CLEAVAGE RESISTANT CD16 CONSTRUCTS AND USES THEREOF

Abstract

Provided herein are cells or populations of cells comprising a polynucleotide encoding a cleavage resistant CD16 polypeptide. Also provided herein are methods of suppressing the proliferation of tumor cells such as HER2+ and methods of treating cancers, such as HER2+ in a subject with populations of placental-derived natural killer cells or placental-derived T cells comprising a cleavage resistant CD16. The natural killer cells, such as CYNK cells, can be placental CD34+ cell-derived natural killer (NK) cells. The placental-derived T cells can be isolated from cord blood or from placental perfusate.

Claims

1. A mammalian cell or population of mammalian cells wherein the mammalian cell comprises, or one or more cells within the population of mammalian cells comprises: a polynucleotide encoding a cleavage resistant CD16 polypeptide.

2. The cell or population of cells of claim 1, wherein the CD16 is selected from the group consisting of a CD16a isoform and a CD16b isoform.

3. The cell or population of cells of claim 2, wherein the CD16b isoform is selected from the group consisting of an NA1 allelic variant and an NA2 allelic variant.

4. The cell or population of cells of any one of claims 1-3, wherein the cleavage resistant CD16 variant comprises a Valine residue at position 176 relative to the wild-type CD16 polypeptide.

5. The cell or population of cells of any one of claims 1-4, wherein the cleavage resistant CD16 variant comprises a residue other than serine or proline at position 197 relative to the wild-type CD16 polypeptide.

6. The cell or population of cells of claim 5, wherein the cleavage resistant CD16 variant comprises a residue selected from the group consisting of cysteine, glycine, threonine, and phenylalanine at position 197 relative to the wild-type CD16 polypeptide.

7. The cell or population of cells of any one of claims 1-4, wherein the cleavage resistant CD16 variant comprises a polypeptide having a sequence identical to a portion of a CD8 polypeptide.

8. The cell or population of cells of claim 7, wherein the cleavage resistant CD16 variant comprises a polypeptide having a sequence identical to the stalk region of CD8a.

9. The cell or population of cells of claim 7, wherein the cleavage resistant CD16 variant comprises a polypeptide having the sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, or a sequence comprising at least 15 consecutive amino acids of the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

10. The cell or population of cells of any one of claims 1-4, wherein the cleavage resistant CD16 variant comprises a polypeptide having a sequence identical to a portion of a CD28 polypeptide.

11. The cell or population of cells of claim 10, wherein the cleavage resistant CD16 variant comprises a polypeptide having a sequence identical to the stalk region of CD28.

12. The cell or population of cells of claim 10, wherein the cleavage resistant CD16 variant comprises a polypeptide having the sequence: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP, or a sequence comprising at least 15 consecutive amino acids of the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

13. The cell or population of cells of any one of claims 1-4, wherein the cleavage resistant CD16 variant comprises a polypeptide having a sequence identical to a portion of a CD64 polypeptide.

14. The cell or population of cells of claim 13, wherein the cleavage resistant CD16 variant comprises a polypeptide having a sequence identical to the stalk region of CD64.

15. The cell or population of cells of claim 10, wherein the cleavage resistant CD16 variant comprises a polypeptide having the sequence: PELELQVLGLQLPTPVWFH, or a sequence comprising at least 15 consecutive amino acids of the sequence PELELQVLGLQLPTPVWFH.

16. The cell or population of cells of any one of claims 1-4, wherein the cleavage resistant CD16 variant comprises a scrambled ADAM17 recognition sequence.

17. The cell or population of cells of claim 13, wherein the scrambled ADAM17 recognition sequence comprises the sequence VITALS.

18. The cell or population of cells of any one of claims 1-6, wherein the cleavage resistant CD16 variant comprises a valine residue at position 195 relative to the wild-type CD16 polypeptide.

19. The cell or population of cells of any one of claims 1-6, or 15, wherein the cleavage resistant CD16 variant comprises a leucine residue at position 195 relative to the wild-type CD16 polypeptide.

20. The cell or population of cells of claim 1, wherein the cleavage resistant CD16 variant comprises a polypeptide having a sequence selected from the group consisting of: TABLE-US-00015 MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVCTIS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVGTIS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVTTIS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVFTIS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVSFCLVMVLLFAVDTG LYFSVKTNIRSSTRDWKDHKFKWRKDPQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITIEVMYPPPYLD NEKSNGTIIHVKGKHLCPSPLFPGPSKPVSFCLVMVLLFAVDTGLYFSVK TNIRSSTRDWKDHKFKWRKDPQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGVITALSS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLVVGTIS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVGTLS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLVVTTIS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVTTLS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK, or MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGA YSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPV QLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKY FHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLVVGTLS SFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKD PQDK.

21. The cell or population of cells of any one of claims 1-20, wherein the cleavage resistant CD16 variant comprises an amino acid tag.

22. The cell or population of cells of claim 21, wherein the amino acid tag is present on the amino terminus of the CD16 polypeptide.

23. The cell or population of cells of claim 21, wherein the amino acid tag is present on the carboxy terminus of the CD16 polypeptide.

24. The cell or population of cells of any one of claims 21-23, wherein the amino acid tag is a 6xHis (HHHHHH) tag or a myc tag (EQKLISEEDL).

25. The cell or population of cells of any one of claims 1-24, wherein the cell or population of cells are natural killer cells.

26. The cell or population of cells of claim 25, wherein the natural killer cells are placental-derived natural killer cells.

27. The cell or population of cells of claim 26, wherein the placental-derived natural killer (NK) cells are CYNK cells.

28. The cell or population of cells of claim 25, wherein the CYNK cells are placental CD34+ cell-derived natural killer (NK) cells.

29. The cell or population of cells of any one of claims 1-28, 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.

30. The cell or population of cells of any one of claims 1-29, 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.

31. The cell or population of cells of any one of claims 1-29, 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.

32. The cell or population of cells of any one of claims 1-31, 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.

33. The cell or population of cells of any one of claims 1-31, 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, C.sub.15orf48, 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.

34. The cell or population of cells of any one of claims 1-33, wherein a nucleic acid encoding the cleavage resistant CD16 has been introduced into the NK cells by transfection.

35. The cell or population of cells of any one of claims 1-33, wherein a nucleic acid encoding the cleavage resistant CD16 has been introduced into the NK cells by transduction.

36. The cell or population of cells of any one of claims 1-35, wherein a nucleic acid encoding the cleavage resistant CD16 has been introduced into the NK cells by retroviral transduction.

37. The cell or population of cells of any one of claims 1-35, wherein a nucleic acid encoding the cleavage resistant CD16 has been introduced into the NK cells by lentiviral transduction.

38. The cell or population of cells of any one of claims 1-37, wherein greater than 90% of the cells in the population are CD56+ and CD3.

39. The cell or population of cells of any one of claims 1-38, wherein less than 1% of the cells in the population are CD3+.

40. The cell or population of cells of any one of claims 1-39, wherein less than 1% of the cells in the population are CD19+.

41. The cell or population of cells of any one of claims 1-40, wherein greater than 65% of the cells in the population are CD16+.

42. The cell or population of cells of any one of claims 1-41, wherein the population of cells comprises cells which express one or more surface markers selected from the group consisting of CD226, NKG2D, CD11a, NKp30, NKp44, NKp46, CD94, and combinations thereof.

43. The cell or population of cells of any one of claims 1-42, wherein the population of cells exhibit greater antibody-dependent cellular cytotoxicity than a population of placental-derived natural killer cells lacking expression of the cleavage resistant CD16.

