selected from the group consisting of hydrogen, phenyl, and fluorophenyl; Y and Z each can be C; and X can be CH. [00326] In some embodiments, when V is S, Ra can be hydrogen or C1-C4 alkyl; Rb can be Rc or -(CH2-CH2)-Rc; Rc can be selected from the group consisting of: -C(=O)NH2; unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, and -O(C1-C4 alkyl); RK can be selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; substituted C1-6 alkyl; -NH(C1-4 alkyl); and -N(C1-4 alkyl)2; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, halo, cyano, and -O-(C1-4 alkyl; RG can be selected from the group consisting of hydrogen, C1-4 alkyl, and -(C1-4 alkyl)-C(=O)NH2; Rf can be selected from the group consisting of hydrogen, C1-4 alkyl, unsubstituted C6-C10 aryl, and C6-C10 aryl substituted with 1-5 halo atoms; U can be CRU; RU can be selected from the group consisting of hydrogen, C1-4 alkyl, halo, and cyano; Y and Z can each be C; and X can be N. [00327] In some embodiments, when V is NRV, Ra can be hydrogen or C1-C4 alkyl; Rb can be Rc or -(CH2-CH2)-Rc; Rc can be selected from the group consisting of: -C(=O)NH2; unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1- C4, and -O(C1-C4 alkyl); RK can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, halo, cyano, and -O-(C1-4 alkyl); RG can be selected from the group consisting of hydrogen, C1-4 alkyl, and -(C1-4 alkyl)-C(=O)NH2; Rf can be hydrogen; U can be N or CRU; RU can be selected from the group consisting of C1-4 alkyl, halo, and cyano; RV can be hydrogen or C1-C4 alkyl; Y and Z can each be C; and X can be N or CH. [00328] In some embodiments, when RJ is –ORb; G can be N; joining G and J can be a double bond; Rb can be –CH2CH2-Rc; Rc can be -C(=O)NH2; RK can unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; U can N; V can be NRv; Rv can be C1-C4 alkyl; Rf can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-B) can be 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- yl)oxy)propanamide. [00329] In some embodiments, when RJ is =O; G can be N substituted with RG; joining G and J can be a single bond; RG can be -(C1-4 alkyl)-C(=O)NH2; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; U can N; V can be NRv; Rv can be C1-C4 alkyl; Rf can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-B) can be 3- (2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide. [00330] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; U can be CRu; Ru can be cyano; V can be NRv; Rv can be C1-C4 alkyl; Rf can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-B) can be 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3- d]pyrimidine-5-carbonitrile. [00331] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be unsubstituted C1-6 alkyl; U can be CRu; Ru can be hydrogen; V can be S; Rf can be phenyl; J can be C; X can be N; Y can be C; Z can be C. In some embodiments, the compound of Formula (I-B) can be N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4- amine. [00332] In some embodiments, when RJ can be –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be hydrogen; U can be CRu; Ru can be hydrogen; V can be S; Rf can be fluorophenyl; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-B) can be N-(2-(1H-indol-3-yl)ethyl)-6- (4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine. [00333] In some embodiments, the compound of Formula (I-B), or a pharmaceutically acceptable salt thereof, can selected from the group consisting of: 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-yl)oxy)propanamide; 3-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide; 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3- d]pyrimidine-5-carbonitrile; N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4-amine; and N-(2-(1H-indol-3-yl)ethyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine. Formula (I-C) [00334] In still other embodiments provided herein, the compound of Formula (I) can have the structure of Formula (I-
Figure imgf000087_0001
ncluding pharmaceutically acceptable salts thereof, wherein: RJ can be –NRaRb; Ra can be hydrogen or C1-C4 alkyl; Rb can be Rc or - (C1-C4 alkyl)-Rc; Rc can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and - O(C1-C4 haloalkyl); RK can be selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl;-NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); A can be N or CH; B can be N or CH; Rg can be selected from the group consisting of hydrogen, C1-4 alkyl, and -N(C1-4 alkyl)2; Y and Z can each be C; and X can be N or CH. [00335] In some embodiments, RK can be -NH(C1-4 alkyl). For example, in some embodiments, RK can be -NH(CH3), -NH(CH2CH3), -NH(isopropyl), or -NH(sec-butyl). In some embodiments, RK can be unsubstituted benzothiophenyl. In other embodiments, RK can be substituted pyridinyl. For example, RK can be methylpyridinyl, ethylpyridinyl, cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl. [00336] In some embodiments, A can be N and B can be N. In other embodiments, A can be N and B can be CH. In still other embodiments, A can be CH and B can be N. In yet still other embodiments, A can be CH and B can be CH. [00337] In some embodiments, Rg can be hydrogen. In other embodiments, Rg can be - N(C1-4 alkyl)2. In certain embodiments, Rg can be -N(CH3)2. [00338] In some embodiments, Ra can be hydrogen; Rb can be -(C1-C4 alkyl)-Rc; Rc can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be selected from the group consisting of: -NH(C1-4 alkyl); unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein the substituted heteroaryl is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); and Rg can be hydrogen or -N(C1-4 alkyl)2. [00339] In some embodiments, Ra can be hydrogen; Rb can be -(C1-C4 alkyl)-Rc; Rc can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be selected from the group consisting of: -NH(C1-4 alkyl); unsubstituted benzothiophenyl; and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); and Rg can be hydrogen or -N(C1-4 alkyl)2. [00340] In some embodiments, Ra can be hydrogen; Rb can be -(CH2CH2)-Rc; Rc can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E can be -OH; RK can be selected from the group consisting of: -NH(sec-butyl); unsubstituted benzothiohenyl, and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: C1-4 alkyl, halo, and cyano; and Rg can be hydrogen or -N(CH3)2. [00341] In some embodiments, when A is C and B is C, RJ can be –NRaRb; G can be N; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; or unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; Rg can be hydrogen; J can be C; X can be N; Y can be C; and Z is C. [00342] In some embodiments, when RJ is –NRaRb; G can be N; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK is unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; A can be N; B can be N; Rg can be -N(C1-4 alkyl)2; J can be C; X can be N; Y can be C; and Z is C. In some embodiments, the compound of Formula (I-C) can be 4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4- yl)amino)ethyl)phenol. [00343] In some embodiments, when RJ is –NRaRb; G can be N; Ra can be hydrogen Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q can be halo; A can be CH; B can be CH; Rg can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-C) can be N-(2-(1H-indol-3-yl)ethyl)-2-(5- fluoropyridin-3-yl)quinazolin-4-amine. [00344] In some embodiments, when RJ is –NRaRb; G is N; joining G and J can be a double bond; Ra can be hydrogen Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more Q, wherein Q can be cyano; A can be CH; B can be CH; Rg can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-C) can be 5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile. [00345] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; RK can be -NH(C1-4 alkyl); A can be CH; B can be CH; Rg can be hydrogen; J can be C; X can be N; Y can be C; and Z can be C. In some embodiments, the compound of Formula (I-C) can be N4- (2-(1H-indol-3-yl)ethyl)-N2-(sec-butyl)quinazoline-2,4-diamine. [00346] In some embodiments, the compound of Formula (I-C), or a pharmaceutically acceptable salt thereof, can selected from the group consisting of: 4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4- yl)amino)ethyl)phenol; N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine; 5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile; and N4-(2-(1H-indol-3-yl)ethyl)-N2-(sec-butyl)quinazoline-2,4-diamine. Formula (I-D) [00347] In yet still other embodiments provided herein, the compound of Formula (I) can have the structure of Formula (I
Figure imgf000090_0001
ncluding pharmaceutically acceptable salts thereof, wherein: RJ can be –NRaRb; Ra can be hydrogen or C1-C4 alkyl; Rb can be Rc or -(C1-4 alkyl)-Rc; Rc can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and - O(C1-C4 haloalkyl); RK can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); Rh can be hydrogen or C1-4 alkyl; D can be N or CH; Y can be N; Z can be C; and X can be N or CH. [00348] In some embodiments, Rh can be hydrogen. In other embodiments, Rh can be C1-4 alkyl. For example, Rh can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. [00349] In some embodiments, D can be N. In other embodiments, D can be CH. [00350] In some embodiments, when D is N, Y can be N, Z can be C, and X can be N. In other embodiments, when D is N, Y can be N, Z can be C, and X can be CH. In some embodiments, when D is CH, Y can be N, Z can be C, and X can be N. In other embodiments, when D is CH, Y can be N, Z can be C, and X can be CH. [00351] In some embodiments, Ra can be hydrogen; Rb can be -(C1-4 alkyl)-Rc; Rc can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); and Rh can be hydrogen or C1-4 alkyl. [00352] In some embodiments, Ra can be hydrogen; Rb can be -(C1-C4 alkyl)-Rc; Rc can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one or more substituents E, wherein each E can be independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl); RK can be unsubstituted benzothiophenyl; and Rh can be hydrogen or C1-4 alkyl. [00353] In some embodiments, Ra can be hydrogen; Rb can be -(CH2-CH2)-Rc; Rc can be selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E can be -OH; RK can be unsubstituted benzothiophenyl; and Rh can be hydrogen or C1-4 alkyl. [00354] In some embodiments, when D is N; RJ is –NRaRb; G can be N; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; or substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; Rh can be C1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms. [00355] In some embodiments, when RJ is –NRaRb; G can be N; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S or substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; D can be N; Rh can be C1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms. In some embodiments, the compound of Formula (I-D) can be N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine. [00356] In some embodiments, when RJ is –NRaRb; G can be N; joining G and J can be a double bond; Ra can be hydrogen; Rb can be –CH2CH2-Rc; Rc can be substituted C6-10 aryl, substituted with one or more E, wherein E is –OH; RK can be unsubstituted five- to ten- membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; D can be N; Rh can be C1-4 alkyl; J can be C; X can be C; Y can be N; and Z can be C; wherein the valency of any carbon atom is filled as needed with hydrogen atoms. In some embodiments, the compound of Formula (I-D) can be 4-(2-((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5- a]pyrazin-8-yl)amino)ethyl)phenol. [00357] In some embodiments, the compound of Formula (I-D), or a pharmaceutically acceptable salt thereof, can selected from the group consisting of: N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine; and 4-(2-((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-yl)amino)ethyl)phenol. [00358] The compounds provided herein may be enantiomerically pure, such as a single enantiomer or a single diastereomer, or be stereoisomeric mixtures, such as a mixture of enantiomers, e.g., a racemic mixture of two enantiomers; or a mixture of two or more diastereomers. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form. Conventional techniques for the preparation/isolation of individual enantiomers include synthesis from a suitable optically pure precursor, asymmetric synthesis from achiral starting materials, or resolution of an enantiomeric mixture, for example, chiral chromatography, recrystallization, resolution, diastereomeric salt formation, or derivatization into diastereomeric adducts followed by separation. 5.4. Isolation of NK Cells [00359] Methods of isolating natural killer cells are known in the art and can be used to isolate the natural killer cells, e.g., NK cells produced using the three-stage method, described herein. For example, NK cells can be isolated or enriched, for example, by staining cells, in one embodiment, with antibodies to CD56 and CD3, and selecting for CD56+CD3– cells. In certain embodiments, the NK cells are enriched for CD56+CD3– cells in comparison with total cells produced using the three-stage method, described herein. NK cells, e.g., cells produced using the three-stage method, described herein, can be isolated using a commercially available kit, for example, the NK Cell Isolation Kit (Miltenyi Biotec). NK cells, e.g., cells produced using the three-stage method, described herein, can also be isolated or enriched by removal of cells other than NK cells in a population of cells that comprise the NK cells, e.g., cells produced using the three-stage method, described herein. For example, NK cells, e.g., cells produced using the three- stage method, described herein, may be isolated or enriched by depletion of cells displaying non- NK cell markers using, e.g., antibodies to one or more of CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA DR and/or CD235a (glycophorin A). Negative isolation can be carried out using a commercially available kit, e.g., the NK Cell Negative Isolation Kit (Dynal Biotech). Cells isolated by these methods may be additionally sorted, e.g., to separate CD11a+ and CD11a- cells, and/or CD117+ and CD117- cells, and/or CD16+ and CD16– cells, and/or CD94+ and CD94–. In certain embodiments, cells, e.g., cells produced by the three-step methods described herein, are sorted to separate CD11a+ and CD11a- cells. In specific embodiments, CD11a+ cells are isolated. In certain embodiments, the cells are enriched for CD11a+ cells in comparison with total cells produced using the three-stage method, described herein. In specific embodiments, CD11a- cells are isolated. In certain embodiments, the cells are enriched for CD11a- cells in comparison with total cells produced using the three-stage method, described herein. In certain embodiments, cells are sorted to separate CD117+ and CD117- cells. In specific embodiments, CD117+ cells are isolated. In certain embodiments, the cells are enriched for CD117+ cells in comparison with total cells produced using the three-stage method, described herein. In specific embodiments, CD117- cells are isolated. In certain embodiments, the cells are enriched for CD117- cells in comparison with total cells produced using the three-stage method, described herein. In certain embodiments, cells are sorted to separate CD16+ and CD16– cells. In specific embodiments, CD16+ cells are isolated. In certain embodiments, the cells are enriched for CD16+ cells in comparison with total cells produced using the three-stage method, described herein. In specific embodiments, CD16– cells are isolated. In certain embodiments, the cells are enriched for CD16- cells in comparison with total cells produced using the three-stage method, described herein. In certain embodiments, cells are sorted to separate CD94+ and CD94– cells. In specific embodiments, CD94+ cells are isolated. In certain embodiments, the cells are enriched for CD94+ cells in comparison with total cells produced using the three-stage method, described herein. In specific embodiments, CD94– cells are isolated. In certain embodiments, the cells are enriched for CD94- cells in comparison with total cells produced using the three-stage method, described herein. In certain embodiments, isolation is performed using magnetic separation. In certain embodiments, isolation is performed using flow cytometry. [00360] Methods of isolating ILC3 cells are known in the art and can be used to isolate the ILC3 cells, e.g., ILC3 cells produced using the three-stage method, described herein. For example, ILC3 cells can be isolated or enriched, for example, by staining cells, in one embodiment, with antibodies to CD56, CD3, and CD11a, and selecting for CD56+CD3–CD11a– cells. ILC3 cells, e.g., cells produced using the three-stage method, described herein, can also be isolated or enriched by removal of cells other than ILC3 cells in a population of cells that comprise the ILC3 cells, e.g., cells produced using the three-stage method, described herein. For example, ILC3 cells, e.g., cells produced using the three-stage method, described herein, may be isolated or enriched by depletion of cells displaying non-ILC3 cell markers using, e.g., antibodies to one or more of CD3, CD4, CD11a, CD14, CD19, CD20, CD36, CD66b, CD94, CD123, HLA DR and/or CD235a (glycophorin A). Cells isolated by these methods may be additionally sorted, e.g., to separate CD117+ and CD117– cells. NK cells can be isolated or enriched, for example, by staining cells, in one embodiment, with antibodies to CD56, CD3, CD94, and CD11a, and selecting for CD56+CD3–CD94+CD11a+ cells. NK cells, e.g., cells produced using the three-stage method, described herein, can also be isolated or enriched by removal of cells other than NK cells in a population of cells that comprise the NK cells, e.g., cells produced using the three-stage method, described herein. In certain embodiments, the NK cells are enriched for CD56+CD3– CD94+CD11a+ cells in comparison with total cells produced using the three-stage method, described herein. [00361] In one embodiment, ILC3 cells are isolated or enriched by selecting for CD56+CD3–CD11a– cells. In certain embodiments, the ILC3 cells are enriched for CD56+CD3– CD11a– cells in comparison with total cells produced using the three-stage method, described herein. In one embodiment, ILC3 cells are isolated or enriched by selecting for CD56+CD3– CD11a–CD117+ cells. In certain embodiments, the ILC3 cells are enriched for CD56+CD3– CD11a–CD117+ cells in comparison with total cells produced using the three-stage method, described herein. In one embodiment, ILC3 cells are isolated or enriched by selecting for CD56+CD3–CD11a–CD117+CDIL1R1+ cells. In certain embodiments, the ILC3 cells are enriched for CD56+CD3–CD11a–CD117+CDIL1R1+ cells in comparison with total cells produced using the three-stage method, described herein. [00362] In one embodiment, NK cells are isolated or enriched by selecting for CD56+CD3– CD94+CD11a+ cells. In certain embodiments, the NK cells are enriched for CD56+CD3– CD94+CD11a+ cells in comparison with total cells produced using the three-stage method, described herein. In one embodiment, NK cells are isolated or enriched by selecting for CD56+CD3–CD94+CD11a+CD117– cells. In certain embodiments, the NK cells are enriched for CD56+CD3–CD94+CD11a+CD117– cells in comparison with total cells produced using the three- stage method, described herein. [00363] Cell separation can be accomplished by, e.g., flow cytometry, fluorescence- activated cell sorting (FACS), or, in one embodiment, magnetic cell sorting using microbeads conjugated with specific antibodies. The cells may be isolated, e.g., using a magnetic activated cell sorting (MACS) technique, a method for separating particles based on their ability to bind magnetic beads (e.g., about 0.5-100 μm diameter) that comprise one or more specific antibodies, e.g., anti-CD56 antibodies. Magnetic cell separation can be performed and automated using, e.g., an AUTOMACS™ Separator (Miltenyi). A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically recognizes a particular cell surface molecule or hapten. The beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker. In one embodiment, these cells can then isolated and re-mixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation. 5.5. Placental Perfusate [00364] NK cells and/or ILC3 cells, e.g., NK cell and/or ILC3 cell populations produced according to the three-stage method described herein may be produced from hematopoietic cells, e.g., hematopoietic stem or progenitors from any source, e.g., placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, or the like. In certain embodiments, the hematopoietic stem cells are combined hematopoietic stem cells from placental perfusate and from cord blood from the same placenta used to generate the placental perfusate. Placental perfusate comprising placental perfusate cells that can be obtained, for example, by the methods disclosed in U.S. Patent Nos.7,045,148 and 7,468,276 and U.S. Patent Application Publication No.2009/0104164, the disclosures of which are hereby incorporated in their entireties. 5.5.1. Cell Collection Composition [00365] The placental perfusate and perfusate cells, from which hematopoietic stem or progenitors may be isolated, or useful in tumor suppression or the treatment of an individual having tumor cells, cancer or a viral infection, e.g., in combination with the NK cells and/or ILC3 cells, e.g., NK cell and/or ILC3 cell populations produced according to the three-stage method provided herein, can be collected by perfusion of a mammalian, e.g., human post-partum placenta using a placental cell collection composition. Perfusate can be collected from the placenta by perfusion of the placenta with any physiologically-acceptable solution, e.g., a saline solution, culture medium, or a more complex cell collection composition. A cell collection composition suitable for perfusing a placenta, and for the collection and preservation of perfusate cells is described in detail in related U.S. Application Publication No.2007/0190042, which is incorporated herein by reference in its entirety. [00366] The cell collection composition can comprise any physiologically-acceptable solution suitable for the collection and/or culture of stem cells, for example, a saline solution (e.g., phosphate-buffered saline, Kreb’s solution, modified Kreb’s solution, Eagle’s solution, 0.9% NaCl. etc.), a culture medium (e.g., DMEM, H.DMEM, etc.), and the like. [00367] The cell collection composition can comprise one or more components that tend to preserve placental cells, that is, prevent the placental cells from dying, or delay the death of the placental cells, reduce the number of placental cells in a population of cells that die, or the like, from the time of collection to the time of culturing. Such components can be, e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesium sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin- releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.); a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor; and/or an oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide, perfluorodecyl bromide, etc.). [00368] The cell collection composition can comprise one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, a hyaluronidase, an RNase, or a DNase, or the like. Such enzymes include, but are not limited to, collagenases (e.g., collagenase I, II, III or IV, a collagenase from Clostridium histolyticum, etc.); dispase, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like. [00369] The cell collection composition can comprise a bacteriocidally or bacteriostatically effective amount of an antibiotic. In certain non-limiting embodiments, the antibiotic is a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc. In a particular embodiment, the antibiotic is active against Gram(+) and/or Gram(–) bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus, and the like. [00370] The cell collection composition can also comprise one or more of the following compounds: adenosine (about 1 mM to about 50 mM); D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50 mM); a macromolecule of molecular weight greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain endothelial integrity and cellular viability (e.g., a synthetic or naturally occurring colloid, a polysaccharide such as dextran or a polyethylene glycol present at about 25 g/l to about 100 g/l, or about 40 g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present at about 25 ^M to about 100 ^M); a reducing agent (e.g., N-acetylcysteine present at about 0.1 mM to about 5 mM); an agent that prevents calcium entry into cells (e.g., verapamil present at about 2 ^M to about 25 ^M); nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant, in one embodiment, present in an amount sufficient to help prevent clotting of residual blood (e.g., heparin or hirudin present at a concentration of about 1000 units/l to about 100,000 units/l); or an amiloride containing compound (e.g., amiloride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride present at about 1.0 ^M to about 5 ^M). 5.5.2. Collection and Handling of Placenta [00371] Generally, a human placenta is recovered shortly after its expulsion after birth. In one embodiment, the placenta is recovered from a patient after informed consent and after a complete medical history of the patient is taken and is associated with the placenta. In one embodiment, the medical history continues after delivery. [00372] Prior to recovery of perfusate, the umbilical cord blood and placental blood are removed. In certain embodiments, after delivery, the cord blood in the placenta is recovered. The placenta can be subjected to a conventional cord blood recovery process. Typically a needle or cannula is used, with the aid of gravity, to exsanguinate the placenta (see, e.g., Anderson, U.S. Patent No.5,372,581; Hessel et 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 aid in draining cord blood from the placenta. Such cord blood recovery may be performed commercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell. In one embodiment, the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery. [00373] Typically, a placenta is transported from the delivery or birthing room to another location, e.g., a laboratory, for recovery of cord blood and collection of perfusate. The placenta can be transported in a sterile, thermally insulated transport device (maintaining the temperature of the placenta between 20-28 °C), for example, by placing the placenta, with clamped proximal umbilical cord, in a sterile zip-lock plastic bag, which is then placed in an insulated container. In another embodiment, the placenta is transported in a cord blood collection kit substantially as described in U.S. Patent No.7,147,626. In one embodiment, the placenta is delivered to the laboratory four to twenty-four hours following delivery. In certain embodiments, the proximal umbilical cord is clamped, for example within 4-5 cm (centimeter) of the insertion into the placental disc prior to cord blood recovery. In other embodiments, the proximal umbilical cord is clamped after cord blood recovery but prior to further processing of the placenta. [00374] The placenta, prior to collection of the perfusate, can be stored under sterile conditions and at either room temperature or at a temperature of 5 to 25 °C (centigrade). The placenta may be stored for a period of longer than forty eight hours, or for a period of four to twenty-four hours prior to perfusing the placenta to remove any residual cord blood. The placenta can be stored in an anticoagulant solution at a temperature of 5 °C to 25 °C (centigrade). Suitable anticoagulant solutions are well known in the art. For example, a solution of heparin or warfarin sodium can be used. In one embodiment, the anticoagulant solution comprises a solution of heparin (e.g., 1% w/w in 1:1000 solution). In some embodiments, the exsanguinated placenta is stored for no more than 36 hours before placental perfusate is collected. 5.5.3. Placental Perfusion [00375] Methods of perfusing mammalian placentae and obtaining placental perfusate are disclosed, e.g., in Hariri, U.S. Patent Nos.7,045,148 and 7,255,879, and in U.S. Application Publication Nos.2009/0104164, 2007/0190042 and 20070275362, issued as U.S. Pat No. 8,057,788, the disclosures of which are hereby incorporated by reference herein in their entireties. [00376] Perfusate can be obtained by passage of perfusion solution, e.g., saline solution, culture medium or cell collection compositions described above, through the placental vasculature. In one embodiment, a mammalian placenta is perfused by passage of perfusion solution through either or both of the umbilical artery and umbilical vein. The flow of perfusion solution through the placenta may be accomplished using, e.g., gravity flow into the placenta. For example, the perfusion solution is forced through the placenta using a pump, e.g., a peristaltic pump. The umbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula, that is connected to a sterile connection apparatus, such as sterile tubing. The sterile connection apparatus is connected to a perfusion manifold. [00377] In preparation for perfusion, the placenta can be oriented in such a manner that the umbilical artery and umbilical vein are located at the highest point of the placenta. The placenta can be perfused by passage of a perfusion solution through the placental vasculature, or through the placental vasculature and surrounding tissue. In one embodiment, the umbilical artery and the umbilical vein are connected simultaneously to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The perfusion solution is passed into the umbilical vein and artery. The perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation. The perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the maternal uterine wall. In another embodiment, the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins, that is, is passed through only the placental vasculature (fetal tissue). [00378] In one embodiment, for example, the umbilical artery and the umbilical vein are connected simultaneously, e.g., to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The perfusion solution is passed into the umbilical vein and artery. The perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation. The perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the maternal uterine wall. Placental cells that are collected by this method, which can be referred to as a “pan” method, are typically a mixture of fetal and maternal cells. [00379] In another embodiment, the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins. Placental cells collected by this method, which can be referred to as a “closed circuit” method, are typically almost exclusively fetal. [00380] The closed circuit perfusion method can, in one embodiment, be performed as follows. A post-partum placenta is obtained within about 48 hours after birth. The umbilical cord is clamped and cut above the clamp. The umbilical cord can be discarded, or can processed to recover, e.g., umbilical cord stem cells, and/or to process the umbilical cord membrane for the production of a biomaterial. The amniotic membrane can be retained during perfusion, or can be separated from the chorion, e.g., using blunt dissection with the fingers. If the amniotic membrane is separated from the chorion prior to perfusion, it can be, e.g., discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce, e.g., an amniotic membrane biomaterial, e.g., the biomaterial described in U.S. Application Publication No.2004/0048796. After cleaning the placenta of all visible blood clots and residual blood, e.g., using sterile gauze, the umbilical cord vessels are exposed, e.g., by partially cutting the umbilical cord membrane to expose a cross-section of the cord. The vessels are identified, and opened, e.g., by advancing a closed alligator clamp through the cut end of each vessel. The apparatus, e.g., plastic tubing connected to a perfusion device or peristaltic pump, is then inserted into each of the placental arteries. The pump can be any pump suitable for the purpose, e.g., a peristaltic pump. Plastic tubing, connected to a sterile collection reservoir, e.g., a blood bag such as a 250 mL collection bag, is then inserted into the placental vein. Alternatively, the tubing connected to the pump is inserted into the placental vein, and tubes to a collection reservoir(s) are inserted into one or both of the placental arteries. The placenta is then perfused with a volume of perfusion solution, e.g., about 750 ml of perfusion solution. Cells in the perfusate are then collected, e.g., by centrifugation. [00381] In one embodiment, the proximal umbilical cord is clamped during perfusion, and, more specifically, can be clamped within 4-5 cm (centimeter) of the cord’s insertion into the placental disc. [00382] The first collection of perfusion fluid from a mammalian placenta during the exsanguination process is generally colored with residual red blood cells of the cord blood and/or placental blood. The perfusion fluid becomes more colorless as perfusion proceeds and the residual cord blood cells are washed out of the placenta. Generally from 30 to 100 mL of perfusion fluid is adequate to initially flush blood from the placenta, but more or less perfusion fluid may be used depending on the observed results. [00383] In certain embodiments, cord blood is removed from the placenta prior to perfusion (e.g., by gravity drainage), but the placenta is not flushed (e.g., perfused) with solution to remove residual blood. In certain embodiments, cord blood is removed from the placenta prior to perfusion (e.g., by gravity drainage), and the placenta is flushed (e.g., perfused) with solution to remove residual blood. [00384] The volume of perfusion liquid used to perfuse the placenta may vary depending upon the number of placental cells to be collected, the size of the placenta, the number of collections to be made from a single placenta, etc. In various embodiments, the volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is perfused with 700-800 mL of perfusion liquid following exsanguination. [00385] The placenta can be perfused a plurality of times over the course of several hours or several days. Where the placenta is to be perfused a plurality of times, it may be maintained or cultured under aseptic conditions in a container or other suitable vessel, and perfused with a cell collection composition, or a standard perfusion solution (e.g., a normal saline solution such as phosphate buffered saline (“PBS”) with or without an anticoagulant (e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g., β- mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 0.5 μg/ml). In one embodiment, an isolated placenta is maintained or cultured for a period of time without collecting the perfusate, such that the placenta is maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days before perfusion and collection of perfusate. The perfused placenta can be maintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a second time with, e.g., 700-800 mL perfusion fluid. The placenta can be perfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In one embodiment, perfusion of the placenta and collection of perfusion solution, e.g., placental cell collection composition, is repeated until the number of recovered nucleated cells falls below 100 cells/ml. The perfusates at different time points can be further processed individually to recover time-dependent populations of cells, e.g., total nucleated cells. Perfusates from different time points can also be pooled. 5.5.4. Placental Perfusate and Placental Perfusate Cells [00386] Typically, placental perfusate from a single placental perfusion comprises about 100 million to about 500 million nucleated cells, including hematopoietic cells from which NK cells and/or ILC3 cells, e.g., NK cells and/or ILC3 cells produced according to the three-stage method described herein, may be produced by the method disclosed herein. In certain embodiments, the placental perfusate or perfusate cells comprise CD34+ cells, e.g., hematopoietic stem or progenitor cells. Such cells can, in a more specific embodiment, comprise CD34+CD45– stem or progenitor cells, CD34+CD45+ stem or progenitor cells, or the like. In certain embodiments, the perfusate or perfusate cells are cryopreserved prior to isolation of hematopoietic cells therefrom. 