44. A method of suppressing the proliferation of tumor cells comprising contacting the tumor cells with a population of placental-derived natural killer cells comprising a cleavage resistant CD16 and an antibody, wherein the tumor cells are HER2+, and wherein the antibody is an anti-HER2 antibody.

45. The method of suppressing the proliferation of tumor cells of claim 44, wherein the placental-derived natural killer (NK) cells are CYNK cells.

46. The method of suppressing the proliferation of tumor cells of claim 45, wherein the CYNK cells are placental CD34+ cell-derived natural killer (NK) cells.

47. The method of suppressing the proliferation of tumor cells of claim 44, wherein the placental-derived natural killer cells are cells of any one of claims 1-43.

48. The method of suppressing the proliferation of tumor cells of any one of claims 44-47, wherein said contacting is contacting in vitro.

49. The method of suppressing the proliferation of tumor cells of any one of claims 44-47, wherein said contacting is contacting in vivo.

50. The method of suppressing the proliferation of tumor cells of claim 46, wherein said contacting is in a human.

51. The method of suppressing the proliferation of tumor cells of any one of claims 44-50, wherein the tumor cells are tumor cells from a cancer selected from the group consisting of Bladder cancers, Breast cancers, Cervical cancers, Cholangiocarcinomas (extrahepatic), Cholangiocarcinomas (intrahepatic), Colorectal cancers, Esophageal or esophagogastric junction cancers, Gallbladder cancers, Gastric adenocarcinomas, Gastrointestinal stromal tumors, Glioblastoma multiforme, high grade gliomas, Gliomas (low grade), Head and neck carcinomas, Hepatocellular carcinomas, Intestinal (small) malignancies, Kidney cancers, Lung cancers (non small cells), Lung cancers (small cells), Melanomas, Melanomas (uveal), Neuroendocrine tumors, Oligodendrogliomas, Ovarian (epithelial) cancers, Ovarian (non-epithelial) cancers, Pancreatic adenocarcinomas, Penile cancers, Pituitary cancers, Prostate cancers, Sarcomas (peritoneal, retroperitoneal), Sarcomas (soft tissues), Solitary fibrous tumors, Testicular cancers, Thymic cancers, Thyroid cancers, Uterine cancers, and combinations thereof.

52. The method of suppressing the proliferation of tumor cells of any one of claims 44-50, wherein the tumor cells are gastric cancer cells.

53. The method of suppressing the proliferation of tumor cells of any one of claims 44-52, wherein the anti-HER2 antibody is Trastuzumab.

54. A method of treating a HER2+ cancer in a subject, comprising administering to the subject a population of placental-derived natural killer cells comprising a cleavage resistant CD16 and an antibody, wherein the antibody is an anti-HER2 antibody.

55. The method of claim 54, wherein the placental-derived natural killer (NK) cells are CYNK cells.

56. The method of claim 55, wherein the CYNK cells are placental CD34+ cell-derived natural killer (NK) cells.

57. The method of any one of claims 54-56, wherein the placental-derived natural killer cells are cells of any one of claims 1-43.

58. The method of any one of claims 54-57, wherein the tumor cells are tumor cells from a cancer selected from the group consisting of Bladder cancers, Breast cancers, Cervical cancers, Cholangiocarcinomas (extrahepatic), Cholangiocarcinomas (intrahepatic), Colorectal cancers, Esophageal or esophagogastric junction cancers, Gallbladder cancers, Gastric adenocarcinomas, Gastrointestinal stromal tumors, Glioblastoma multiforme, high grade gliomas, Gliomas (low grade), Head and neck carcinomas, Hepatocellular carcinomas, Intestinal (small) malignancies, Kidney cancers, Lung cancers (non small cells), Lung cancers (small cells), Melanomas, Melanomas (uveal), Neuroendocrine tumors, Oligodendrogliomas, Ovarian (epithelial) cancers, Ovarian (non-epithelial) cancers, Pancreatic adenocarcinomas, Penile cancers, Pituitary cancers, Prostate cancers, Sarcomas (peritoneal, retroperitoneal), Sarcomas (soft tissues), Solitary fibrous tumors, Testicular cancers, Thymic cancers, Thyroid cancers, Uterine cancers, and combinations thereof.

59. The method of any one of claims 54-57, wherein the tumor cells are gastric cancer cells.

60. The method of any one of claims 54-59, wherein the anti-HER2 antibody is Trastuzumab.

61. The method of claim 60, wherein said population of placental-derived natural killer cells comprising a cleavage resistant CD16 and/or said anti-HER2 antibody are administered intravenously.

62. The method of claim 60 or claim 61, wherein the population of placental-derived natural killer cells comprising a cleavage resistant CD16 and the anti-HER2 antibody are administered sequentially.

63. The method of claim 60 or claim 61, wherein the population of placental-derived natural killer cells comprising a cleavage resistant CD16 and the anti-HER2 antibody are administered concurrently.

64. A population of human placental-derived natural killer cells comprising a cleavage resistant CD16 for use in the manufacture of a medicament for treatment of a HER2+ cancer in a subject.

65. Use of a composition comprising a population of human placental-derived natural killer cells comprising a cleavage resistant CD16 for treatment of a HER2+ cancer in a subject.

66. The cell or population of cells of any one of claims 1-24, wherein the cell or population of cells are T cells.

67. The cell or population of cells of claim 66, wherein the T cells are placental-derived T cells.

68. The cell or population of cells of claim 67, wherein the placental-derived T cells are cord blood T cells.

69. The cell or population of cells of claim 67, wherein the placental-derived T cells are placental perfusate T cells.

70. The cell or population of cells of any one of claims 66-69, wherein, in said population of T cells, the predominant subpopulation of CAR+ T cells has a T scm/naive phenotype.

71. The cell or population of cells of any one of claims 66-70, wherein said population of T cells has a greater percentage of cells expressing CD45RA than a population of peripheral blood mononuclear cell T cells.

72. The cell or population of cells of any one of claims 66-71, wherein said population of T cells has a greater percentage of cells expressing CD27 than a population of peripheral blood mononuclear cell T cells.

73. The cell or population of cells of any one of claims 66-72, wherein said population of T cells has a greater percentage of cells expressing CCR7 than a population of peripheral blood mononuclear cell T cells.

74. The cell or population of cells of any one of claims 66-73, wherein said population of T cells has a greater percentage of cells expressing CD 127 than a population of peripheral blood mononuclear cell T cells.

75. The cell or population of cells of any one of claims 66-74, wherein said population of T cells has a lower percentage of cells expressing CD57 than a population of peripheral blood mononuclear cell T cells.

76. The cell or population of cells of any one of claims 66-75, wherein said population of T cells has a greater percentage of cells expressing CD62L than a population of peripheral blood mononuclear cell T cells.

77. The cell or population of cells of any one of claims 66-76, wherein said population of T cells has a lower percentage of cells expressing CD25 than a population of peripheral blood mononuclear cell T cells.

78. The cell or population of cells of any one of claims 66-77, wherein said population of T cells has a greater percentage of cells expressing Lag-3+ than a population of peripheral blood mononuclear cell T cells.

79. The cell or population of cells of any one of claims 66-78, wherein said population of T cells has a lower percentage of cells expressing Tim-3 than a population of peripheral blood mononuclear cell T cells.

80. The present invention also provides methods of suppressing the proliferation of tumor cells comprising contacting the tumor cells with a population of placental-derived T cells comprising a cleavage resistant CD16 and an antibody, wherein the tumor cells are HER2+, and wherein the antibody is an anti-HER2 antibody.

81. The present invention also provides methods of treating a HER2+ cancer in a subject, comprising administering to the subject a population of placental-derived T cells comprising a cleavage resistant CD16 and an antibody, wherein the antibody is an anti-HER2 antibody.