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. 5.6. NK Cells 5.6.1. NK Cells Produced by Three-Stage Method [00387] In another embodiment, provided herein is an isolated NK cell population, wherein said NK cells are produced according to the three-stage method described above. [00388] In one embodiment, provided herein is an isolated NK cell population produced by a three-stage method described herein, wherein said NK cell population comprises a greater percentage of CD3–CD56+ cells than an NK progenitor cell population produced by a three-stage method described herein, e.g., an NK progenitor cell population produced by the same three-stage method with the exception that the third culture step used to produce the NK progenitor cell population was of shorter duration than the third culture step used to produce the NK cell population. In a specific embodiment, said NK cell population comprises about 70% or more, in some embodiments, 75%, 80%, 85%, 90%, 95%, 98%, or 99% CD3–CD56+ cells. In another specific embodiment, said NK cell population comprises no less than 80%, 85%, 90%, 95%, 98%, or 99% CD3–CD56+ cells. In another specific embodiment, said NK cell population comprises between 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-99% CD3–CD56+ cells. [00389] In certain embodiments, said CD3–CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally NKp46+. In certain embodiments, said CD3– CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally CD16-. In certain embodiments, said CD3–CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally CD16+. In certain embodiments, said CD3–CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally CD94-. In certain embodiments, said CD3–CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally CD94+. In certain embodiments, said CD3–CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally CD11a+. In certain embodiments, said CD3– CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally NKp30+. In certain embodiments, said CD3–CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally CD161+. In certain embodiments, said CD3–CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally DNAM-1+. In certain embodiments, said CD3–CD56+ cells in said NK cell population comprises CD3–CD56+ cells that are additionally T-bet+. [00390] In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are CD117+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are NKG2D+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are NKp44+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are CD244+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which express perforin. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which express EOMES. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which express granzyme B. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which secrete IFNγ, GM-CSF and/or TNFα. 5.7. ILC3 Cells 5.7.1. ILC3 Cells Produced by Three-Stage Method [00391] In another embodiment, provided herein is an isolated ILC3 cell population, wherein said ILC3 cells are produced according to the three-stage method described above. [00392] In one embodiment, provided herein is an isolated ILC3 cell population produced by a three-stage method described herein, wherein said ILC3 cell population comprises a greater percentage of CD3–CD56+ cells than an ILC3 progenitor cell population produced by a three- stage method described herein, e.g., an ILC3 progenitor cell population produced by the same three-stage method with the exception that the third culture step used to produce the ILC3 progenitor cell population was of shorter duration than the third culture step used to produce the ILC3 cell population. In a specific embodiment, said ILC3 cell population comprises about 70% or more, in some embodiments, 75%, 80%, 85%, 90%, 95%, 98%, or 99% CD3–CD56+ cells. In another specific embodiment, said ILC3 cell population comprises no less than 80%, 85%, 90%, 95%, 98%, or 99% CD3–CD56+ cells. In another specific embodiment, said ILC3 cell population comprises between 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%- 99% CD3–CD56+ cells. [00393] In certain embodiments, said CD3–CD56+ cells in said ILC3 cell population comprises CD3–CD56+ cells that are additionally NKp46–. In certain embodiments, said CD3– CD56+ cells in said ILC3 cell population comprises CD3–CD56+ cells that are additionally CD16-. In certain embodiments, said CD3–CD56+ cells in said ILC3 cell population comprises CD3–CD56+ cells that are additionally IL1R1+. In certain embodiments, said CD3–CD56+ cells in said ILC3 cell population comprises CD3–CD56+ cells that are additionally CD94-. In certain embodiments, said CD3–CD56+ cells in said ILC3 cell population comprises CD3–CD56+ cells that are additionally RORγt+. In certain embodiments, said CD3–CD56+ cells in said ILC3 cell population comprises CD3–CD56+ cells that are additionally CD11a–. In certain embodiments, said CD3–CD56+ cells in said ILC3 cell population comprises CD3–CD56+ cells that are additionally T-bet+. [00394] In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are CD117+. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are NKG2D–. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are NKp30–. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are CD244+. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are DNAM-1+. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which express AHR. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which do not express perforin. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which do not express EOMES. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which do not express granzyme B. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which secrete IL-22 and/or IL-8. [00395] In certain aspects, cell populations produced by the three-stage method described herein comprise CD11a+ cells and CD11a– cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 50:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 20:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 10:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 5:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:1. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:5. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:10. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:20. In certain aspects, a population of cells described herein comprises CD11a+ cells and CD11a– cells in a ratio of 1:50. [00396] In certain aspects, cell populations described herein are produced by combining the CD11a+ cells with the CD11a– cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50 to produce a combined population of cells. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 50:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 20:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 10:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 5:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:1. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:5. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:10. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:20. In certain aspects, a combined population of cells described herein comprises CD11a+ cells and CD11a– cells combined in a ratio of 1:50. [00397] In certain aspects, cell populations produced by the three-stage method described herein comprise NK cells and ILC3 cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 50:1. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 20:1. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 10:1. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 5:1. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:1. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:5. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:10. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:20. In certain aspects, a population of cells described herein comprises NK cells and ILC3 cells in a ratio of 1:50. [00398] In certain aspects, cell populations described herein are produced by combining the NK cells with the ILC3 cells in a ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50 to produce a combined population of cells. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 50:1. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 20:1. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 10:1. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 5:1. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:1. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:5. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:10. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:20. In certain aspects, a combined population of cells described herein comprises NK cells and ILC3 cells combined in a ratio of 1:50. 5.8. Compositions Comprising NK Cells and/or ILC3 Cells 5.8.1. NK Cells and/or ILC3 Cells Produced Using The Three-Stage Method [00399] In some embodiments, provided herein is a composition, e.g., a pharmaceutical composition, comprising an isolated NK cell and/or ILC3 cell population produced using the three-stage method described herein. In a specific embodiment, said isolated NK cell and/or ILC3 cell population is produced from hematopoietic cells, e.g., hematopoietic stem or progenitor cells isolated from placental perfusate, umbilical cord blood, and/or peripheral blood. In another specific embodiment, said isolated NK cell and/or ILC3 cell population comprises at least 50% of cells in the composition. In another specific embodiment, said isolated NK cell and/or ILC3 cell population, e.g., CD3–CD56+ cells, comprises at least 80%, 85%, 90%.95%, 98% or 99% of cells in the composition. In certain embodiments, no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the cells in said isolated NK cell and/or ILC3 cell population are CD3–CD56+ cells. In certain embodiments, said CD3–CD56+ cells are CD16-. [00400] NK cell and/or ILC3 cell populations produced using the three-stage method described herein, can be formulated into pharmaceutical compositions for use in vivo. Such pharmaceutical compositions comprise a population of NK cells and/or ILC3 cells in a pharmaceutically-acceptable carrier, e.g., a saline solution or other accepted physiologically- acceptable solution for in vivo administration. Pharmaceutical compositions of the invention can comprise any of the NK cell and/or ILC3 cell populations described elsewhere herein. [00401] The pharmaceutical compositions of the invention comprise populations of cells that comprise 50% viable cells or more (that is, at least 50% of the cells in the population are functional or living). Preferably, at least 60% of the cells in the population are viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the cells in the population in the pharmaceutical composition are viable. [00402] The pharmaceutical compositions of the invention can comprise one or more compounds that, e.g., facilitate engraftment; stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, and the like. [00403] When formulated as an injectable solution, in one embodiment, the pharmaceutical composition of the invention comprises about 1.25% HSA and about 2.5% dextran. Other injectable formulations, suitable for the administration of cellular products, may be used. [00404] In one embodiment, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for systemic or local administration. In specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for parenteral administration. In specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for injection, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor administration. In specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for administration via a device, a matrix, or a scaffold. In specific embodiments, the compositions, e.g., pharmaceutical compositions provided herein are suitable for injection. In specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for administration via a catheter. In specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for local injection. In more specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for local injection directly into a solid tumor (e.g., a sarcoma). In specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for injection by syringe. In specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for administration via guided delivery. In specific embodiments, the compositions, e.g., pharmaceutical compositions, provided herein are suitable for injection aided by laparoscopy, endoscopy, ultrasound, computed tomography, magnetic resonance, or radiology. [00405] In certain embodiments, the compositions, e.g., pharmaceutical compositions provided herein, comprising NK cells and/or ILC3 cells produced using the methods described herein, are provided as pharmaceutical grade administrable units. Such units can be provided in discrete volumes, e.g., 15 mL, 20 mL, 25 mL, 30 nL.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 so as to contain a specified number of cells, e.g., NK cells and/or ILC3 cells, e.g., 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 can comprise about, at least about, or at most about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106 or more NK cells and/or ILC3 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 specified numbers of NK cells and/or ILC3 cells or NK cell and/or ILC3 cell populations and/or any of the other cells. In specific embodiments, the NK cells and ILC3 cells are present in ratios provided herein. [00406] In another specific embodiment, said isolated NK cells and/or ILC3 cells in said composition are from a single individual. In a more specific embodiment, said isolated NK cells and/or ILC3 cells comprise NK cells and/or ILC3 cells from at least two different individuals. In another specific embodiment, said isolated NK cells and/or ILC3 cells in said composition are from a different individual than the individual for whom treatment with the NK cells and/or ILC3 cells is intended. In another specific embodiment, said NK cells have been contacted or brought into proximity with an immunomodulatory compound or thalidomide in an amount and for a time sufficient for said NK cells to express detectably more granzyme B or perforin than an equivalent number of natural killer cells, i.e. NK cells not contacted or brought into proximity with said immunomodulatory compound or thalidomide. In another specific embodiment, said composition additionally comprises an immunomodulatory compound or thalidomide. In certain embodiments, the immunomodulatory compound is a compound described below. See, e.g., U.S. Patent No.7,498,171, the disclosure of which is hereby incorporated by reference in its entirety. In certain embodiments, the immunomodulatory compound is an amino-substituted isoindoline. In one embodiment, the immunomodulatory compound is 3-(4-amino-1-oxo-1,3-dihydroisoindol- 2-yl)-piperidine-2,6-dione; 3-(4'aminoisolindoline-1'-one)-1-piperidine-2,6-dione; 4-(amino)-2- (2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; or 4-Amino-2-(2,6-dioxopiperidin-3-yl)isoindole- 1,3-dione. In another embodiment, the immunomodulatory compound is pomalidomide, or lenalidomide. In another embodiment, said immunomodulatory compound is a compound having the structure
Figure imgf000110_0001
, wherein one of X and Y is C=O, the other of X and Y is C=O or CH2 , and R2 is hydrogen or lower alkyl, or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. In another embodiment, said immunomodulatory compound is a compound having the structure
Figure imgf000111_0001
wherein one of X and Y is C=O and the other is CH2 or C=O; R1 is H, (C1–C8 )alkyl, (C3–C7)cycloalkyl, (C2–C8)alkenyl, (C2–C8)alkynyl, benzyl, aryl, (C0-C4)alkyl–(C1-C6)heterocycloalkyl, (C0-C4)alkyl–(C2-C5)heteroaryl, C(O)R3, C(S)R3, C(O)OR4, (C1–C8)alkyl–N(R6)2, (C1–C8)alkyl–OR5, (C1–C8)alkyl–C(O)OR5, C(O)NHR3, C(S)NHR3, C(O)NR3R3’, C(S)NR3R3’ or (C1–C8)alkyl–O(CO)R5; R2 is H, F, benzyl, (C1-C8)alkyl, (C2-C8)alkenyl, or (C2-C8)alkynyl; R3 and R3’ are independently (C1-C8)alkyl, (C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2- C8)alkynyl, benzyl, aryl, (C0-C4)alkyl–(C1-C6)heterocycloalkyl, (C0-C4)alkyl–(C2-C5)heteroaryl, (C0-C8)alkyl–N(R6)2, (C1-C8)alkyl–OR5, (C1-C8)alkyl–C(O)OR5, (C1-C8)alkyl–O(CO)R5, or C(O)OR5; R4 is (C1–C8)alkyl, (C2–C8)alkenyl, (C2–C8)alkynyl, (C1-C4)alkyl–OR5, benzyl, aryl, (C0- C4)alkyl–(C1-C6)heterocycloalkyl, or (C0-C4)alkyl–(C2-C5)heteroaryl; R5 is (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl, or (C2-C5)heteroaryl; each occurrence of R6 is independently H, (C1–C8)alkyl, (C2–C8)alkenyl, (C2–C8)alkynyl, benzyl, aryl, (C2-C5)heteroaryl, or (C0-C8)alkyl–C(O)O–R5 or the R6 groups can join to form a heterocycloalkyl group; n is 0 or 1; and * represents a chiral-carbon center; or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. In another embodiment, said immunomodulatory compound is a compound having the structure R R
Figure imgf000112_0001
wherein: one of X and Y is C=O and the other is CH2 or C=O; R is H or CH2OCOR’; (i) each of R1, R2, R3, or R4, independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R1, R2, R3, or R4 is nitro or -NHR5 and the remaining of R1, R2, R3, or R4 are hydrogen; R5 is hydrogen or alkyl of 1 to 8 carbons R6 hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro; R’ is R7-CHR10-N(R8R9); R7 is m-phenylene or p-phenylene or -(CnH2n)- in which n has a value of 0 to 4; each of R8 and R9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R8 and R9 taken together are tetramethylene, pentamethylene, hexamethylene, or -CH2CH2X1CH2CH2– in which X1 is -O-, -S-, or -NH-; R10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and * represents a chiral-carbon center; or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. [00407] In another specific embodiment, the composition additionally comprises one or more anticancer compounds, e.g., one or more of the anticancer compounds described below. [00408] In a more specific embodiment, the composition comprises NK cells and/or ILC3 cells from another source, or made by another method. In a specific embodiment, said other source is placental blood and/or umbilical cord blood. In another specific embodiment, said other source is peripheral blood. In more specific embodiments, the NK cell and/or ILC3 cell population in said composition is combined with NK cells and/or ILC3 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 the like. [00409] In another specific embodiment, the composition comprises an NK cell and/or ILC3 cell population produced using the three-stage method described herein and either isolated placental perfusate or isolated placental perfusate cells. In a more specific embodiment, said placental perfusate is from the same individual as said NK cell and/or ILC3 cell population. In another more specific embodiment, said placental perfusate comprises placental perfusate from a different individual than said NK cell and/or ILC3 cell population. In another specific embodiment, all, or substantially all (e.g., greater than 90%, 95%, 98% or 99%) of cells in said placental perfusate are fetal cells. In another specific embodiment, the placental perfusate or placental perfusate cells, comprise fetal and maternal cells. In a more specific embodiment, the fetal cells in said placental perfusate comprise less than about 90%, 80%, 70%, 60% or 50% of the cells in said perfusate. In another specific embodiment, said perfusate is obtained by passage of a 0.9% NaCl solution through the placental vasculature. In another specific embodiment, said perfusate comprises a culture medium. In another specific embodiment, said perfusate has been treated to remove erythrocytes. In another specific embodiment, said composition comprises an immunomodulatory compound, e.g., an immunomodulatory compound described below, e.g., an amino-substituted isoindoline compound. In another specific embodiment, the composition additionally comprises one or more anticancer compounds, e.g., one or more of the anticancer compounds described below. [00410] In another specific embodiment, the composition comprises an NK cell and/or ILC3 cell population and placental perfusate cells. In a more specific embodiment, said placental perfusate cells are from the same individual as said NK cell and/or ILC3 cell population. In another more specific embodiment, said placental perfusate cells are from a different individual than said NK cell and/or ILC3 cell population. In another specific embodiment, the composition comprises isolated placental perfusate and isolated placental perfusate cells, wherein said isolated perfusate and said isolated placental perfusate cells are from different individuals. In another more specific embodiment of any of the above embodiments comprising placental perfusate, said placental perfusate comprises placental perfusate from at least two individuals. In another more specific embodiment of any of the above embodiments comprising placental perfusate cells, said isolated placental perfusate cells are from at least two individuals. In another specific embodiment, said composition comprises an immunomodulatory compound. In another specific embodiment, the composition additionally comprises one or more anticancer compounds, e.g., one or more of the anticancer compounds described below. [00411] 6. KITS [00412] Provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the compositions described herein, e.g., a composition comprising NK cells and/or ILC3 cells produced by a method described herein, e.g., NK cell and/or ILC3 cell populations produced using the three-stage method described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. [00413] The kits encompassed herein can be used in accordance with the methods described herein, e.g., methods of suppressing the growth of tumor cells and/or methods of treating cancer, e.g., hematologic cancer, and/or methods of treating viral infection. In one embodiment, a kit comprises NK cells and/or ILC3 cells produced by a method described herein or a composition thereof, in one or more containers. In a specific embodiment, provided herein is a kit comprising an NK cell and/or ILC3 cell population produced by a three-stage method described herein, or a composition thereof. 7. EXAMPLES 7.1. Example 1: Three-stage method of producing natural killer cells from hematopoietic stem or progenitor cells [00414] CD34+ cells are cultured in the following medium formulations for the indicated number of days, and aliquots of cells are taken for assessment of cell count, cell viability, characterization of natural killer cell differentiation and functional evaluation. [00415] Stage 1 medium: 90% Stem Cell Growth Medium (SCGM) (CellGro®), 10% Human Serum-AB, supplemented with 25 ng/mL or 250 ng/mL recombinant human thrombopoietin (TPO), 25 ng/mL recombinant human Flt3L, 27 ng/mL recombinant human stem cell factor (SCF), 25 ng/mL recombinant human IL-7, 0.05 ng/mL or 0.025 ng/mL recombinant human IL-6, 0.25 ng/mL or 0.125 ng/mL recombinant human granulocyte colony-stimulating factor (G-CSF), 0.01 ng/mL or 0.025 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), 0.10% gentamicin, and 1 to 10µm StemRegenin-1 (SR-1) or other stem cell mobilizing agent. [00416] Stage 2 medium: 90% SCGM, 10% Human Serum-AB, supplemented with 25 ng/mL recombinant human Flt3L, 27 ng/mL recombinant human SCF, 25 ng/mL recombinant human IL-7, 20 ng/mL recombinant human IL-15, 0.05 ng/mL or 0.025 ng/mL recombinant human IL-6, 0.25 ng/mL or 0.125 ng/mL recombinant human granulocyte colony-stimulating factor (G-CSF), 0.01 ng/mL or 0.025 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), 0.10% gentamicin, and 1 to 10µm SR1 or other stem cell mobilizing agent. [00417] Stage 3 medium: 90% STEMMACSTM, 10% Human Serum-AB, 0.025 mM 2- mercaptoethanol (55 mM), supplemented with 22 ng/mL recombinant human SCF, 1000 U/mL recombinant human IL-2, 20 ng/mL recombinant human IL-7, 20 ng/mL recombinant human IL- 15, 0.05 ng/mL or 0.025 ng/mL recombinant human IL-6, 0.25 ng/mL or 0.125 ng/mL recombinant human granulocyte colony-stimulating factor (G-CSF), 0.01 ng/mL or 0.025 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), and 0.10% gentamicin. [00418] Cells are seeded at Day 0 at 3×104 cells/mL in Stage 1 media, and cells are tested for purity by a CD34+ and CD45+ count and viability by 7AAD staining. At Day 5 cells are counted and seeded to a concentration of 1×105 cells/mL with Stage 1 medium. At Day 7 cells are counted and seeded to a concentration of 1×105 cells/mL with Stage 1 medium. [00419] At Day 10, cells are counted and seeded to a concentration of 1×105 cells/mL in Stage 2 medium. At Day 12, cells are counted and seeded to a concentration of 3×105 cells/mL in Stage 2 medium. At Day 14, cells are counted and seeded in Stage 3 medium. Cells are maintained in Stage 3 media until day 35. [00420] Alternatively, the following protocol is used through Day 14: Cells seeded at Day 0 at 7.5×103 cells/mL in Stage 1 media, and cells are tested for purity by a CD34+ and CD45+ count and viability by 7AAD staining. At Day 7 cells are counted and seeded to a concentration of 3×105 cells/mL with Stage 1 medium. At Day 9 cells are counted and seeded to a concentration of 3×105 cells/mL with Stage 2 medium. At Day 12, cells are counted and seeded to a concentration of 3×105 cells/mL in Stage 2 medium. At Day 14, cells are counted and seeded to a concentration of 3×105 cells/mL in Stage 2 medium. [00421] Seeding of cells into at passage is performed either by dilution of the culture with fresh media or by centrifugation of cells and resuspension / addition of fresh media. [00422] For harvest, cells are spun at 400×g for seven minutes, followed by suspension of the pellet in an equal volume of Plasmalyte A. The suspension is spun at 400×g for seven minutes, and the resulting pellet is suspended in 10% HSA (w/v), 60% Plasmalyte A (v/v) at the target cell concentration. The cells are then strained through a 70 µm mesh, the final container is filled, an aliquot of the cells are tested for viability, cytotoxicity, purity, and cell count, and the remainder is packaged. 7.2. Example 2: Selection of stem cell mobilizing agents for the expansion of NK cells [00423] The following compounds were investigated for their ability to promote the expansion of NK cell populations in vitro: 4-(2-((2-(benzo[b]thiophen-3-yl)-6-(isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol) (“CRL1”)
Figure imgf000116_0001
4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol)) (“CRL2”)
Figure imgf000117_0001
2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-5,7-dihydro-6H- pyrrolo[2,3-d]pyrimidin-6-one (“CRL4”) 3
Figure imgf000117_0002
4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4- yl)amino)ethyl)phenol (“CRL6”) 5
Figure imgf000118_0001
N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4-amine (“CRL8”) N
Figure imgf000118_0002
3-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide (“CRL10”)
Figure imgf000119_0001
2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3- d]pyrimidine-5-carbonitrile (“CRL14”)
Figure imgf000120_0001
N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine (“CRL15”)
Figure imgf000120_0002
4-(2-((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-yl)amino)ethyl)phenol (“CRL16”)
Figure imgf000120_0003
5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7-isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile (“CRL17”)
Figure imgf000121_0001
N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine (“CRL18”) N
Figure imgf000121_0003
N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine (“CRL20”)
Figure imgf000121_0002
N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine (“CRL21”) 5
Figure imgf000122_0001
. 7.3. Example 3: Characterization of three-stage NK cells METHODS [00424] UCB CD34+ cells were cultivated in presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 for 35 days to produce three-stage NK cells, as described in Example 1. Multi-color flow cytometry was used to determine the phenotypic characteristics of three-stage NK cells. [00425] For biological testing, the compounds were provided to culture to evaluate their effects on NK cell expansion and differentiation. Specifically, donors of CD34+ cells (StemCell Technology) were thawed and expanded in vitro following NK culture protocol. During the first 14 days of the culture, each CRL compounds was dissolved in DMSO and added to the culture at 10 µM concentration. SR1 (at 10 µM) served as a positive control compound, while DMSO alone without any compound served as a negative control. At the end of the culture on Day 35, cell expansion, natural killer (NK) cell differentiation and cytotoxicity of the cells against K562 tumor cell line were characterized. Due to the large number of the compounds, the testing was performed in two experiments, CRL1-11 and CRL 12-22. The same donors were used for each experiment. Positive and negative controls were also included in both experiments. Results [00426] Cell expansion data showed that 20 out of the 22 compounds supported NK expansion at 10 µM concentration. Except for CRL7 and CRL13, the rest of the compounds all resulted in a NK expansion of 2,000 ~ 15,000 fold over 35 days (FIG.1 and FIG.2). Among all the compounds, CRL19, 20 and 22 supported cell expansion the best, and they demonstrated a similar level of expansion compared to SR1 at Day 35 (FIG.3). CD34 cell expansion at Day 14 of the culture showed a similar trend that most of the compounds supported CD34 cells expansion, and CRL19, 20 and 22 achieved the highest CD34 cell expansion at Day 14 (FIG.4). [00427] Cytotoxicity assay was run using compound cultured cells against K562 tumor cells at 10:1 effector to target ratio (FIG.5) to evaluate cell functions. The results showed that the cells cultured with compounds killed 30~60% of K562 cells at 10:1 E:T ratio, indicating that the cells present NK functions. For both donors, cells cultured with CRL17, 18, 19 and 21 demonstrated similar or greater killing activities compared to those cultured with SR1. Conclusions: [00428] In summary, we found that all the compounds except CRL7 and CRL13 supported PNK-007 expansion and differentiation. Expansion with the compounds ranged from 2,000 ~ 15, 000 fold over 35 days, and the culture achieved more than 70% of NK cells. Among these compounds, CRL 19, 20 and 22 demonstrated very similar expansion, differentiation and cytotoxicity profiles as SR1 for PNK-007 culture. CRL 17, 18, and 21 resulted in slightly less expansion compared to SR1 but increased CD56+/CD11a+ subpopulation, and also increased killing activities of the cells. 7.4 Example 4: Further characterization of three-stage NK cells METHODS [00429] Cells: Frozen PBMC were acquired from Stem Cell Technologies. Peripheral blood derived NKs (PB-NK) cells were isolated from fresh blood of healthy donors using the Human NK Cell Enrichment Kit (Stem Cell Technologies) according to manufacturer’s instructions. CYNK cells were generated from umbilical cord blood-derived CD34+ stem cells (Ref: Zhang et al. J Immunother Cancer.2015). Briefly, the CD34+ cells were cultivated in the presence of cytokines including thromobopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 for 35 days. PBNK and CYNK cells were cryopreserved until analysis. [00430] Magnetic-activated cell sorting: PNK cells were stained with PE Mouse Anti- Human CD11a (BD) and CD11a+ PNK cells concentrated using anti-PE MicroBeads according to manufacturer’s instructions (Miltenyi Biotec). [00431] Single cell RNA sequencing: CYNK cells were combined with PB-NK at 1:1 ratio and gene expression analyzed on single cell level using 10X Genomics Chromium platform and Illumina sequencing. Bioinformatics analysis utilized 10X Genomics Cell Ranger analysis pipeline. [00432] Flow Cytometry: Cryopreserved cells were rapidly thawed in a 37°C water bath and washed once in RPMI1640 + 10% hiFBS (heat inactivated Fetal Bovine Serum, Gibco), followed by LIVE/DEAD™ Fixable Aqua Stain in PBS. Cells were washed with FACS buffer (PBS + 2% FBS) followed by incubation in blocking solution (Brilliant Stain buffer, Mouse IgG2a isotype k control and Human BD Fc Block (all from BD)). Cells were washed with FACS buffer and incubated with fluorophore-coupled antibodies in FACS buffer for 25 min on ice. Cells were washed with FACS buffer before analysis on Fortessa X20 flow cytometer (BD). [00433] qRT-PCR: RNA was isolated from cells using Quick-RNA Miniprep kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) in a standard reaction. RT-PCR was performed using Taqman Gene expression assays (Applied Biosystems). Expression levels were calculated relative to GAPDH (Hs02758991) using the ΔΔCt method. RESULTS [00434] CYNK cells efficiently kill various tumor cell lines in vitro, however, the mechanisms CYNK cells use to induce cell death remains poorly understood (ref). To elucidate on the activating NK cell receptors, the intracellular signaling pathways and molecular mechanisms CYNK cells employ to carry out their functional roles, we used single-cell RNA sequencing (scRNAseq) as an unbiased approach to compare CYNK cells to peripheral blood NK cells (PB-NK) (FIG.6A). Unbiased transcriptional clustering revealed two distinct signatures differentiating between CYNK and PB-NK cells (FIG.6B). Tables 1 and 2 list top 50 upregulated genes per cluster in PB-NK and CYNK cells, respectively. The gene set expressed higher in PB- NK cells included genes associated with NK cell functional roles, including FGFBP2, granzymes (GZMH, GZMM), CXCR4, KLRF1, KLF2, IFNG (Table 1). ^ FGFBP2, encoding fibroblast growth factor-binding protein, is known to be secreted by cytotoxic lymphocytes. ^ Granzymes are a group of serine proteases which are stored in the cytotoxic granules of NK cells and cytotoxic T lymphocytes (ref). While GzmA and GzmB induce target cell death upon release to their cytoplasm and have been extensively studied, less is known about the functional role of GzmH, GzmK and GzmM. ^ CXCR4 regulates NK cell homing to bone marrow. ^ KLRF1 encodes NKp80, an activating C-type lectin-like immunoreceptor that is activated upon binding to activation-induced C-type lectin (AICL), inducing NK cell cytotoxicity and cytokine secretion. ^ Transcription factor KLF2 that regulates both NK cell proliferation and survival. ^ NK cell-derived IFN-γ (IFNG gene) is a key immunoregulatory factor secreted from activated NK cells that promotes adaptive immune response by modulating dendritic cell and T cell responses.
Figure imgf000125_0001
Figure imgf000126_0001
[00435] Top differentially expressed genes in CYNK cluster that are encode factors associated with NK cell functional role include surface receptors and co-receptors (CD96, NCR3, CD59, KLRC1), TNFSF10, immune checkpoint genes (TNFRSF18, TNFRSF4, HAVCR2), NK cell receptor adaptor molecule genes (FCER1G and LAT2) (Table 2). Table 2. Top 50 upregulated genes per CYNK cluster.
Figure imgf000127_0001
Figure imgf000128_0001
[00436] To better understand how the cytotoxic response is initiated in CYNK cells, we specifically analyzed the expression of manually chosen genes encoding well characterized proteins leading from target detection to a cytolytic response, with main focus on NK cell receptors and adaptor molecule (Table 3). Differential gene expression analysis showed high expression of the two key cytotoxic molecules perforin (PRF1) and granzyme B (GZMB) in CYNK cells. Similarly, most receptors that were differentially expressed between CYNK and PB- NK cells, with the exception of KLRF1 (encoding NKp80), were higher expressed on CYNK cells. Expression of selected NK cell effector and receptor genes is visualized on tSNE plots in FIG.6C. Elevated expression of genes encoding components of the NK cell cytotoxic machinery correlate well with the high cytotoxic activity of CYNK cells against a broad range of target cells.