82. The present invention also provides methods of suppressing the proliferation of tumor cells comprising contacting the tumor cells with a population of placental-derived T cells comprising a cleavage resistant CD16 and an antibody, wherein the antibody is an anti-PD-L1 antibody.

83. The present invention also provides methods of treating a cancer in a subject, comprising administering to the subject a population of placental-derived T cells comprising a cleavage resistant CD16 and an antibody, wherein the antibody is an anti-PD-L1 antibody.

84. The method of suppressing the proliferation of tumor cells of claim 80 or claim 82 or the method of treating a cancer in a subject of claim 81 or claim 83, wherein the population of T cells is a population of any one of claims 66-79.

Description

4. BRIEF DESCRIPTION OF THE FIGURES

[0071] FIG. 1 shows expansion of NK cells for compounds CRL1-CRL11.

[0072] FIG. 2 shows expansion of NK cells for compounds CRL12-CRL22.

[0073] FIG. 3 shows expansion of NK cells relative to SR1 positive control.

[0074] FIG. 4 shows expansion of CD34+ cells from which the NK cells were derived.

[0075] FIGS. 5A-5B show cytotoxicity of the expanded NK cultures.

[0076] FIGS. 6A-6C show 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 10 Genomics Chromium platform and Illumina sequencing. Bioinformatics analysis utilized 10 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.

[0077] 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 meanSD of three donors for CYNK and PBNK cells (n=3). * p<0.05, ** p<0.005, *** p<0.001.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] FIG. 11A shows a schema of placental CD34+ cells expanded and differentiated to NK cells. FIG. 11B and FIG. 11C show total fold expansion of 35-day cultures and confirmation of NK phenotype of CD16VP cells (n=8 donors). FIG. 11D shows examples of phenotypic characterization of CD16VP cells.

[0082] FIG. 12A shows expression of CD 16 on non transduced cells (NT) and CD16 VP groups after transduction and on day 35 (n=8 donors). FIG. 12B and FIG. 12C show quantitative assessment of CD 16 shedding (n=4) and representative flow cytometry diagrams showing CD 16 shedding of NT and CD 16 VP cells upon activation by PMA/ionomycin.

[0083] FIG. 13A shows representative examples of ADCC activities of CD 16 VP cells compared to NT cells against Daratumumab or Rituximab opsonized lymphoma cells lines (line graphs) and average ADCC activities of CD 16 VP cells compared to NT cells against Daratumumab opsonized lymphoma cells lines at 10 1 E/T ratio (n=3 to 5) * indicates significant higher activities compared to NT control (bar graphs, p<0 05). FIG. 13B shows ADCC of CD 16 VP cells before and after PMA/ionomycin treatment against Daratumumab opsonized Daudi cells.

[0084] FIG. 14A shows a schematic study plan to test in vivo ADCC activities of CD16VP cells using a disseminated Daudi Xenograft model. FIG. 14B shows bioluminescence images of mice from each group after 10 days of cell infusion. FIG. 14C shows analysis of BLI after 10 days of cell infusion. * indicates a significant difference in between groups (n=6, p<0.05). FIG. 14D shows survival of mice through the course of the study.

[0085] FIGS. 15A-15B show expression of cell surface markers on CYNK CD16-VP cells.

[0086] FIG. 16 shows expression of CD16 on CYNK CD16-VP cells.

[0087] FIG. 17 shows resistance to shedding of CD16 on activated CYNK CD16-VP cells from multiple donors.

[0088] FIG. 18 shows a schema of CD16 mediated lysis of opsonized tumor cell.

[0089] FIG. 19 shows a schema of CD16 with the high affinity (158V) modification and an exemplary shedding resistance (197P) modification.

[0090] FIG. 20 shows a study schema of an in vivo efficacy and safety study.

[0091] FIGS. 21A and 21B show that CYNK-101 cells exert enhanced cytotoxic activity in combination with Herceptin against HER2 overexpressing gastric tumor cell lines.

[0092] FIG. 22 shows CYNK-101 in combination with Herceptin specifically kill HER2 overexpressing gastric tumor cells and spare low HER2 expression normal cells. FIG. 22A shows the expression of HER2 on NHDF, PBMC and NCI-N87. FIG. 22B shows the average cytotoxicity of CYNK-101 (n=3 donors) in combination with Herceptin against mixed targets of NCI-N87 and NHDF. CYNK-101 plus IgG, IgG alone and Herceptin alone served as control.

[0093] FIG. 23 shows that CYNK-101 cells express high expression of CD16. FIG. 23 shows CD16 expression on CD56+/CD3 CYNK-101 cells (meanSD, n=7 donors) with a representative CYNK-101 donor CD16 flow cytometry graph shown on the left.

[0094] FIGS. 24A-24B show that CYNK-101 is resistant to CD16 shedding post PMAi stimulation. FIG. 24A shows representative flow cytometry graphs showing CD16 loss following cleavage on NT cells but cleavage resistance on CYNK-101 cells following 2 h activation by PMAi. FIG. 24B shows quantification of CD16 cleavage resistance in CYNK-101 cells (n=7 donors) and lack of cleavage resistance in a NT (non-transduced) donor.

[0095] FIGS. 25A-25B show that CYNK-101 cells exert enhanced cytotoxic activity in combination with Herceptin against HER2+ breast cancer cell lines. FIGS. 25A-25B shows the average cytotoxic activity of CYNK-101 cells in combination with Herceptin or a control IgG against the indicated breast cancer cell lines, AU565, BT-474, HCC-1954, SK-BR-3 and ZR-75-30. The error bars represent the SD from the mean calculated from seven different CYNK-101 donors (unless specified). Cytotoxicity from Herceptin or IgG alone is included for reference. * indicate significantly higher activity of CYNK-101 in combination with Herceptin compared to that of IgG (***p<0.001, **p<0.005, and *p<0.05).

[0096] FIG. 26 shows increased IFN- production of CYNK-101 in combination with Herceptin against HER2+ breast cancer cell lines. FIG. 26 shows the average of IFN- production of target cell alone, CYNK-101 alone, or CYNK-101 coculture with target cells in the presence of Herceptin or IgG control after 24 h incubation. The error bars represent the SD from the mean calculated from different CYNK-101 donors (*p<0.05).

[0097] FIG. 27 shows the purity of CYNK-101 enriched from mouse liver post injection. FIG. 27 shows percent NK purity of the cells from the livers of NSG mice pre and post mouse cell depletion in ex vivo study. Representative data from total of three studies.

[0098] FIG. 28 shows ex vivo CYNK-101 phenotype characterization. FIG. 28 shows NK surface marker expression on the in vitro CYNK-101 donor (prior infusion CYNK-101) compared to the ex vivo CYNK-101.

[0099] FIG. 29 shows ex vivo CYNK-101 is resistant to PMAi stimulated CD16 shedding. FIG. 29 shows in vitro CYNK-101 and ex vivo CYNK-101 shedding resistance post PMAi stimulation: CD16 expression on control, in vitro CYNK-101 and ex vivo CYNK-101 without stimulation (top graphs). CD16 expression on control, in vitro CYNK-101 and ex vivo CYNK-101 stimulated by PMAi for 2 h (bottom graphs).

[0100] FIG. 30 shows ex vivo CYNK-101 displays ADCC activity against NCI-N87 at E:T ratio of 0.5:1. FIG. 30 shows cytotoxicity of ex vivo CYNK-101 or in vitro CYNK-101 in combination with Herceptin against NCI-N87 at E:T ratio of 0.5:1. CYNK-101+IgG, Herceptin alone or IgG alone served as control.