Figure imgf000128_0002
Figure imgf000129_0001
[00437] We next analyzed the transcriptional profile of CYNK and PB-NK cells by quantitative real-time PCR (qRT-PCR) focusing on selected NK cell-associated genes that were highly and/or differentially expressed in the scRNAseq dataset (FIG.7). RNA was extracted from freshly thawed naïve cells post isolation or culture. qRT-PCR demonstrated high expression of CD69, KLRK1 and KLRB1 relative to the housekeeping gene GAPDH in both CYNK and PB- NK cells, whereas, KLRK1 and KLRB1, encoding for NKG2D and CD161/KLRB1, respectively, were significantly higher expressed in PB-NK cells. Significant differential expression of NKp80, encoded by KLRF1 gene, earlier seen by scRNAseq (Table 3), was confirmed by qRT-PCR. Similarly, KLRD1 was higher expressed on PB-NK compared to CYNK cells. Together, the data show higher expression of the inhibitory killer cell lectin-like receptor (KLRB1, KLRD1, KLRF1) expression on PB-NK cells when compared to CYNK cells. The two C-type lectin receptor genes KLRC1 and KLRC2, encoding the inhibitory NKG2A and the activating NKG2C, were higher expressed in CYNK cells. Of the natural cytotoxicity receptors (NCRs), only NCR2 (encoding NKp44) was differentially expressed with high expression in CYNK cells and almost no expression in PB-NK cells. Two co-activating NK cell receptor genes CD244 (2B4) and CD226 (DNAM-1) were slightly higher expressed in PB-NK compared to CYNK cells. Alongside the typical ligand-activated NK cell receptor genes, we also analyzed the expression of FCGR3A encoding an Fc receptor CD16 that is required for antibody-dependent cell-mediated cytotoxicity. Whereas scRNAseq data demonstrated no significant differential expression of FCGR3A, by qRT-PCR it was highly expressed in the PB-NK cells and at a very low level in CYNK cells. The expression of two genes TNFRSF18 and TNFSF10 that were highly differentially expressed by scRNAseq and elevated in the CYNK cluster, were also analyzed by qRT-PCR. The PCR data confirms high expression of these genes encoding for GITR and TRAIL, respectively, on CYNK cells relative to low level expression in PB-NK cells. [00438] Lastly, we characterized CYNK cells relative to PB-NK by surface protein expression using flow cytometry. Antibodies targeting various NK cell receptors were chosen based on the transcriptional characterization by scRNAseq and qRT-PCR (Tables 1-3, GIG.6 and FIG.7). NK cells express high level of the NK cell marker CD56 and lack the expression of T cell, B cell and myeloid cell markers CD3, CD19 and CD14, respectively (FIG.8). Whereas a majority of PB-NK cells express CD56 at a low level, a small subset of PB-NK cells express CD56 at a level seen in CYNK cells (FIG.9). NCR analysis demonstrated a high expression of NKp44 in CYNK cells, whereas, NKp44 was expressed at a low level in PB-NK, corresponding well to our transcriptional analysis (FIG.7). NKp80, on the other hand, was expressed on PB-NK cell and little on CYNK, also confirming the transcriptional data of KLRF1 expression (Table 1 and FIG.7). CD16 was virtually not expressed on CYNK cells, whereas the majority of PB-NK cells expressed CD16 at a high level. CD16 protein expression, therefore, also corresponds well to transcriptional analysis (Table 1 and FIG.7). The expression of killer cell lectin-like receptors was comparable between CYNK and PB-NK cells, with CYNK cells demonstrating higher mean fluorescence intensity compared to PB-NK cells for NKG2D, NKG2C, CD94 (NKG2C) and NKG2A. GITR, a checkpoint inhibitor molecule, encoded by TNFRSF18, was not expressed on PB-NK cells but highly on all CYNK cells, correlating well to qRT-PCR data. [00439] We used the flow cytometry dataset (FIG.8 and FIG.9) to perform an unbiased analysis of the surface marker expression on CYNK and PB-NK cell populations (FIG.10). Antibody-stained CYNK and PBMC cells were mixed for acquisition and analyzed by flow cytometry. It is evident from the tSNE plots that CYNK and PB-NK cells cluster separately from each other and other peripheral blood cells when looking at the localization of CD56- and CD3/CD14/CD19-positive cells on the plot. High expression of NKp44 (CD336) and GITR (CD357) enable the identification of CYNK cells as GITR is virtually not expressed in any cell type in the PBMC subsets. PB-NK cells on the other hand, highly express CD16 and NKp80 that are not expressed on CYNK cells. Altogether, we have identified cell surface markers that allow to distinguish CYNK cells from PB-NK with high confidence. 7.5 Example 5: Treatment of AML Table 4: Synopsis of protocol
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
1. TRIAL OBJECTIVES AND PURPOSE [00440] Primary Objective: The primary objective of the proposed study is to determine the maximum tolerated dose (MTD) or maximum planned dose (MPD) of CYNK-001 and to assess the safety of multiple infusions of CYNK-001 administered using a flat, non-weight based dose with or without rhIL-2 as assessed by the frequency and severity of adverse events (AE). [00441] Secondary Objectives: The secondary objective is to assess the clinical efficacy of CYNK-001 in AML subjects in Morphological CR with or without hematological recovery by assessing the MRD Response [conversion from MRD positive (i.e. MRD ≥ 0.1%) to MRD negative (i.e. no MRD identified, 0% blasts) or MRD indeterminate as measured by multiparameter flow cytometry (MFC) with assay lower limit of detection at 1:104 or lower, time to MRD Response, duration of MRD Response, progression-free survival (PFS), duration of morphologic CR, time to progression (TTP), and overall survival (OS). [00442] For the R/R subjects, the secondary objectives are to assess Overall Response Rate which consists of Complete Remission (CR), Complete Remission with incomplete hematologic recovery (CRi) and Morphologic leukemic-free state (MLFS) (CR + CRi+ MLFS) in the study Additional secondary endpoints for subjects with relapsed or refractory AML include Duration of Response (DoR), and Overall Survival (OS). [00443] Exploratory Objectives: Exploratory objectives include monitoring immune reconstitution following CYNK-001 dosing, in vivo persistence and expansion of CYNK-001 cells during treatment and up to 60 days following the first CYNK-001 infusion, characterization of immune cell populations in the bone marrow and peripheral blood, serum analysis of immune correlates, alloreactivity characterization, anti-HLA antibody analysis, and transcriptome analysis of bone marrow immune microenvironment. Depth of MRD Response (for both MRD positive population and R/R population who achieve CR, CRi, or MLFS) with CYNK-001 treatment will be evaluated. In the R/R AML population, changes in blast count will be assessed. Aggregated data will be used to determine biomarker correlations to MRD Response. 2. INVESTIGATIONAL PLAN 2.1. Overall Study Design [00444] The proposed study will enroll and treat up to approximately 94 subjects, which includes the option of further dose escalation based on the DMC recommendation. The study is divided into 3 study periods: Treatment Eligibility Period, Treatment Period, and Follow-up Period. Subjects will have a BMA Collection to determine AML disease status either following signing the Pre-Screening BMA Collection ICF prior to the potential subject signing the main Study ICF, or after signing the main Study ICF as part of the collective Treatment Eligibility screening activities. Each period has associated evaluations and procedures that must be performed at specific timepoints. [00445] Subject participation is dependent on slot availability based on time of entry into the study. Error! Reference source not found. shows the cohort escalation which includes both the MRD Positive and R/R Subject population. Dose escalation as outlined in Error! Reference source not found. will follow a standard 3+3 design, with 2 separate arms (MRD+ arm and R/R arm) starting with Cohort 4. Starting with Cohorts 5a and 5b rhIL-2 will be administered in combination with CYNK-001 cells. The decision to administer rhIL-2 in Cohorts 6a, 6b, 7a and 7b will be based on review of safety, efficacy, and translational data in Cohorts 5a and 5b, respectively, after thorough review and recommendation from the DMC and based on Sponsor decision. [00446] Pre-Screening BMA Collection (not required): Clinical sites have the option to collect and screen BMA samples prior to subjects signing the main Study ICF, approximately 2 weeks prior to the start of the treatment eligibility screening period. Subjects must consent to the Pre-Screening BMA Collection by signing the Pre-Screening BMA Collection ICF. Thereafter, pre-screening BMA will be collected and sent to the MRD central laboratory for flow cytometric analysis. A local morphological assessment should be performed as well. [00447] Treatment Eligibility Screening Period: If the pre-screening BMA collection was not previously collected as part of pre-screening BMA Collection, this BMA collection will be done as part of main Treatment Eligibility Screening after signing the main Study ICF. [00448] MRD positive subjects: The Treatment Eligibility Screening Period is defined as the period from Day -28 to Day -7 (or Day -6 for Cohorts 1, 2, and 3 only) in which subjects will be confirmed to be in morphological CR (or CRi, MLFS) with MRD positivity as measured on BMA by MFC and assessed for inclusion/exclusion criteria to confirm eligibility to participate in the study. For the purposes of this study, MRD positivity is defined as greater than or equal to 0.1% blasts detected by MFC on BMA by the Sponsor-selected Central MRD analysis laboratory, where assay sensitivity allows for a Lower Limit of Detection (LOD) of 1 x 10-4 (i.e., 0.01%) or lower. [00449] R/R subjects: The Treatment Eligibility Screening Period is defined as the period from Day -28 to Day -7 in which subjects will have a confirmed diagnosis of R/R AML based on local disease assessment. [00450] During this period, a 14-day washout of prior chemotherapy will occur before each subject receives the Lymphodepletion Regimen (Cy-Flu). Consultation with the Medical Monitor is encouraged when determining appropriate washout period. Treatment Eligibility Criteria are provided. A Treatment Eligibility Checklist will be provided to aide in confirmation of eligibility. [00451] Only AEs associated with study-related procedures which are not considered standard of care will be reported in this study period. [00452] Subjects may be rescreened for Treatment Eligibility up to two times for a total of 3 screens at a maximum. Subjects who are found to have relapsed upon central MRD analysis would be eligible to screen for R/R cohorts dependent on slot availability. [00453] Central BMA analysis for R/R subjects is not required for eligibility, however, a sample will be obtained for analyses. R/R subjects who have had local BMA assessment confirming R/R disease within 1 month prior to signing the main ICF are eligible. [00454] Treatment Period: The Treatment Period will consist of 2 parts: [00455] Part 1: Lymphodepletion Regimen (Cy-Flu) starting on Study Day -6 or -5 through Study Day -3, followed by two days with no treatment on Study Days -2 and -1. Mesna shall be administered, for Cohorts 4 and above, on days of Lymphodepletion for the inhibition of hemorrhagic cystitis induced by cyclophosphamide. Route of administration, dosage, and frequency of Mesna should be based on institutional standards. o Cy 300 Flu 25 Regimen (Cohorts 1, 2, 3 only): Cyclophosphamide dose is 300 mg/m2 to be administered on Study Days -5, -4, and -3 Fludarabine dose is 25 mg/m2 to be administered on Study Days -5, -4, and -3 o Cy 900 Flu 30 Regimen (all other cohorts): Cyclophosphamide dose is 900 mg/m2 to be administered on Study Days -6, -5, -4, and -3 Fludarabine dose is 30 mg/m2 to be administered on Study Days -6, -5, -4, and -3 [00456] Subjects enrolled to the expansion portion of a treatment arm may receive Cy 900 Flu 30 for 3 days only (-5, -4 and -3) per Sponsor decision based on analysis of safety, efficacy and translational data. [00457] Part 2: o CYNK-001 Treatment period begins on Study Day 0 with the first of up to 4 CYNK-001 IV infusions. Three CYNK-001 infusions occur on Study Days 0, 7, and 14, followed by 2 weeks of no treatment for Cohorts 1 through 5 and Study Days 1, 7, 14, and 21 for Cohorts 6a, 6b, 7a and 7b followed by one week of no treatment through the end of the 28-day DLT period o rhIL-2 Treatment: Subjects treated in Cohorts 5a and 5b will receive rhIL-2 injections SC at least 1 to 3 hours prior CYNK-001 infusion. Treatment with 6M IU rhIL-2 begins on the day of the first CYNK-001 infusion with the first of 7 total rhIL-2 injections. rhIL-2 injections should occur on each CYNK-001 infusion day and every other day in between CYNK-001 infusions (Days 0, 2, 4, 7, 9, 11, and 14). On non-CYNK-001 days, rhIL-2 may be delayed or skipped due to logistical constraints or adverse events at the PI’s discretion. rhIL-2 must not be administered on 2 consecutive days. Consultation with the Medical Monitor is encouraged when determining the rhIL-2 dosing schedule. The use of rhIL-2 in Cohorts 6a, 6b, 7a and 7b will be determined based on careful review of safety, efficacy, and translational data from subjects treated in Cohorts 5a and 5b. Those subjects will receive injections on the same days as Cohorts 5a and 5b and have one additional rhIL-2 injection on Day 21. The decision to include rhIL-2 will be based on DMC recommendation and Sponsor decision. o Pre- and Post- medications: Pre- and post-medication of acetaminophen 650 mg orally (PO) and diphenhydramine 25 mg (PO/IV) is to be administered at the following schedule: ■ Cohorts 1, 2, 3 only: approximately 1 hour prior to each CYNK-001 infusion and approximately 4 hours after each infusion. ■ All other cohorts on rhIL-2 plus CYNK-001 infusion days: approximately 1 hour prior to rhIL-2 injection and approximately 2 hours after CYNK-001 infusion. ■ All other cohorts on rhIL-2 only days: approximately 1 hour prior to rhIL-2 injection and approximately 3 hours after rhIL-2 injection. ■ Meperidine may also be administered to control rigors, if clinically indicated. [00458] Subjects will undergo optional BMA collection during the first 28 days on study and a protocol-mandated BMA collection on Study Day 28, both including MRD analysis by MFC by the Sponsor-selected Central MRD analysis laboratory (for MRD positive subjects only) and local Response Assessment (for MRD positive subjects and R/R subjects). [00459] Initially, 3 MRD positive subjects will be treated with CYNK-001 at 1.2 x 109 cells which will be administered on Study Days 0, 7, and 14. Subjects will be followed for a 28- day DLT period. Subjects in each cohort may be treated concurrently. Thereafter, dose escalation or cohort expansion will follow the rules outlined below. [00460] During this time, all AEs irrespective of relatedness will be collected through the end of the Treatment period which ends on Study Day 28. Subjects will be monitored for DLTs for 28 days after the first CYNK-001 infusion. [00461] Follow-up Period: The follow-up Period will start on Study Day 29 and will continue until 12 months after the first CYNK-001 infusion, Progressive Disease (PD), loss to follow-up, death or withdrawal from study whichever occurs first. During the Follow-up Period, all AEs irrespective of relatedness will be collected. Follow-up on this study ends at Month 12 with assessments outlined in the Table of Events. [00462] Day 29 through Day 60: [00463] All subjects will have a bone marrow assessment (BMA collection) at Study Day 60, which includes BMA analysis by MFC measured by the study Central Analyst for MRD positive subjects and local response assessment for all subjects (MRD positive and R/R). Thereafter, subjects will undergo bone marrow assessments (BMA collection) every other month, including MRD assessments by MFC by the study Central Analyst for MRD positive subjects and local response assessment for all subjects (MRD positive and R/R). If clinically indicated, subjects may receive aSCT at any time on or after Study Day 29, provided they have completed the 28-day DLT period and Study Day 28 BMA sample collection. If subjects receive aSCT at any time after completion of the 28-day DLT period, Bone Marrow assessments should occur approximately 90 to 100 days after aSCT and every other month through Month 12. [00464] Day 61 to Month 12 or Early Termination: [00465] For subjects with Morphologic CR and MRD positive disease at study entry (“MRD subjects”), it is anticipated that subjects who are deemed good candidates for allogeneic stem cell transplant (aSCT) may opt for this treatment, at the discretion of the treating physician. Subjects may receive aSCT no sooner than 60 days after the first dose of CYNK-001 on Study Day 0, unless clinically indicated. If clinically indicated prior to Study Day 60, aSCT must occur after completion of the 28-day DLT period, provided that the Day 28 BMA has been collected for MRD Central Laboratory assessment. [00466] Following aSCT, if a Bone Marrow assessment (BMA collection) has not occurred by Day 100 (per standard of care practices), a bone marrow assessment (BMA collection) including MRD analysis by MFC will occur at approximately 90 to 100 days after the aSCT and every other month thereafter through Month 12. Medications for the prevention of GVHD following aSCT are permitted. [00467] For MRD positive subjects: After Study Day subjects may receive medication with the intent to maintain AML disease status (i.e., maintenance therapy) after Study Day 28. Should a subject in the MRD positive population require additional anti-leukemic therapy after treatment with CYNK-001 such as cytotoxic chemotherapy or medications for debulking of disease burden, the subject will be taken off the study after consulting with the Medical Monitor and followed for survival only, unless lost to follow up or consent is withdrawn. [00468] For R/R subjects: They should not receive additional anti-leukemic therapy until after day 28 post first CYNK-001 infusion, unless clinically indicated. [00469] The study will be conducted in compliance with International Council for Harmonisation (ICH) Good Clinical Practices (GCPs) and in concordance with local Health Authority regulations. [00470] Early Termination for PD: Subjects who experience PD should have Early Termination visit completed (if possible) and will be followed for survival, every three months from early termination up to and including Month 12, loss to follow-up, death, or withdrawal from study, whichever occurs first. Survival follow-up may be done by telephone calls every three months. [00471] Number of Subjects: The study will enroll up to approximately 94 subjects, including a total