[0101] FIG. 31 shows cytokine secretion profiling of ex vivo CYNK-101 in combination with Herceptin against NCI-N87. FIG. 31 shows cytokine secretion profiling of ex vivo CYNK-101 or in vitro CYNK-101 in combination with Herceptin against NCI-N87 at E:T ratio of 0.5:1.

[0102] FIG. 32 shows in vivo efficacy of CYNK-101 in combination with Trastuzumab in NCI-N87 xenograft NSG-Tg-hIL15 mouse model. FIG. 32 shows percentage of tumor volume change over Day 7 baseline. Animals were preconditioned with busulfan on Day 3. NCI-N87 cells were subcutaneously injected on Day 0. Trastuzumab was intraperitonially injected on Day 7. Vehicle or CYNK-101 was intravenously administered on Days 7, 14 and 21. The data are presented as the meanSEM; Multiple t tests were performed to compare Trastuzumab with Vehicle and Trastuzumab with CYNK-101+Trastuzumab. ** P<0.01, *** P<0.001, ****P<0.0001.

[0103] FIG. 33 shows a schema of CD16 mediated lysis of opsonized tumor cell.

[0104] FIG. 34 shows a schema of CD16 with the high affinity (158V) and shedding resistance (197P) modifications.

[0105] FIG. 35 shows the structure of proposed amino acid substitutions.

[0106] FIG. 36 shows a schema of a strategy to replace the membrane proximal stalk sequence with proposed sequences.

[0107] FIG. 37 shows a schema of a strategy to replace multiple amino acids in the cleavage region.

[0108] FIG. 38 shows Day 10 CD16 expression from a titration of CD16 variant constructs. Percentage of CD16 expression on Day 10 cells transduced with CD16 variants at indicated MOI, CD16VP generated by Lentigen with MOI 150 was served as control.

[0109] FIGS. 39A-39B show CD16 expression of CD16 variants at different time points. Representative CD16 expression of CD16 variants with Myc tag generated by Sirion Bio at different time points as indicated. CD16VP generated by Lentigen and NT (nontransduced) CYNK cells were served as control. A) CD16 expression at Day 10. B) CD16 expression at Day 21, 28, 35 and 40.

[0110] FIG. 40 shows CD16 shedding resistance evaluation of CD16 variants transduced CYNK cells. CD16 expression of CD16 variants transduced CYNK cells in the presence or absence of PMAi stimulation. The NT CYNK cell were served as a control.

[0111] FIGS. 41A-41B show Cytotoxicity evaluation of CD16 variants transduced CYNK cells in combination with trastuzumab against HER2+ NCI-N87 gastric cancer cells. Cytotoxicity of CD16 variants transduced CYNK cells in combination with trastuzumab against HER2+ NCI-N87 gastric cancer cells at E:T ratios from 10:1 down to 0.6:1. A) Cytotoxicity of Construct #5 transduced CYNK in combination with trastuzumab against NCI-N87 at 4 h (top) and 24 h (bottom). B) Cytotoxicity of CD16VP transduced CYNK in combination with trastuzumab against NCI-N87 at 4 h (top) and 24 h (bottom).

[0112] FIG. 42 shows fold expansion of CD16 variants transduced CYNK cells.

[0113] FIG. 43 shows CD16 expression of CD16 variants transduced CYNK cells at different timepoints. CD16 expression of constructs #1, 5, 10 and CD16VP on CYNK cells at different timepoints as indicated. Each symbol represents one donor.

[0114] FIGS. 44A-44B show CD16 shedding evaluation of CD16 variants transduced CYNK cells post PMAi stimulation. A) Representative CD16 expression of CD16 variants transduced CYNK cells in the presence or absence of PMAi stimulation. A NT CYNK cells were served as control. For each histogram, the X-axis shows CD16 PE fluorescence, and the Y-axis shows CD56 APC fluorescence. B) The shedding percentage of CD16 from CD16 variants transduced CYNK cell and NT controls (n=6 donors). Each symbol represents one donor.

[0115] FIG. 45 shows the fold expansion of CD16 variants transduced CYNK cells. Each symbol represents one donor (n=6 donors).

[0116] FIG. 46 shows ADCC activity of CD16 variants transduced CYNK cells in combination with Trastuzumab against NCI-N87. Change of cytotoxicity from CYNK cells (n=6 donors) at 4 h (top) and 24 h (bottom) in combination with trastuzumab against NCI-N87 at E:T ratio of 2:1. Change of cytotoxicity=Cytotoxicity(CYNK+Trastuzumab)Cytotoxicity(CYNK+IgG) * P<0.05.

[0117] FIG. 47 shows cytokine secretion of CD16 variant transduced CYNK cells in combination with Trastuzumab against NCI-N87.

[0118] FIG. 48 shows 24-hour ADCC results of PT-CD16VS and NT cells against RT-112.

[0119] FIG. 49 shows 24-hour ADCC results of PT-CD16VS and NT cells against T-24.

[0120] FIG. 50 shows 24-hour ADCC results of PT-CD16VS and NT cells against MDA-MB-231.

[0121] FIG. 51 shows 24-hour ADCC results of PT-CD16VS and NT cells against NCI-H1975.

[0122] FIG. 52 shows 24-hour ADCC results of PT-CD16VS and NT cells against 5637.

5. DETAILED DESCRIPTION

[0123] 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.

[0124] 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

[0125] 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.

[0126] 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%).

[0127] 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.

[0128] 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.

[0129] In certain embodiments, the hematopoietic cells are CD34.sup.+ cells. In specific embodiments, the hematopoietic cells useful in the methods disclosed herein are CD34.sup.+CD38.sup.+ or CD34.sup.+CD38.sup.. In a more specific embodiment, the hematopoietic cells are CD34.sup.+CD38.sup.Lin.sup.. In another specific embodiment, the hematopoietic cells are one or more of CD2.sup., CD3.sup., CD11b.sup., CD11c.sup., CD14.sup., CD16.sup., CD19.sup., CD24.sup., CD56.sup., CD66b.sup. and/or glycophorin A.sup.. In another specific embodiment, the hematopoietic cells are CD2.sup., CD3.sup., CDTTb.sup., CD11c.sup., CD14.sup., CD16.sup., CD19.sup., CD24.sup., CD56.sup., CD66b.sup. and glycophorin A.sup.. In another more specific embodiment, the hematopoietic cells are CD34.sup.+CD38.sup.CD33.sup.CD117.sup.. In another more specific embodiment, the hematopoietic cells are CD34.sup.+CD38.sup.CD33.sup.CD117.sup.CD235.sup.CD36.sup..

[0130] In another embodiment, the hematopoietic cells are CD45.sup.+. In another specific embodiment, the hematopoietic cells are CD34.sup.+CD45.sup.+. In another embodiment, the hematopoietic cell is Thy-1.sup.+. In a specific embodiment, the hematopoietic cell is CD34.sup.+Thy-1.sup.+. In another embodiment, the hematopoietic cells are CD133.sup.+. In specific embodiments, the hematopoietic cells are CD34.sup.+CD133.sup.+ or CD133.sup.+Thy-1.sup.+. In another specific embodiment, the CD34.sup.+ hematopoietic cells are CXCR4.sup.+. In another specific embodiment, the CD34.sup.+ hematopoietic cells are CXCR4.sup.. In another embodiment, the hematopoietic cells are positive for KDR (vascular growth factor receptor 2). In specific embodiments, the hematopoietic cells are CD34.sup.+KDR.sup.+, CD133.sup.+KDR.sup.+ or Thy-1.sup.+KDR.sup.+. In certain other embodiments, the hematopoietic cells are positive for aldehyde dehydrogenase (ALDH.sup.+), e.g., the cells are CD34.sup.+ALDH.sup.+.

[0131] In certain other embodiments, the CD34.sup.+ cells are CD45.sup.. In specific embodiments, the CD34.sup.+ cells, e.g., CD34.sup.+, CD45.sup. 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.

[0132] In certain embodiments, the hematopoietic cells are CD34.sup..

[0133] The hematopoietic cells can also lack certain markers that indicate lineage commitment, or a lack of developmental naivet. For example, in another embodiment, the hematopoietic cells are HLA-DR.sup.. In specific embodiments, the hematopoietic cells are CD34.sup.+HLA-DR.sup., CD133.sup.+HLA-DR.sup., Thy-1.sup.+HLA-DR.sup. or ALDH.sup.+HLA-DR.sup. 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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

[0138] In certain embodiments, the hematopoietic cells used in the methods provided herein are placental hematopoietic cells. In one embodiment, placental hematopoietic cells are CD34V. 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.sup.+CD38.sup. 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.sup.+CD38.sup.+ 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.

[0139] In another embodiment, the placental hematopoietic cell is CD45.sup.. In a specific embodiment, the hematopoietic cell is CD34.sup.+CD45.sup.. In another specific embodiment, the placental hematopoietic cells are CD34.sup.+CD45.sup.+.

5.2. Production of Natural Killer and/or ILC3 Cells and Natural Killer Cell and/or ILC3 Cell Populations

[0140] 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.sup.+ 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0141] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0142] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0143] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0144] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0145] 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 RORt or IL1R1.

[0146] 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 CDTTa. 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). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0147] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0148] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0149] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0150] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, and Flt3L. In certain embodiments, none of the first medium, second medium or third medium comprises heparin, e.g., low-molecular weight heparin.

[0151] In certain embodiments, said ILC3 cells express RORt 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

[0152] 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 210.sup.4 and about 610.sup.6 cells per milliliter. In certain aspects, said hematopoietic stem or progenitor cells are initially inoculated into said first medium from 110.sup.4 to 110.sup.5 cells/mL. In a specific aspect, said hematopoietic stem or progenitor cells are initially inoculated into said first medium at about 310.sup.4 cells/mL.

[0153] In certain aspects, said first population of cells are initially inoculated into said second medium from 510.sup.4 to 510.sup.5 cells/mL. In a specific aspect, said first population of cells is initially inoculated into said second medium at about 110.sup.5 cells/mL.

[0154] In certain aspects said second population of cells is initially inoculated into said third medium from 110.sup.5 to 510.sup.6 cells/mL. In certain aspects, said second population of cells is initially inoculated into said third medium from 110.sup.5 to 110.sup.6 cells/mL. In a specific aspect, said second population of cells is initially inoculated into said third medium at about 510.sup.5 cells/mL. In a more specific aspect, said second population of cells is initially inoculated into said third medium at about 510.sup.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 310.sup.5 cells/mL. In a more specific aspect, said second population of cells is initially inoculated into said third medium at about 310.sup.5 cells/mL in a static culture.

[0155] 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.sup.+ 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-1) or both.

[0156] 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-1) or both.

[0157] 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-1, 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.

[0158] 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).

[0159] 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 1 U/mL to 10 U/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 4 U/mL to 5 U/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 4.5 U/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 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 0.25 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 SCGM, STEMMACS, GBGM, AIM-V, X-VIVO 10, X-VIVO 15, OPTMIZER, STEMSPAN H3000, CELLGRO COMPLETE, 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-VIVO 10, X-VIVO 15, OPTMIZER, STEMSPAN H3000, CELLGRO COMPLETE 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-1, or both.

[0160] 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 1 U/mL to 10 U/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 4 U/mL to 5 U/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 4 U/mL to 5 U/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 4.5 U/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 SCGM, STEMMACS, GBGM, AIM-V, X-VIVO 10, X-VIVO 15, OPTMIZER, STEMSPAN H3000, CELLGRO COMPLETE 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-VIVO 10, X-VIVO 15, OPTMIZER, STEMSPAN H3000, CELLGRO COMPLETE, 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-1, or both.

[0161] 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-1, Flt-3L.

[0162] 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 SCGM, STEMMACS, GBGM, AIM-V, X-VIVO 10, X-VIVO 15, OPTMIZER, STEMSPAN H3000, CELLGRO COMPLETE, 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-VIVO 10, X-VIVO 15, OPTMIZER, STEMSPAN H3000, CELLGRO COMPLETE 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] In certain aspects, the three-stage method disclosed herein produces natural killer cells that comprise at least 20% CD56+CD3CDTTa+ 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] In certain aspects, provided herein is a population of natural killer cells that is CD56+CD3 CD117+CD11a+, wherein said natural killer cells express perform and/or EOMES, and do not express one or more of RORt, aryl hydrocarbon receptor (AHR), and IL1R1. In certain aspects, said natural killer cells express perform and EOMES, and do not express any of RORt, 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.

[0179] 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 RORt, aryl hydrocarbon receptor, and IL1R1, and do not express one or more of CD94, perform, and EOMES. In certain aspects, said ILC3 cells express RORt, aryl hydrocarbon receptor, and IL1R1, and do not express any of CD94, perform, 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, TNF, and DNAM-1. In certain aspects, said ILC3 cells express CD226, 2B4, IL-22, TNF, and DNAM-1.

[0180] 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-1). In certain embodiments, said third medium lacks LIF, MIP-1, and FMS-like tyrosine kinase-3 ligand (Flt-3L). In specific embodiments, said first medium and said second medium lack LIF and MIP-1, and said third medium lacks LIF, MIP-1, 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 CDTTa+ 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 CDT 1a+ cells and CD11a cells are combined in a ratio of 1:5. In certain aspects, in the fourth population of cells, the CDT 1a+ 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

[0181] To facilitate understanding of the disclosure of stem cell mobilizing factors set forth herein, a number of terms are defined below.

[0182] 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.

[0183] 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.

[0184] As used herein, any R group(s) such as, without limitation, R.sup.a, R.sup.b, R.sup.c, R.sup.d, R.sup.e, R.sup.f, R.sup.g, R.sup.h, R.sup.m, R.sup.G, R.sup.J, R.sup.K, R.sup.U, R.sup.V, R.sup.Y, and R.sup.Z 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 R.sup.a and R.sup.b of an NR.sup.aR.sup.b group are indicated to be taken together, it means that they are covalently bonded to one another to form a ring:

##STR00001##

[0185] 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.

[0186] 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.

[0187] As used herein, C.sub.a to C.sub.b 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 C.sub.1 to C.sub.4 alkyl group refers to all alkyl groups having from 1 to 4 carbons, that is, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, (CH.sub.3).sub.2CH, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH(CH.sub.3) and (CH.sub.3).sub.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.

[0188] 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 C.sub.1-C.sub.4 alkyl or similar designations. By way of example only, C.sub.1-C.sub.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. 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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 C.sub.6-C.sub.14 aryl group, a C.sub.6-C.sub.10 aryl group, or a C.sub.6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. An aryl group may be substituted or unsubstituted.

[0194] 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.

[0195] 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).

[0196] 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.

[0197] 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.

[0198] 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). [00200]Lower alkylene groups are straight-chained CH.sub.2 tethering groups, forming bonds to connect molecular fragments via their terminal carbon atoms. Examples include but are not limited to methylene (CH.sub.2), ethylene (CH.sub.2CH.sub.2), propylene (CH.sub.2CH.sub.2CH.sub.2), and butylene (CH.sub.2CH.sub.2CH.sub.2CH.sub.2). 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.