of 10 subjects treated at the selected MTD/MPD for each population (MRD positive and R/R). The Sponsor may open the enrollment to 10 additional subjects for a total of 20 per expansion cohort based on review of safety and efficacy data. [00472] Dose Limiting Toxicity: Adverse events occurring up to Study Day 28 will be included in the dose-limiting toxicity (DLT) determination. Known toxicities associated with cyclophosphamide and fludarabine will be carefully considered and differentiated from CYNK- 001 in order to identify CYNK-001-related toxicities. [00473] Dose Limiting Toxicity (DLT) Definition: A DLT is defined as the development of any new (not pre-existing) event that is deemed related to CYNK-001 and meets one of the following criteria: ■ Grade 4 or 5 event in any organ system with the following exceptions: o hematologic and infectious events o disease progression/relapse ■ Grade 3 AE for > 24 hour duration in the following organ systems: cardiac (excluding hypertension), pulmonary, hepatic, renal, central nervous system (CNS). ■ Grade ≥ 3 allergic reaction that is suspected to be related to CYNK-001. ■ Grade ≥ 3 hypertension event for > 48 hour duration. ■ Grade ≥ 3 GVHD event occurring within the first 28 days following CYNK-001 infusion. o GVHD grading [Harris, 201625]. ■ Grade ≥ 3 CRS event for > 24 hour duration occurring within the first 28 days following the first CYNK-001 infusion. o CRS management and grading guidelines [Lee, 201933]. [00474] In the event that 2 separate subjects within a dosing cohort experience a DLT that is suspected to be related to CYNK-001, the events will be forwarded to the DMC for review and confirmation as to whether or not the MTD has been exceeded. If the MTD is confirmed exceeded by the DMC, no further CYNK-001 administration will occur within that dose level or at any higher dose level. [00475] DLTs will be evaluated in the MRD positive and R/R populations independently. Dose escalation within each population (MRD positive and R/R populations) will occur simultaneously and independently according to the dose escalation rules outlined. [00476] Treatment Assignment: Upon confirmation of eligibility during the Treatment Eligibility Screening Period, eligible subjects may be sequentially assigned to one of the following dose level cohorts based on time of eligibility and treatment slot availability. [00477] There will be dose escalation of CYNK-001 cells in two patient populations with CYNK-001 in escalating doses alone or with CYNK-001 and rhIL-2. Doses and schedules are as listed in Table 5 and Table 6 for MRD Positive subjects and Table 7 and Table 8 for R/R subjects. [00478] The study will utilize a 3 + 3 dose escalation design with 3 to 6 subjects enrolled into each dose cohort. The final dose level cohort (either MTD or MPD) will enroll an additional 4 subjects to bring the total to 10 subjects treated at MTD/MPD. The Sponsor may open the enrollment to 10 additional subjects for a total of 20 per expansion cohort based on review of safety and efficacy data. Dose de-escalations are shown in Table 6 and Table 6. [00479] On each day of rhIL-2 plus CYNK-001 infusion and rhIL-2 only, Subjects in Cohorts 5a, 5b, 6a, 6b, 7a and 7b will receive pre- and post- medication of acetaminophen (650 mg PO) and diphenhydramine (25 mg PO/IV) administered at the following schedule: ■ Cohorts 1, 2, 3, 4a, 4b only: approximately 1 hour prior to each CYNK-001 infusion and approximately 4 hours after each infusion. ■ All other cohorts on rhIL-2 plus CYNK-001 infusion days: approximately 1 hour prior to rhIL-2 injection and approximately 2 hours after CYNK-001 infusion. ■ All other cohorts on rhIL-2 only days: approximately 1 hour prior to rhIL-2 injection and approximately 3 hours after rhIL-2 injection. [00480] Meperidine may be administered to control rigors if clinically indicated. Vital signs will be taken during the infusion. Subjects must be monitored for at least 4 hours after completion of each CYNK-001 infusion. [00481] The decision to increase the size of the cohort or proceed to the next cohort will follow criteria outlined in Table 9. Cohort 3 (1.8 x 109 CYNK-001) will be completed prior to the beginning of Cohort 4a and 4b. After Cohort 3, enrollment amongst the MRD positive population and R/R population will occur simultaneously and dose escalation and DLT evaluation in each population will occur independently. Table 5: MRD Positive Subjects
Figure imgf000149_0001
a Cy 300 Flu 25 = Cyclophosphamide 300mg/m2/day; Fludarabine 25 mg/m2/day on Days -5, -4, -3 Cy 900 Flu 30 = Cy 900 mg/m2/day; Fludarabine 30 mg/m2/day on Days -6, -5, -4, -3 b The use of rhIL-2 in MRD cohorts 6a, and 7a will be determined based on careful review of safety, efficacy, and translational data from subjects treated in Cohorts 5a. The decision to include rhIL-2 will be based on DMC recommendation and Sponsor decision. Subjects enrolled to the expansion portion of a treatment arm may receive Cy 900 Flu 30 for 3 days only (-5, -4 and -3) per Sponsor decision based on analysis of safety, efficacy and translational data. Abbreviations: IU= international units; M= million; N/A= not applicable; QOD= once every other day; rhIL-2= recombinant human interleukin 2 (aldesleukin)
Table 6: MRD Positive Subjects with Possible De-escalation a
Figure imgf000150_0001
4a de-escalation: The 4a cohort is intended to determine safety at 1.8x 109 cells at an increased lymphodepletion dose and schedule than in Cohort 3. If this 4a cohort is not tolerated, the de-escalation plan is to determine if the increased lymphodepletion regimen will be tolerated at a 1.2 x 109 CYNK-001 cell dose to determine if increased lymphodepletion would be tolerable in this population. b 5a de-escalation: The 5a cohort is intended to determine safety at 4a dose plus inclusion of rhIL-2. If this 5a cohort is not tolerated, the de-escalation plan is to determine if rhIL-2 will be tolerated at a 1.2 x 109 CYNK-001 cell dose before concluding that the rh-IL2 is tolerable in this population. Table 7: R/R Subjects
Figure imgf000150_0002
a Cy 300 Flu 25 = Cyclophosphamide 900mg/m2/day; Fludarabine 25 mg/m2/day on Days -5, -4, - 3 Cy 900 Flu 30 = Cy 900 mg/m2/day; Fludarabine 30 mg/m2/day on Days -6, -5, -4, -3 b The use of rhIL-2 in MRD cohorts 6b and 7b will be determined based on careful review of safety, efficacy, and translational data from subjects treated in Cohorts 5b. The decision to include rhIL-2 will be based on DMC recommendation and Sponsor decision. Subjects enrolled to the expansion portion of a treatment arm may receive Cy 900 Flu 30 for 3 days only (-5, -4 and -3) per Sponsor decision based on analysis of safety, efficacy and translational data. Abbreviations: IU= international units; M= million; N/A= not applicable; QOD= once every other day; rhIL-2= recombinant human interleukin 2 (aldesleukin) Table 8: R/R Subjects with Possible De-escalation a
Figure imgf000151_0001
4b de-escalation: The 4b cohort is intended to determine safety at 1.8x 109 cells at with an increased lymphodepletion dose and schedule than in Cohort 3. If this 4b cohort is not tolerated, the de-escalation plan is to determine if the increased lymphodepletion regimen will be tolerated at a 1.2 x 109 CYNK-001 cell dose to determine if increased lymphodepletion would be tolerable in this population. b 5b de-escalation: The 5b cohort is intended to determine safety at 4b dose plus inclusion of rhIL-2. If this 5b cohort is not tolerated, the de-escalation plan is to determine if rhIL-2 will be tolerated at a 1.2 x 109 CYNK-001 cell dose before concluding that the rh-IL2 is tolerable in this population. [00482] CYNK-001 cells are administered to all subjects on Study Days 0, 7, and 14 for Cohorts 1 through 5 and Study Days 0, 7, 14 and 21 for Cohorts 6a, 6b, 7a and 7b at the dose amount indicated by the appropriate cohort (listed in Table 5, Table 6, Table 7, and Table 8). [00483] Subjects treated in Cohort 5a and Cohort 5b, will receive rhIL-2 injections SC at least 1 to 3 hours prior CYNK-001 infusion. Treatment with 6M IU rhIL-2 begins on the day of the first CYNK-001 infusion with the first of 7 total rhIL-2 injections. rhIL-2 injections should occur on each CYNK-001 infusion day and every other day in between CYNK-001 infusions (Days 0, 2, 4, 7, 9, 11, and 14). On non-CYNK-001 days, rhIL-2 may be delayed or skipped due to logistical constraints or clinically significant adverse events at the PI’s discretion. rhIL-2 must not be administered on 2 consecutive days. Consultation with the Medical Monitor is encouraged when determining the rhIL-2 dosing schedule. The use of rhIL-2 in R/R cohorts 6a, 6b, 7a and 7b will be determined based on careful review of safety, efficacy, and translational data from subjects treated in Cohorts 5a and 5b. Those subjects will receive injections on the same days as Cohorts 5a and 5b and have one additional dose on Day 21. The decision to include rhIL-2 will be based on DMC recommendation and Sponsor decision. Table 9: Dose Escalation Scheme
Figure imgf000152_0001
Abbreviations: DLT = dose-limiting toxicity; DMC = Data Monitoring Committee; MTD = maximum tolerated dose. Note: DLTs will be evaluated separately amongst the MRD positive arm and the R/R arm. Therefore, dose escalation and enrollment to each arm will occur independent of the other arm. [00484] Dose Adjustment Criteria [00485] CYNK-001 Dose Adjustments: CYNK-001 Dose adjustments may occur if clinically indicated by the treating physician. In general, the following should be followed: ■ Dose reductions are not permitted in this study. ■ Should dose delays for CYNK-001 be required: o Day 0 dose may not be delayed for longer than 48 hours o Day 7 dose may not be started early or delayed for longer than 48 hours ■ If delayed longer than 48 hours, the dose will be skipped. ■ A subject that skips Day 7 dosing may receive Day 14 dosing. o Day 14 dose may not be started early or delayed for longer than 48 hours ■ If delayed longer than 48 hours, the dose will be skipped. o If applicable, Day 21 dose may not be started early or delayed for longer than 48 hours ■ If delayed longer than 48 hours, the dose will be skipped. [00486] rhIL-2 Dose Adjustments: rhIL-2 Dose Adjustments may occur if the subject weighs < 45 kgs. The dose of rhIL-2 should be reduced to 3M IU rhIL-2. rhIL-2 injections should not be given on 2 consecutive days or on the day prior to planned CYNK-001 infusion. On non-CYNK-001 days, rhIL-2 may be delayed or skipped due to logistical constraints or clinically significant adverse events at the PI’s discretion. Consultation with the Medical Monitor is encouraged when determining the rhIL-2 dosing schedule. [00487] MRD Definitions: For the purposes of this study, MRD definitions are based on the consensus document from the European LeukemiaNet MRD Working Party [Schuurhuis, 201850] and in accordance with the assay specifications from the Sponsor-selected Central MRD analysis laboratory, as follows: ■ MRD positive greater than or equal to 0.1%: MRD positivity is defined as greater than or equal to 0.1% blasts detected by MFC on BMA by the Sponsor-selected central MRD analysis laboratory, where assay sensitivity allows for a Lower Limit of Detection (LOD) of 1 x 10-4 (0.01%) or lower. ■ MRD positive less than 0.1%: MRD positivity less than the protocol-defined threshold for eligibility, detected by MFC on BMA by the Sponsor-selected central MRD analysis laboratory, where assay sensitivity allows for a Lower Limit of Detection (LOD) of 1x10-4 (0.01%) or lower. ■ MRD Indeterminate: major phenotype shift or mild change from normal (regardless of quantity). ■ MRD negative: No MFC MRD identified (i.e., 0% blasts). ■ MRD MFC not possible: sample could not be analyzed (e.g., insufficient sample volume or total cell number to determine MRD status, compromised sample quality, etc.). [00488] Endpoint Definitions [00489] MRD Response: defined as conversion of MRD status from positive (≥ 0.1 blasts) to either negative (no MRD identified; i.e., 0% blasts), MRD positive less than 0.1%, or MRD Indeterminate as measured by MFC on BMA with assay lower limit of detection at 1:104 or lower. [00490] Time to MRD Response: defined as time from first CYNK-001 infusion to MRD Response. [00491] Duration of MRD Response: defined as date of first MRD response after CYNK-001 infusion to date of MRD positivity (≥ 0.1% blasts). If subject experiences hematologic relapse before MRD assessment yields positive MRD results, date of hematologic relapse as defined by Dohner, 201716 should be used to calculate Duration of MRD Response. [00492] Duration of Morphologic CR: for MRD positive cohorts, defined as duration from first morphologic CR (or CRi, MLFS) observation measured during front-line setting therapy to the time of disease progression per AML Response Criteria (Error! Reference source not found.), with deaths from causes other than progression censored. (The rationale for using first observation of morphologic CR is to allow for comparison to clinical response in AML as described in literature. For the purposes of this study, morphologic CR is defined as bone marrow blasts < 5% with other associated criteria outlined in ELN response criteria guidelines based on type of response (CR, CRi, MLFS) [Dohner, 201716] and may include subjects with MRD positivity, negativity, or unknown MRD status) [00493] Progression-free Survival (PFS): defined as the time from the date of the first CYNK-001 infusion to date of disease progression per AML Response Criteria (Error! Reference source not found.) or death (regardless of cause of death), whichever comes first. [00494] Time to Progression (TTP): defined as the time from the date of the first CYNK-001 infusion to the date of disease progression per AML Response Criteria (Error! Reference source not found.), with deaths from causes other than progression censored. [00495] Overall Survival (OS): defined as date of the first CYNK-001 infusion to the date of death. [00496] Duration of Response, DoR: applicable for R/R cohorts, defined only for subjects who experience an objective response, as the time from the first CR (MLFS or CRi) after CYNK-001 to relapse or death due to disease progression. [00497] Time to Response, TTR: for R/R cohorts, defined as the length of time from first CYNK-001 treatment to the time of response of CR, CRi, or MLFS per AML Response Criteria. [00498] Overall Response Rate, ORR: defined as the percentage of subjects whose AML disease have a response of CR, CRi, or MLFS per AML Response Criteria.
Table 10: Overall Study Design – Screening
Figure imgf000156_0001
Table 11: Overall Study Design – Treatment Period
Figure imgf000157_0001
Table 12: Overall Study Design - Follow-up Period
Figure imgf000158_0001
e 77htno Mriod days -8htno M -9htno M -01htno M -11htno M - -21htno M - -noitanimreTylra E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Figure imgf000159_0001
pU-wolloFlavivru S
Pe ± 77htno Mriod days - -8htno M -9htno M -01htno M -11htno M -21htno M -noitanimreTylra E - X X X X X X X - X X X X X X X O - - - - - - - - X X X X X X X - X X X X X X X O C C C C C C C - C C C C C C C - C C C C C C C -
Figure imgf000160_0001
pU-wolloFlavivru S
Mp Pe ± 77htno Mriod days - -8htno M -9htno M -01htno M -11htno M -21htno M -noitanimreTylra E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L g - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - XpU-wolloFlavivru Sr
Figure imgf000161_0001
Mp Pe ± 77htno Mriod days - -8htno M -9htno M -01htno M -11htno M -21htno M - - - - - - - - - - - -noitanimreTylra E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Figure imgf000162_0001
pU-wolloFlavivru S
±82ya D 3 da24ya Dys06ya D3htno M4htno M Fo5htno Mllow-6htno Mup Pe ± 77htno Mriod days - - - - - - - -8htno M -9htno M -01htno M -11htno M -21htno M XnoitanimreTylra E -ssments - X - X - X - X - X - X O - - C - C - C - C - C - C O - - L - L - L - L - L - L O -
Figure imgf000163_0001
pU-wolloFlavivru S
Pe 7 o7htno Mriod days D test by nth BM8htno M MFC) A as9htno M appro sessm01htno Mxima ents e Assessment by MFC by rmed at each BM aspirate oratory Biomarkers must o11htno Mtely ccur and on the same schedule as - - - - - -21htno M O -noitanimreTylra E - performed before this m - - C - C - C O - m - - - - - - - -
Figure imgf000164_0001
pU-wolloFlavivru S
C4htno M Fo - -5htno Mllow- - C6htno Mup Pe ± 7 - -7htno Mriod days - C8htno M - -9htno M - C01htno M - -11htno M - C21htno M O OnoitanimreTylra E - m C - C - C - C - C O - m - m - - - - - - - - - - C - C - C - C - C O - m - - - - - - - - - -
Figure imgf000165_0001
pU-wolloFlavivru S
Pe ± 77htno Mriod days othromb8htno Moplast9htno Min tim01htno Me aSC11htno MT = al21htno MlogenenoitanimreTylra Eic stepU-wolloFlavivru Sm encephalography; FACS = fluorescenceeukocyte antigen; ICF = Informed Consent RD = minimal residual disease; O = Optional Health Organization; nt, hemoglobin, hematocrit, white blood ritin, and Coagulation panel). n. In the event that locally drawn the same day, only the most clinically r Day -5 or -4 and recorded accordingly 3 central collection is done on Day -4, se, albumin, total protein, alkalineOT), alanine aminotransferase/serum dication of acetaminophen (650 mg PO)ately 2 hours after each CYNK-001 fusion. Subjects must be monitored for at on acetaminophen (650 mg PO) and ter CYNK-001 infusion. For subjects
Figure imgf000166_0001
etaminophen (650 mg PO) and -2 subcutaneous injections, should be ic gonadotropin (β-hCG) pregnancy test l be performed within 72 hours prior to the bility for CYNK-001 treatment. At the Day e performed. Core Antibody (HBcAB), Hepatitis B T, SARS-CoV-2. -1. Mesna shall be administered, for rrhagic cystitis induced by ds. Subjects enrolled to the expansionnalysis of safety, efficacy and translational nt (including during Treatment Eligibilityurs of collection; 3 mL send to infusion; BMA samples must be collected practice allow, a separate puncture is to-Chex tube for Immune phenotyping and um heparin tubes at each designated d will be collected in marbled red top urs (optional), and 24 hours after 2, 4, 9, and 11) a pre-infusion blood and egrees Celsius until shipment of all m separation tubes for serum analysis at eachd samples at the 24-hour timepoint. Note: y be skipped provided the 24-hour ns, this collection may be skipped. CYNK-001. ndicated during the treatment period if e does not need to be repeated. usions for a total of 7 doses. An ECG should ormed at each follow-up visit. Dose y -3 during the Lymphodepletion Regimen,
Figure imgf000167_0001
ct’s disease progresses, survival status should be collected. Survival follow-up can be done by visit or telephone call every three months until ubject withdraws or is lost to follow up. a, 6b, 7a and 7b: Subjects will receive a total of 4 CYNK-001 infusions on Study Days 0, 7, 14, and 21.