[0199] 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.

[0200] 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.

[0201] As used herein, acylalkyl refers to an acyl connected, as a substituent, via a lower alkylene group. Examples include aryl-C(O)(CH.sub.2).sub.n and heteroaryl-C(O)(CH.sub.2).sub.n, where n is an integer in the range of 1 to 6.

[0202] As used herein, alkoxyalkyl refers to an alkoxy group connected, as a substituent, via a lower alkylene group. Examples include C.sub.1-4 alkyl-O(CH.sub.2).sub.n, wherein n is an integer in the range of 1 to 6.

[0203] As used herein, aminoalkyl refers to an optionally substituted amino group connected, as a substituent, via a lower alkylene group. Examples include H.sub.2N(CH.sub.2).sub.n, wherein n is an integer in the range of 1 to 6.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] A sulfonyl group refers to an SO.sub.2R group in which R can be the same as defined with respect to sulfenyl. A sulfonyl may be substituted or unsubstituted.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] A trihalomethanesulfonyl group refers to an X.sub.3CSO.sub.2 group wherein each X is a halogen.

[0214] A trihalomethanesulfonamido group refers to an X.sub.3CS(O).sub.2N(R.sub.A) 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).

[0215] The term amino as used herein refers to a NH.sub.2 group.

[0216] As used herein, the term hydroxy refers to a OH group.

[0217] A cyano group refers to a CN group.

[0218] The term azido as used herein refers to a N.sub.3 group.

[0219] An isocyanato group refers to a NCO group.

[0220] A thiocyanato group refers to a CNS group.

[0221] An isothiocyanato group refers to an NCS group.

[0222] A carbonyl group refers to a CO group.

[0223] An S-sulfonamido group refers to a SO.sub.2N(R.sub.AR.sub.B) 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.

[0224] An N-sulfonamido group refers to a RSO.sub.2N(R.sub.A) 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.

[0225] An O-carbamyl group refers to a OC(O)N(R.sub.AR.sub.B) 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.

[0226] An N-carbamyl group refers to an ROC(O)N(R.sub.A) 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.

[0227] An O-thiocarbamyl group refers to a OC(S)N(R.sub.AR.sub.B) 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.

[0228] An N-thiocarbamyl group refers to an ROC(S)N(R.sub.A) 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.

[0229] A C-amido group refers to a C(O)N(R.sub.AR.sub.B) 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.

[0230] An N-amido group refers to a RC(O)N(R.sub.A) 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.

[0231] A urea group refers to N(R)C(O)NR.sub.AR.sub.B 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.

[0232] 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.

[0233] As used herein, indicates a single or double bond, unless stated otherwise.

[0234] 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, C.sub.1-C.sub.3 alkoxyphenyl may include one or more of the same or different alkoxy groups containing one, two or three atoms.

[0235] 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)).

[0236] 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.

[0237] 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.

[0238] 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 (.sup.1H), deuterium (.sup.2H), tritium (H), carbon-11 (.sup.11C), carbon-12 (.sup.12C), carbon-13 (.sup.13C), carbon-14 (.sup.14C), nitrogen-13 (.sup.13N), nitrogen-14 (.sup.14N), nitrogen-15 (.sup.15N), oxygen-14 (.sup.14O), oxygen-15 (.sup.15O), oxygen-16 (.sup.16O), oxygen-17 (.sup.17O), oxygen-18 (.sup.18O), fluorine-17 (.sup.17F), fluorine-18 (.sup.18F), phosphorus-31 (.sup.31P) phosphorus-32 (.sup.32P), phosphorus-33 (.sup.33P), sulfur-32 (.sup.32S), sulfur-33 (.sup.33S), sulfur-34 (.sup.34S), sulfur-35 (.sup.35S), sulfur-36 (.sup.36S), chlorine-35 (.sup.35Cl), chlorine-36 (.sup.36Cl), chlorine-37 (.sup.37Cl), bromine-79 (.sup.79Br), bromine-81 (.sup.81Br), iodine-123 (.sup.123I), iodine-125 (.sup.125I), iodine-127 (.sup.127I), iodine-129 (.sup.129I), and iodine-131 (.sup.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 (.sup.1H), deuterium (.sup.2H), carbon-12 (.sup.12C), carbon-13 (.sup.13C), nitrogen-14 (.sup.14N), nitrogen-15 (.sup.15N), oxygen-16 (.sup.16O), oxygen-17 (.sup.17O), oxygen-18 (.sup.18O), fluorine-17 (.sup.17F), phosphorus-31 (.sup.31P), sulfur-32 (32S), sulfur-33 (33S), sulfur-34 (34S), sulfur-36 (36S), chlorine-35 (.sup.35C.sub.1), chlorine-37 (.sup.37C.sub.1), bromine-79 (.sup.79Br), bromine-81 (.sup.81Br), and iodine-127 (.sup.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 (.sup.3H), carbon-11 (.sup.11C), carbon-14 (.sup.14C), nitrogen-13 (.sup.13N), oxygen-14 (.sup.14O), oxygen-15 (.sup.15O), fluorine-18 (.sup.18F), phosphorus-32 (.sup.32P), phosphorus-33 (.sup.33P), sulfur-35 (.sup.35S), chlorine-36 (.sup.36Cl), iodine-123 (.sup.123I), iodine-125 (.sup.125I), iodine-129 (.sup.129I), and iodine-131 (.sup.131I). It will be understood that, in a compound as provided herein, any hydrogen can be .sup.2H, for example, or any carbon can be .sup.13C, for example, or any nitrogen can be .sup.15N, for example, or any oxygen can be .sup.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).

[0239] 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.

[0240] 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

[0241] 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)

[0242] Some embodiments disclosed herein relate to a compound of Formula (I), or a pharmaceutically acceptable salt thereof, having the structure:

##STR00002##

wherein: each can independently represent a single bond or a double bond; R.sup.J can be selected from the group consisting of NR.sup.aR.sup.b, OR.sup.b, and O; wherein if R.sup.J is O, then joining G and J represents a single bond and G is N and the N is substituted with R.sup.G; otherwise joining G and J represents a double bond and G is N; R.sup.a can be hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.b can be R.sup.c or (C.sub.1-C.sub.4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of: OH, O(C.sub.1-C.sub.4 alkyl), O(C.sub.1-C.sub.4 haloalkyl); C(O)NH.sub.2; unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.c 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, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: hydrogen, unsubstituted C.sub.1-6 alkyl; substituted C.sub.1-6 alkyl; NH(C.sub.1-4 alkyl); N(C.sub.1-4 alkyl).sub.2, unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.K 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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4 haloalkyl); R.sup.G can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, and (C.sub.1-4 alkyl)-C(O)NH.sub.2; R.sup.Y and R.sup.Z can each independently be absent or be selected from the group consisting of: hydrogen, halo, C.sub.1-6 alkyl, OH, O(C.sub.1-4 alkyl), NH(C.sub.1-4 alkyl), and N(C.sub.1-4 alkyl).sub.2; or R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can joined together to form a ring selected from:

##STR00003##

wherein said ring can be optionally substituted with one, two, or three groups independently selected from C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, OH, O(C.sub.1-4 alkyl), N(C.sub.1-4 alkyl).sub.2, unsubstituted C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10 aryl substituted with 1-5 halo atoms, and O(C.sup.1-4 haloalkyl); and wherein if R.sup.Y and R.sup.Z taken together forms

##STR00004##

then R.sup.J can be OR.sup.b or O; R.sup.d can be hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.m can be selected from the group consisting of C.sub.1-4 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.

[0243] 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 represents a single bond, G can be N and the N is substituted with R.sup.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 represents a double bond, then joining J and R.sup.J can be a single bond. In some embodiments, when joining G and J represents a double bond, then joining J and R.sup.J can not be a double bond. In some embodiments, when joining J and R.sup.J represents a double bond, then joining G and J can be a single bond. In some embodiments, when joining J and R.sup.J represents a double bond, then joining G and J can not be a double bond.

[0244] In some embodiments, R.sup.J can be NR.sup.aR.sup.b. In other embodiments, R.sup.J can be OR.sup.b. In still other embodiments, R.sup.J can be O. In some embodiments, when R.sup.J is O, then joining G and J represents a single bond and G is N and the N is substituted with R.sup.G. In some embodiments, R.sup.G is CH.sub.2CH.sub.2C(O)NH.sub.2.

[0245] In some embodiments, R.sub.a can be hydrogen. In some embodiments, R.sup.a can be C.sub.1-C.sub.4 alkyl. For example, R.sup.a can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl.

[0246] In some embodiments, R.sup.b can be R.sup.c. In some embodiments, R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c. For example, R.sup.b can be CH.sub.2R.sup.c, CH.sub.2CH.sub.2R.sup.c, CH.sub.2CH.sub.2CH.sub.2R.sup.c, or CH.sub.2CH.sub.2CH.sub.2CH.sub.2R.sup.c. In some embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be O(C.sub.1-C.sub.4 alkyl). In other embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be O(C.sub.1-C.sub.4 haloalkyl). In still other embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be C(O)NH.sub.2.

[0247] In some embodiments, R.sup.c can be OH. In some embodiments, R.sup.c can be O(C.sub.1-C.sub.4 alkyl). In some embodiments, R.sup.c can be O(C.sub.1-C.sub.4 haloalkyl). In some embodiments, R.sup.c can be C(O)NH.sub.2. In some embodiments, R.sup.c can be unsubstituted C.sub.6-10 aryl. In some embodiments, R.sup.c can be substituted C.sub.6-10 aryl. In some embodiments, R.sup.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.sup.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.sup.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.sub.1-C.sub.4 alkyl. In some embodiments, E can be C.sub.1-C.sub.4 haloalkyl. In some embodiments, E can be O(C.sub.1-C.sub.4 alkyl). In some embodiments, E can be O(C.sub.1-C.sub.4 haloalkyl).

[0248] In some embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be unsubstituted C.sub.6-10 aryl. In other embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be substituted C.sub.6-10 aryl. In still other embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c 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, R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c and R.sup.c can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. When a R.sup.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 other embodiments, E can be C.sub.1-C.sub.4 alkyl. In still other embodiments, E can be C.sub.1-C.sub.4 haloalkyl. In still other embodiments, E can be O(C.sub.1-C.sub.4 alkyl). In still other embodiments, E can be O(C.sub.1-C.sub.4 haloalkyl).

[0249] In some embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be phenyl. In other embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be naphthyl. In still other embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be hydroxyphenyl. In still other embodiments, when R.sup.b is CH.sub.2CH.sub.2R.sup.c, R.sup.c can be indolyl.

[0250] In some embodiments, R.sup.K can be hydrogen. In other embodiments, R.sup.K can be unsubstituted C.sub.1-6 alkyl. For example, in some embodiments, R.sup.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). In other embodiments, R.sup.K can be substituted C.sub.1-6 alkyl. In other embodiments, R.sup.K can be NH(C.sub.1-4 alkyl). For example, in some embodiments, R.sup.K can be NH(CH.sub.3), NH(CH.sub.2CH.sub.3), NH(isopropyl), or NH(sec-butyl). In other embodiments, R.sup.K can be N(C.sub.1-4 alkyl).sub.2.

[0251] In some embodiments, R.sup.K can be unsubstituted C.sub.6-10 aryl. In other embodiments, R.sup.K can be substituted C.sub.6-10 aryl. In other embodiments, R.sup.K 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, R.sup.K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. When a R.sup.K moiety is indicated as substituted, the moiety can be substituted with one or more, for example, one, two, three, or four substituents Q. In some embodiments, Q can be OH. In other embodiments, Q can be C.sub.1-4 alkyl. In still other embodiments, Q can be C.sub.1-4 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(C.sub.1-4 alkyl). In still other embodiments, Q can be O(C.sub.1-4 haloalkyl).

[0252] In some embodiments, R.sup.K can be phenyl or naphthyl. In other embodiments, R.sup.K can be benzothiophenyl. In other embodiments, R.sup.K can be benzothiophenyl. In other embodiments, R.sup.K can be benzothiophenyl. In still other embodiments, R.sup.K can be pyridinyl. In yet still other embodiments, R.sup.K can be pyridinyl substituted with one or more substituents Q. For example, R.sup.K can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl.

[0253] In some embodiments, R.sup.G can be hydrogen. In some embodiments, R.sup.G can be C.sub.1-4 alkyl. In some embodiments, R.sup.G can be (C.sub.1-4 alkyl)-C(O)NH.sub.2.

[0254] In some embodiments, R.sup.Y and R.sup.Z can independently be absent. In other embodiments, R.sup.Y and R.sup.Z can independently be hydrogen. In other embodiments, R.sup.Y and R.sup.Z can independently be halo. In other embodiments, R.sup.Y and R.sup.Z can independently be C.sub.1-6 alkyl. In other embodiments, R.sup.Y and R.sup.Z can independently be OH. In still other embodiments, R.sup.Y and R.sup.Z can independently be O(C.sub.1-4 alkyl). In other embodiments, R.sup.Y and R.sup.Z can independently be NH(C.sub.1-4 alkyl). For example, R.sup.Y and R.sup.Z can independently be NH(CH.sub.3), NH(CH.sub.2CH.sub.3), NH(isopropyl), or NH(sec-butyl). In other embodiments, R.sup.Y and R.sup.Z can independently be N(C.sub.1-4 alkyl).sub.2.

[0255] In some embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form a ring. In some embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00005##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00006##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00007##

In still other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00008##

In yet still other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00009##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00010##

In yet other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00011##

In yet still other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00012##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00013##

In still other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form and

##STR00014##

In some embodiments, when R.sup.Y and R.sup.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.sub.1-C.sub.4 alkyl, N(C.sub.1-C.sub.4 alkyl).sub.2, cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms.

[0256] In some embodiments, when R.sup.Y and R.sup.Z taken together forms

##STR00015##

then R.sup.J can be OR.sup.b or O.

[0257] In some embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00016##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00017##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00018##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00019##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00020##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00021##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00022##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00023##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00024##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form

##STR00025##

In some embodiments, when R.sup.Y and R.sup.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.sub.1-C.sub.4 alkyl, N(C.sub.1-C.sub.4 alkyl).sub.2, cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms. In some embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be

##STR00026##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be

##STR00027##

In still other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be

##STR00028##

In yet still other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be

##STR00029##

In other embodiments, R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be

##STR00030##

[0258] In some embodiments, R.sup.d can be hydrogen. In other embodiments, R.sup.d can be C.sub.1-C.sub.4 alkyl. For example R.sup.d can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In still other embodiments, R.sup.d can be halo. In other embodiments, R.sup.d can be cyano.

[0259] In some embodiments, R.sup.m can be hydrogen. In other embodiments, R.sup.m can be C.sub.1-C.sub.4 alkyl. For example R.sup.m can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In still other embodiments, R.sup.m can be halo. For example, R.sup.m can be fluoro, chloro, bromo, or iodo. In other embodiments, R.sup.m can be cyano.

[0260] 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.