166
Figure imgf000168_0001
[00499] Selection and Withdrawal of Subjects [00500] Optional Pre-Screening BMA Collection [00501] Subjects who will have their Pre-Screening BMA Collected to pre-screen for AML disease eligibility must meet the following criteria: a. Subject has AML. b. Subject is ≥ 18 and ≤ 80 years of age at the time of signing the Pre-Screening BMA Collection informed consent form (ICF). c. Subject understands and voluntarily signs the Pre-Screening BMA Collection ICF prior to any BMA Collection. d. The Pre-Screening BMA collection must occur approximately 2 weeks prior to the start of the Treatment Eligibility Period [00502] Treatment Eligibility Screening Period [00503] Treatment Eligibility Screening Period Subject Inclusion Criteria (all subjects): Subjects must satisfy the following criteria to be enrolled in the study: 1. Subject has eligible disease status: MRD positive population: Primary or Secondary AML subjects in first or second Morphological Complete Remission (CR), Morphological Complete Remission with incomplete hematologic recovery (CRi), or Morphologic Leukemia-free State (MLFS) as defined by the European LeukemiaNet (ELN) recommendations for AML Response Criteria [Dohner, 201716]. R/R population: R/R diagnosis based on confirmed diagnosis with local pathology report following any re- induction/salvage therapy ELN guidelines. ■ Relapsed AML are defined as having relapsed after achieving ≥ 1 CR, including relapse after allogeneic stem cell transplantation (≥ 2 months after transplant). ■ Refractory AML, defined as not achieving CR, CRi, or MLFS after 2 or more cycles of induction therapy (primary refractory) or not achieving CR after treatment for relapsed AML. ■ Secondary AML (MDS transformation): Secondary AML subjects are eligible to participate if they have received a minimum of one prior line of treatment for AML. ■ Treatment-related AML: Treatment-related AML subjects are eligible to participate if they have received a minimum of one prior line of treatment for AML. 2. Subjects with prior central nervous system involvement by malignancy are eligible provided that it has been treated and cerebral spinal fluid sampled at least two weeks prior to the start of the lymphodepletion regimen was negative for AML by both cytology and flow cytometry. 3. MRD positive population only: Subject is MRD positive, as assessed on BMA by Multiparameter Flow Cytometry (MFC) at time of Treatment Eligibility assessment. a. For the purposes of this study, MRD positivity is defined as greater than or equal to 0.1% blasts detected by MFC on BMA by the Sponsor-selected Central analysis laboratory, where assay sensitivity allows for a Lower Limit of Detection (LOD) of 1 x 10-4 (0.01%) or lower. 4. Subject is ≥ 18 and ≤ 80 years of age at the time of signing the Study informed consent form (ICF). 5. Subject understands and voluntarily signs the Study ICF prior to any study-related assessments/procedures are conducted. 6. Subject is willing and able to adhere to the study schedule and other protocol requirements. 7. Performance status of Eastern Cooperative Oncology Group (ECOG) ≤ 2. 8. Ability to be off immunosuppressive drugs for at least 4 weeks prior to the first planned CYNK-001 infusion (only up to 7.5 mg of prednisone per day is permissible). Furthermore, these subjects should NOT have any signs and/or symptoms of acute GVHD or chronic GVHD during this 4-week wash out period and at the time of enrollment into the study. Consultation with Medical Monitor is encouraged when considering immunosuppressive drug washout period. 9. Female of childbearing potential (FCBP)* must not be pregnant and must agree to not become pregnant for at least 28 days following the CYNK-001. FCBP must agree to use an adequate method of contraception during the treatment period. a. *FCBP is a female who: 1) has achieved menarche at some point, 2) has not undergone a hysterectomy or bilateral oophorectomy and 3) has not been naturally postmenopausal (amenorrhea following cancer therapy does not rule out childbearing potential) for at least 24 consecutive months (i.e., has had menses at any time in the preceding 24 consecutive months). 10. Male subject must agree to use a condom during sexual contact for at least 28 days following the last infusion of CYNK-001, even if he has undergone a successful vasectomy. [00504] Treatment Eligibility Screening Period Subject Exclusion Criteria (all subjects): The presence of any of the following will exclude the subject from enrollment: 1. Subject has any significant medical condition, laboratory abnormality, or psychiatric illness that would prevent the subject from participating in the study. 2. Subject has any condition including the presence of laboratory abnormalities which places the subject at unacceptable risk if he or she were to participate in the study. 3. Subject has any condition that confounds the ability to interpret data from the study. 4. Subject has bi-phenotypic acute leukemia. 5. Subject has acute promyelocytic leukemia (APL). 6. Exclusion criterion 6 was removed with Protocol Amendment 3.0. 7. Subject has inadequate organ function as defined below at time of Treatment Eligibility Period: a. Subject has aspartate aminotransferase (AST), alanine aminotransferase (ALT), or alkaline phosphatase ≥ 2.5 x the upper limit of normal (ULN). b. Creatinine clearance less than 40 mL/minute. c. Subject has a bilirubin level > 2 mg/dL (unless subject has known Gilbert’s disease). 8. Subject has had prior treatment with biologic antineoplastic agents less than 7 days or 5 half-lives before the first CYNK-001 infusion, whichever is longer. (Exception will be granted for monoclonal antibodies that are known to have long half-lives, in which case a minimum of 2 weeks from last dose will be required). For agents that have known AEs occurring beyond these specified days after administration, this period must be extended beyond the time during which acute AEs are known to occur. Treating physicians are encouraged to discuss cases with the Medical Monitor. 9. Subject is pregnant or breastfeeding. 10. Subject has new or progressive pulmonary infiltrates or pleural effusion large enough to be detected by chest x-ray or CT scan within 2 weeks of first CYNK-001 infusion. 11. Subject has active autoimmune disease other than controlled connective tissue disorder or those who are not on active therapy. 12. Subject has had a ASCT < 60 days prior to Treatment Eligibility Screening Visit or plans to have transplant within the 28 day period following first CYNK-001 infusion. Consultation with the Medical Monitor is encouraged when considering enrollment of subjects with prior ASCT. 13. Subject has a history of malignancy other than AML or other underlying hematologic conditions such as MDS or MPN, unless the subject has been in remission or free of disease for greater than 1 year prior to CYNK-001 infusion. Other exceptions will include the following malignancies: a. Basal cell carcinoma of the skin b. Squamous cell carcinoma of the skin c. Carcinoma in situ of the cervix d. Carcinoma in situ of the breast e. Incidental biological finding of prostate cancer (TNM stage of T1a or T1b) f. Superficial Bladder Cancer g. For subjects with therapy-related AML, the underlying malignancy which led to secondary AML must have no evidence of the underlying malignant disease as of the last surveillance and the subject must not be planned for further treatment of the underlying malignant disease. 14. Subject has a history of severe asthma for which the subject is presently on chronic medications or has a history of other symptomatic pulmonary disease. 15. Subjects with the following prior history of GVHD will be excluded: ■ Acute GVHD: Subjects with prior history of acute GVHD where signs and/or symptoms did not completely resolve (no clinical signs/symptoms and not on more than 7.5 mg of prednisone per day) within 90 days of ongoing immunosuppression. ■ Chronic GVHD: Subjects with prior history of chronic GVHD where signs and/or symptoms did not completely resolve (no clinical signs/symptoms and not on more than 7.5 mg of prednisone per day) within 90 days of ongoing immunosuppression. 16. Subject has an untreated chronic infection or has received treatment of any uncontrolled or progressive infection with systemic antibiotics within 2 weeks prior to first CYNK-001 infusion. Prophylactic antibiotic, antiviral, and antifungal medication are permissible (and required prior to or at start of lymphodepletion regimen). 17. Subject has any other organ dysfunction (CTCAE Version 5.0 Grade 3 or greater) that will interfere with the administration of the therapy according to this protocol. 18. Subject has a resting left ventricular ejection fraction (LVEF) of < 40% obtained by echocardiography or multi-gated acquisition scan (MUGA). 19. Subject was treated with an investigational product within 28 days of first CYNK-001 infusion. Subject must no longer be a participant in the previous interventional study at the time of CYNK-001 infusion. (Subjects who are under survival follow-up or observation associated with a study are permitted, and if treatment information is collected for this period, “Investigational Study” must be used to capture the study treatment.). [00505] Treatment of Subjects [00506] Description of Study Drug: For full description of CYNK-001, refer to the Investigator’s Brochure (IB). Celularity will supply CYNK-001 for IV administration. Subjects will receive CYNK-001 according to the protocol-specified treatment plan. [00507] Commercially available acetaminophen and diphenhydramine will be used for pre-and post- medication. Subjects enrolled should obtain commercially available product through the local hospital pharmacy or licensed distributor. [00508] Commercially available cyclophosphamide and fludarabine will be used as Lymphodepletion Regimen as outlined in this protocol. [00509] Commercially available rhIL-2 will be used as outlined in this protocol. [00510] Lymphodepletion Regimen: Each subject will undergo a Lymphodepletion Regimen beginning on Study Day -5 and ending on Study Day -3 followed by two days with no treatment on Study Days -2 and -1 (where Study Day 0 is the day of the first CYNK-001 infusion). [00511] Cy 300 Flu 25 Regimen (Cohorts 1, 2, 3 only): Cyclophosphamide dose is 300 mg/m2 to be administered on Study Days -5, -4, and -3. Fludarabine dose is 25 mg/m2 to be administered on Study Days -5, -4, and -3. [00512] Cy 900 Flu 30 Regimen (all other cohorts): Cyclophosphamide dose is 900 mg/m2 to be administered on Study Days -6, -5, -4, and -3. Fludarabine dose is 30 mg/m2 to be administered on Study Days -6, -5, -4, and -3. [00513] Dose calculations for cyclophosphamide and fludarabine may be based on actual or adjusted body weight based at the treating physician’s discretion and per institutional practices; doses may be rounded to the nearest 5% per institutional practices. [00514] Mesna shall be administered, for Cohorts 4 and above, at the start of Lymphodepletion (e.g. on Study Days -6, -5, -4, and -3) for the inhibition of hemorrhagic cystitis induced by cyclophosphamide. Route of administration, dosage, and frequency of Mesna should be based on institutional standards. [00515] Subjects enrolled to the expansion portion of a treatment arm may receive Cy 900 Flu 30 for 3 days only (-5, -4 and -3) per Sponsor decision based on analysis of safety, efficacy and translational data. [00516] CYNK-001: CYNK-001 is an allogeneic off the shelf cell therapy enriched for CD56+/CD3- NK cells culture-expanded from human placental CD34+ cells. Culture- expanded cells are harvested, washed in Plasma-Lyte A and then packaged at 30 x 106 cells/mL in a total volume of 20 mL of cryopreservation solution containing 10% (w/v) HSA, 5.5% (w/v) Dextran 40, 0.21% NaCl (w/v), 32% (v/v) Plasma-Lyte A, and 5% (v/v) DMSO. It is filled into the container closure, frozen using a controlled rate freezer, and cryopreserved. Prior to releasing to the site, all release and characterization testing will be complete. When required by site, CYNK-001 is shipped in vapor phase LN2 to the designated clinical site where it will be processed for dose preparation in a standardized manner just prior to IV administration. [00517] On Study Days 0, 7, and 14 (and Day 21 for subjects treated in Cohorts 6a, 6b, 7a and 7b), subjects will receive acetaminophen 650 mg PO and diphenhydramine 25 mg as outlined. [00518] CYNK-001 will be administered at either 6 x 108 cells per dose, 1.2 x 109 cells per dose, 1.8 x 109 cells per dose, or 3.0 x 109 cells per dose (depending on Dose Cohort assignment), with or without rhIL-2. CYNK-001 is administered IV, using a gravity IV administration set with a 16- to 22-gauge (or equivalent) needle or catheter with no filters. A central line may be used to infuse CYNK-001 after confirming that the catheter diameter is 16- to 22-gauge (or equivalent) needle. For substantial deviation from this catheter diameter, consultation with the medical monitor is required. The recommended infusion rate is approximately 240 mL per hour. No other medications or blood products should be in the IV line at the time of CYNK-001 infusion. Vital signs should be taken during CYNK-001 infusion if clinically indicated and any abnormal clinically significant findings should be documented. Immediately following the infusion, the infusion line will be flushed with 30 to 60 mL of normal saline. Table 14: Investigational Product
Figure imgf000175_0001
[00519] Overdose: Overdose as defined for this protocol, refers to CYNK-001, cyclophosphamide, fludarabine, and rhIL-2. On a per dose basis, an overdose is defined as the following amount over the protocol-specified dose of CYNK-001 assigned to a given subject, regardless of any associated AEs or sequalae: [00520] CYNK-001: 30% over the assigned protocol-specified dose of 6 x 108 cells, 1.2 x 109 cells, 1.8 x 109 cells, or 3.0 x 109 cells. [00521] Refer to cyclophosphamide, fludarabine, and rhIL-2 package inserts for overdose information. [00522] On a schedule or frequency basis, an overdose is defined as anything more frequent than the protocol required schedule. Complete data about drug administration, including any overdose, regardless of whether the overdose was accidental or intentional, should be reported in the eCRF. [00523] rhIL-2 to Facilitate CYNK-001 Cell Survival and Expansion: Subjects treated in Cohort 5a, Cohort 5b, Cohort 5a De-escalation, Cohort 5b De-escalation, Cohort 6a*, Cohort 6b*, Cohort 7a*, and Cohort 7b* will receive rhIL-2 SC injections which will be administered according to the following instructions: ■ On CYNK-001 infusion days (Days 0, 7, 14 [and 21 for Cohorts 6a, 7b, 7a and 7b]), start subcutaneous rhIL-2 injections 1 to 3 hours prior to CYNK-001 cell infusion in the absence of Grade 4 infusion-related toxicity. ■ *Cohorts 6a, 6b, 7a and 7b may receive rhIL-2 after thorough review of all safety, efficacy, and translational data in Cohorts 5a and 5b, respectively. The decision to administer or hold rhIL-2 will be made by the Sponsor after review and DMC recommendation. ■ The location of the injection site is at the discretion of the treating physician following local institutional practices. Suggested locations for subcutaneous rhIL- 2 injections include upper arm, upper thigh, or abdomen. The location of each injection is to be collected in the electronic case report form (eCRF). ■ If subject is experiencing a Grade 4 AE on days where rhIL-2 will be given, then administration of rhIL-2 can be skipped, according to PI discretion. ■ rhIL-2 injections should not be given on 2 consecutive days or on the day prior to planned CYNK-001 infusion. If rhIL-2 dose delays occur where rhIL-2 is to be administered on the day prior to planned CYNK-001 infusion, that rhIL-2 dose should be skipped and the next rhIL-2 dose would be given on the same day of CYNK-001 infusion, 1 to 3 hours prior to CYNK-001 administration. ■ If there is an immediate clinically significant adverse reaction to rhIL-2 administration, CYNK-001 infusion can be delayed until recovered, according to PI discretion. ■ If subject is experiencing a Grade 4 AE on study days that both rhIL-2 and CYNK-001 will be administered, then the rhIL-2 and subsequent CYNK-001 administration can be delayed according to PI discretion. ■ rhIL-2 administration: rhIL-2 will be given at a dose of 6M approximately every other day (e.g., 0, 2, 4, 7, 9, 11, and 14 [and Cohorts 6a, 6b, 7a and 7b: additional rhIL-2 dose on Day 21]) for a total of up to 8 doses ■ For subjects weighing less than 45 kilograms, the rhIL-2 will be given at rhIL-2 3M IU on planned rhIL-2 dosing days. ■ An ECG should be administered prior to each CYNK-001 infusion and after the third rhIL-2 injection. ECGs should continue to be performed at each follow-up visit. ■ Pulmonary function tests should be performed if clinically indicated during the treatment period if there is a history of or ongoing pulmonary condition. ■ Meperidine may be administered to control rigors if clinically indicated. ■ Pre- and post- IL-2 medications for each rhIL-2 injection are outlined. [00524] Treatment Compliance: CYNK-001 is to be administered IV at the clinical study site. Study personnel will review the dosing treatment allocation and ensure treatment is administered according to the subject’s treatment plan. Treatment compliance will be noted on the appropriate CRFs and source records based on administration records. ■ CYNK-001 Dose reductions are not permitted in this study. ■ Should dose delays for CYNK-001 be required o Day 0 dose may not be delayed for longer than 48 hours o Day 7 dose may not be started early or delayed for longer than 48 hours ■ If delayed longer than 48 hours, the dose will be skipped. ■ A subject that skips Day 7 dosing may receive Day 14 dosing. o Day 14 dose may not be started early or delayed for longer than 48 hours ■ If delayed longer than 48 hours, the dose will be skipped. o Cohorts 6a, 6b, 7a and 7b: Day 21 dose may not be started early or delayed for longer than 48 hours ■ If delayed longer than 48 hours, the dose will be skipped. ■ Concomitant Therapies o Blood product transfusions should not occur within 24 hours prior to and/or 24 hours after CYNK-001 infusion, if possible. Transfusions within this window may be permitted if clinically indicated and upon consultation with the medical monitor. o Use of steroids greater than the equivalent of 7.5 mg prednisone per day is prohibited for 4 weeks prior to infusion and avoided until the end of the treatment period. Should steroids be clinically required, dosing delays and/or skipping may occur after consultation with the Medical Monitor. ■ Subjects assigned to treatment in Cohort 5a, Cohort 5b, Cohort 6a*, Cohort 6b*, Cohort 7a*, and Cohort 7b* are to receive rhIL-2, approximately 1 to 3 hours prior to CYNK-001 infusion. rhIL-2 Dose Adjustments to 3M IU may occur if the subject weighs < 45 kgs. *Note: decision to include rhIL-2 in cohorts 6a, 6b, 7a and 7b are based on DMC recommendation and Sponsor decision. [00525] Assessment of Efficacy: This study will explore the potential clinical efficacy of CYNK-001 by evaluating the following: [00526] MRD positive population: MRD Response (as defined herein) as assessed centrally by MFC at defined time points after CYNK-001 infusion. Additionally, time to MRD Response, duration of MRD Response, PFS, duration of morphologic CR, TTP, and OS will be evaluated. [00527] R/R population: Overall Response Rate (ORR) defined as achievement of Complete Remission (CR), Complete Remission with incomplete (CRi) hematologic recovery, or Morphologic leukemic-free state (MLFS), Duration of Response (DoR), and Overall (OS). [00528] The data will be adjudicated by a Data Monitoring Committee (DMC) to confirm the clinical efficacy data. [00529] Assessment of Safety: Safety Parameters: Subject safety will be assessed in all subjects who receive any amount of CYNK-001 and will include AEs, vital signs, body weight measurements, physical examination findings, clinical laboratory test results, infusion site assessments, x-ray, magnetic resonance imaging (MRI) or computerized tomography (CT) scan results, electrocardiogram (ECG) interpretations, electroencephalography (EEG) if clinically indicated, pregnancy testing for FCBP, and concomitant medications and procedures will be tabulated and summarized by cohort. Timing of evaluations will be assessed as outlined in the Table of Events. [00530] All AEs will be reported and recorded in the electronic case report form (eCRF). For serious adverse events (SAEs), an expedited reporting procedure will be used. The rate of AEs, SAEs, abnormal laboratory AEs and vital signs (graded according to the NCI CTCAE Version 5.0) will be measured while the subject is on study. [00531] The ASTCT Consensus Grading for CRS and Neurologic Toxicity Associated with Immune Effector Cells will be used for the purposes of grading of CRS considered associated with CYNK-001 by the Investigator. CRS at any grade is an expected event and immediately reportable. [00532] Subjects will be monitored for AEs associated with study-related sample collections and procedures only from the time of signing the ICF through the Treatment Eligibility Screening Period. All AEs will be collected during the start of the Treatment Period through the end of the study. [00533] Exploratory Assessments: The translational and biomarker assays for this study will require obtaining peripheral blood samples, along with serum and BMA samples. ■ Blood o Immune phenotyping by flow cytometry o CYNK-001 cell evaluation for expansion and persistence o Serum Collection: cytokine and correlates evaluation o Serum Collection: Anti-human leukocyte antigen (HLA) testing and anti-panel reactive antibodies (PRA) antibodies o T Cell receptor analysis o KIR genotyping (screen) ■ Bone Marrow Aspirate o Cellular immune panels by flow cytometry o T Cell receptor analysis o Transcriptome analysis o AML genomic mutation analysis (if sample is collected at time of diagnosis) [00534] These tests will require obtaining serum, blood and BMA samples as specified in the Table of Events. List of References 1. Actemra®. [Package Insert]. South San Francisco, United States of America: Genentech, Inc; 2019. 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IL-2- based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial. Bone Marrow Transplant 2003;32(2):177-86. 6. Caligiuri MA, Murray C, Robertson MJ, Wang E, Cochran K, Cameron C. Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin 2. J Clin Invest 1993;91:123-32. 7. Chen X, Xie H, Wood B, Walter R, Pagel J et al. Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia. J Clin Onc.2015;33(11):1258-64. 8. Cheson BD, Bennett JM, Kopecky KJ, Büchner T, Willman CL, Estey EH, et al. Revised recommendations of the International Working Group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol.2003;21(24):4642-9. 9. 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Options in Oncol.21, 66 (2020). 55. Trinchieri G, Matsumoto-Kobayashi M, Clark SC, Seehra J, London L, Perussia B. Response of resting human peripheral blood natural killer cells to interleukin 2. J Exp Med 1984 Apr;160:1147-69. 56. Trinchieri G. Natural killer cells wear different hats: effector cells of innate resistance and regulatory cells of adaptive immunity and of hematopoiesis. Semin Immunol 1995;7(2):83-8. Equivalents: [00535] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. [00536] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Claims

WHAT IS CLAIMED IS: 1. A method of treating acute myeloid leukemia (AML) in a subject comprising administering to the subject an effective amount of CYNK cells to the subject so as thereby to provide an effective treatment of the AML in the subject.
2. The method of claim 1, wherein the CYNK cells are placental-derived natural killer (NK) cells.
3. The method of claim 1, wherein the CYNK cells are placental CD34+ cell- derived natural killer (NK) cells. 4. The method of any one of claims 1-3, wherein the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells and / or expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.
4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 which is higher than expression of said markers in peripheral blood natural killer cells.
5. The method of any one of claims 1-4, wherein the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells.
6. The method of claim 4 or claim 5, wherein expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS is lower than expression of said markers in peripheral blood natural killer cells. 7. The method of any one of claims 1-6, wherein the CYNK cells are characterized by expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 which is higher than expression of said markers in peripheral blood natural killer cells. 8. The method of any one of claims 1-7, wherein expression of 2, 3, 4, 5, 6,
7,
8, 9, 10, or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 is higher than expression of said markers in peripheral blood natural killer cells.
9. The natural killer cell of any one of claims 1-8, wherein the CYNK cells are prepared by the methods presented herein.
10. The method of any one of claims 1-9, wherein the acute myeloid leukemia is primary acute myeloid leukemia or secondary acute myeloid leukemia.
11. The method of any one of claims 1-10, wherein the acute myeloid leukemia is in remission with minimal residual disease.
12. The method of any one of claims 1-10, wherein the acute myeloid leukemia is relapsed/refractory AML.
13. The method of any one of claims 1-12, wherein providing an effective treatment comprises reducing the rate of minimal residual disease (MRD) relative to placebo.
14. The method of any one of claims 1-12, wherein providing an effective treatment comprises converting the subject to MRD negative.
15. The method of any one of claims 1-12, wherein providing an effective treatment comprises converting the subject to MRD positive less than 0.1%.
16. The method of any one of claims 1-15, wherein the MRD is measured by flow cytometry.
17. The method of any one of claims 1-15, wherein the MRD is measured by nucleic acid sequencing, preferably by next generation sequencing.
18. The method of any one of claims 1-17, wherein providing an effective treatment comprises reducing the time to minimal residual disease (MRD) response relative to placebo.
19. The method of any one of claims 1-18, wherein providing an effective treatment comprises increasing the duration of minimal residual disease (MRD) response relative to placebo.
20. The method of any one of claims 1-19, wherein providing an effective treatment comprises reducing the incidence, severity, or duration of the disease as measured by one or more International Myeloma Working Group (IMWG) response criteria relative to placebo.
21. The method of any one of claims 1-20, wherein providing an effective treatment comprises reducing the incidence, severity, or duration of the disease as measured by the Eastern Cooperative Oncology Group (ECOG) Performance Status relative to placebo.
22. The method of any one of claims 1-21, wherein providing an effective treatment comprises increasing the duration of clinical response relative to placebo.
23. The method of any one of claims 1-22, wherein providing an effective treatment comprises increasing the rate of progression free survival, the rate of front-line progression free survival, or the rate of survival relative to placebo.
24. The method of any one of claims 1-23, wherein providing an effective treatment comprises increasing the time to progression, the front-line time to progression or the time to death relative to placebo.
25. The method of any one of claims 1-24, wherein providing an effective treatment comprises increasing the overall survival or front-line overall survival relative to placebo.
26. The method of any one of claims 1-25, wherein providing an effective treatment comprises increasing the patient reported outcome relative to placebo or relative to pretreatment.
27. The method of any one of claims 1-26, wherein administering the cells to the subject is performed intravenously.
28. The method of any one of claims 1-27, wherein from about 6 x 108 to about 3.0 x 109 cells are administered per administration.
29. The method of any one of claims 1-27, wherein from about 9 x 108 to about 1.8 x 109 cells are administered per administration.
30. The method of any one of claims 1-27, wherein about about 6 x 108 cells are administered per administration.
31. The method of any one of claims 1-27, wherein about about 1.2 x 109 cells are administered per administration.
32. The method of any one of claims 1-27, wherein about about 1.8 x 109 cells are administered per administration.
33. The method of any one of claims 1-27, wherein about about 3.0 x 109 cells are administered per administration.
34. The method of any one of claims 1-33, wherein the treatment comprises 1 to 5 administrations of cells.
35. The method of any one of claims 1-33, wherein the treatment comprises 3 administrations of cells.
36. The method of any one of claims 1-33, wherein the treatment comprises 4 administrations of cells.
37. The method of any one of claims 1-36, wherein the administrations occur approximately 1 week apart.
38. The method of any one of claims 1-37, wherein one administration of cells occurs at approximately day 0 of the treatment.
39. The method of any one of claims 1-38, wherein one administration of cells occurs at approximately day 7 of the treatment.
40. The method of any one of claims 1-39, wherein one administration of cells occurs at approximately day 14 of the treatment.
41. The method of any one of claims 1-40, wherein one administration of cells occurs at approximately day 21 of the treatment.
42. The method of any one of claims 1-41, wherein the treatment comprises about 3 administrations of cells occurring at about days 0, 7, and 14 of the treatment.
43. The method of any one of claims 1-41, wherein the treatment comprises about 3 administrations of cells occurring at about days 0, 7, 14, and 21 of the treatment.
44. The method of any one of claims 1-43, wherein the treatment further comprises a lymphodepletion regimen.
45. The method of claim 44, wherein the lymphodepletion regimen comprises administering cyclophosphamide and fludarabine to the subject.
46. The method of claim 44 or claim 45, wherein the lymphodepletion regimen comprises administering about 300 mg/m2/day cyclophosphamide and 25 mg/m2/day fludarabine to the subject.
47. The method of claim 44 or claim 45, wherein the lymphodepletion regimen comprises administering about 900 mg/m2/day cyclophosphamide and 30 mg/m2/day fludarabine to the subject.
48. The method of any one of claims 44-47, wherein the cyclophosphamide and fludarabine are administered to the subject on days -5, -4, and -3.
49. The method of any one of claims 44-47, wherein the cyclophosphamide and fludarabine are administered to the subject on days -6, -5, -4, and -3.
50. The method of any one of claims 1-49, wherein the treatment further comprises administering recombinant human interleukin-2 (rhIL-2) to the subject.
51. The method of claim 50, wherein the administration of rhIL-2 occurrs on each day that CYNK cells are administered.
52. The method of claim 50, wherein the administration of rhIL-2 occurrs on days 0, 7, and 14.
53. The method of claim 50, wherein the administration of rhIL-2 occurrs on days 0, 7, 14, and 21.
54. The method of any one of claims 50-53, wherein the rhIL-2 is additionally administered every other day between each administration of CYNK cells.
55. The method of any one of claims 50-54, wherein about 4M IU to about 8MIU of rhIL-2 is administered to the subject for each administration.
56. The method of any one of claims 50-54, wherein about 6M IU of rhIL-2 is administered to the subject for each administration.
57. The method of any one of claims 1-56, wherein the study days are measured relative to administration fo the first dose of CYNK cells at day 0.
58. A composition comprising human CYNK cells for use in the treatment of acute myeloid leukemia (AML) in a subject.
59. Use of a composition comprising human CYNK cells for use in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML) in a subject.
60. The composition of claim 58 or use of claim 59, wherein the cancer is multiple myeloma.
61. The composition of claim 60 or use claim 61, wherein the acute myeloid leukemia is primary acute myeloid leukemia or secondary acute myeloid leukemia.
62. The composition of claim 60 or use claim 61, wherein the acute myeloid leukemia is in remission with minimal residual disease.
63. The composition of claim 60 or use claim 61, wherein the acute myeloid leukemia is relapsed/refractory AML.
64. The composition or use of any one of claims 59-63, wherein the CYNK cells are placental-derived natural killer (NK) cells.
65. The composition or use of any one of claims 59-64, wherein the CYNK cells are placental CD34+ cell-derived natural killer (NK) cells.
66. The composition or use of any one of claims 59-65, wherein the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells and / or expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 which is higher than expression of said markers in peripheral blood natural killer cells.
67. The composition or use of any one of claims 59-66, wherein the CYNK cells are characterized by expression of one or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS which is lower than expression of said markers in peripheral blood natural killer cells.
68. The composition or use of any one of claims 59-67, wherein expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers selected from the group consisting of FGFBP2, GZMH, CCL3L3, GZMM, CXCR4, ZEB2, KLF2, LITAF, RORA, LYAR, CNOT1, IFNG, DUSP2, ATG2A, CD7, PMAIP1, PPP2R5C, NR4A2, ZFP36L2, PIK3R1, KLRF1, SNHG9, MT2A, RGS2, CHD1, DUSP1, EML4, ZFP36, ZC3H12A, DNAJB6, SBDS, IRF1, TSC22D3, TSPYL2, PNRC1, ISCA1, JUNB, WHAMM, RICTOR, TNFAIP3, EPC1, MVD, CLK1, ARL4C, REL, KMT2E, YPEL5, AMD1, BTG2, and IDS is lower than expression of said markers in peripheral blood natural killer cells.
69. The composition or use of any one of claims 59-68, wherein the CYNK cells are characterized by expression of one or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 which is higher than expression of said markers in peripheral blood natural killer cells.
70. The composition or use of any one of claims 59-69, wherein expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10, TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5, FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 is higher than expression of said markers in peripheral blood natural killer cells.
71. The composition or use of any one of claims 59-70, wherein the CYNK cells are prepared by the methods presented herein.
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