[0261] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of C(O)NH.sub.2; unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: hydrogen, unsubstituted C.sub.1-6 alkyl; NH(C.sub.1-4 alkyl); N(C.sub.1-4 alkyl).sub.2, unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.K 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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4 haloalkyl); R.sup.G can be (C.sub.1-4 alkyl)-C(O)NH.sub.2; R.sup.Y and R.sup.Z can each be independently absent or be selected from the group consisting of: hydrogen, C.sub.1-6 alkyl, and NH(C.sub.1-4 alkyl); or R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form a ring selected from:

##STR00031##

wherein said ring can be optionally substituted with one, two, or three groups independently selected from C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, OH, O(C.sub.1-4 alkyl), N(C.sub.1-4 alkyl).sub.2, unsubstituted C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10 aryl substituted with 1-5 halo atoms, and O(C.sub.1-4haloalkyl); R.sup.d can be C.sub.1-C.sub.4 alkyl; R.sup.m 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.

[0262] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be selected from the group consisting of: unsubstituted phenyl, substituted phenyl, indolyl, and C(O)NH.sub.2; R.sup.K can be selected from the group consisting of: hydrogen, methyl, substituted pyridinyl, unsubstituted benzothiophenyl, and NH(C.sub.1-C.sub.4 alkyl); R.sup.G can be CH.sub.2CH.sub.2C(O)NH.sub.2; R.sup.Y can be NH(C.sub.1-C.sub.4 alkyl); R.sup.Z can be absent or hydrogen; or R.sup.Y and R.sup.Z taken together with the atoms to which they are attached can be joined together to form a ring selected from

##STR00032##

wherein said ring can be optionally substituted with one, two, or three groups independently selected from C.sub.1-C.sub.4 alkyl, N(C.sub.1-C.sub.4 alkyl).sub.2, cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms; R.sup.d can be C.sub.1-C.sub.4 alkyl; R.sup.m can be cyano; and X can be N or CH.

[0263] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.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.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.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.sup.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.sub.1-C.sub.4 alkyl; R.sup.Y and R.sup.Z taken together can be

##STR00033##

[0264] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.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.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.K can be hydrogen, C.sub.1-4 alkyl, or unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and R.sup.Y and R.sup.Z taken together can be or

##STR00034##

[0265] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.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.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.K can be hydrogen, C.sub.1-4 alkyl, or unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and R.sup.Y and R.sup.Z taken together can be

##STR00035##

[0266] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond, R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be substituted C.sub.6-10 aryl; substituted with one or more E, wherein E can be OH; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y can be NH(C.sub.1-4 alkyl); R.sup.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. 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.

[0267] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c, R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E can be OH; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y and R.sup.Z taken together is

##STR00036##

wherein the ring is substituted with C.sub.1-C.sub.4 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.

[0268] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c, R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E can be OH; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y and R.sup.Z taken together is

##STR00037##

R.sup.d can be C.sub.1-C.sub.4 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.

[0269] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c, R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E can be OH; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y and R.sup.Z taken together is

##STR00038##

R.sup.d can be C.sub.1-C.sub.4 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.

[0270] In some embodiments, when R.sup.J is OR.sup.b; G can be N; joining G and J can be a double bond; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be C(O)NH.sub.2; R.sup.K can unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y and R.sup.Z taken together can be

##STR00039##

R.sup.d can be C.sub.1-C.sub.4 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.

[0271] In some embodiments, when R.sup.1 is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.K is unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y and R.sup.Z taken together can be

##STR00040##

wherein said ring is substituted with N(C.sub.1-4 alkyl).sub.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.

[0272] In some embodiments, when R.sup.1 is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; R.sup.Y can be NH(C.sub.1-4 alkyl); R.sup.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. 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.

[0273] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.K can be unsubstituted C.sub.1-6 alkyl; R.sup.Y and R.sup.Z taken together can

##STR00041##

wherein the ring is substituted with unsubstituted C.sub.6-C.sub.10 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

[0274] In some embodiments, when R.sup.J can be NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.K can be hydrogen; R.sup.Y and R.sup.Z taken together can be

##STR00042##

wherein the ring is substituted with substituted C.sub.6-C.sub.10 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

[0275] In some embodiments, when R.sup.J is O; G can be N substituted with R.sup.G; joining G and J can be a single bond; R.sup.G can be (C.sub.1-4 alkyl)-C(O)NH.sub.2; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of 0, N, and S; R.sup.Y and R.sup.Z taken together can be

##STR00043##

R.sup.d can be C.sub.1-C.sub.4 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.

[0276] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond R.sup.a can be hydrogen R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of 0, N, and S; R.sup.K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of 0, N, and S; wherein a R.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q can be halo; R.sup.Y and R.sup.Z taken together can be

##STR00044##

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)quinazolin-4-amine.

[0277] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G is N; joining G and J can be a double bond; R.sup.a can be hydrogen R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of 0, N, and S; R.sup.K can be substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of 0, N, and S; wherein a R.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q can be cyano; R.sup.Y and R.sup.Z taken together is

##STR00045##

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)quinazolin-2-yl)nicotinonitrile.

[0278] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.K can be NH(C.sub.1-4 alkyl); R.sup.Y and R.sup.Z taken together can be

##STR00046##

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.sup.4-(2-(1H-indol-3-yl)ethyl)-N.sup.2-(sec-butyl)quinazoline-2,4-diamine.

[0279] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y and R.sup.Z taken together can be

##STR00047##

wherein the ring is substituted with cyano; R.sup.d can be C.sub.1-C.sub.4 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.

[0280] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y and R.sup.Z taken together can be

##STR00048##

wherein the ring is substituted with C.sub.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. 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.

[0281] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.Y and R.sup.Z taken together can be

##STR00049##

wherein the ring can be substituted with C.sub.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. 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.

[0282] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J represents a double bond; R.sup.a can be hydrogen R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; R.sup.Y and R.sup.Z taken together is

##STR00050##

wherein the ring is substituted with C.sub.1-C.sub.4 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.

[0283] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J represents a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; R.sup.Y and R.sup.Z taken together can be

##STR00051##

wherein the ring is substituted with C.sub.1-C.sub.4 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.

[0284] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; R.sup.Y and R.sup.Z taken together can be

##STR00052##

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)furo[3,2-d]pyrimidin-4-amine.

[0285] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is C.sub.1-C.sub.4 alkyl; R, and R.sup.Z taken together can be

##STR00053##

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-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine.

[0286] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is C.sub.1-C.sub.4 alkyl; R.sup.Y and R.sup.Z taken together can be

##STR00054##

wherein the ring is substituted with C.sub.1-C.sub.4 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.

[0287] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G is N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; R.sup.Y and R.sup.Z taken together can be

##STR00055##

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)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile.

[0288] In some embodiments, provided herein is compound of Formula (I), wherein the compound can be selected from: [0289] 4-(2-((2-(benzo[b]thiophen-3-yl)-6-(isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol; [0290] 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol; [0291] 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)ethyl)phenol; [0292] 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one; [0293] 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-yl)oxy)propanamide; [0294] 4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4-yl)amino)ethyl)phenol; [0295] 5-(2-((2-(1H-indol-3-yl)ethyl)amino)-6-(sec-butylamino)pyrimidin-4-yl)nicotinonitrile; [0296] N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4-amine; [0297] N-(2-(1H-indol-3-yl)ethyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine; [0298] 3-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide; [0299] N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine; [0300] 5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile; [0301] N.sup.4-(2-(1H-indol-3-yl)ethyl)-N.sup.2-(sec-butyl)quinazoline-2,4-diamine; [0302] 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile; [0303] N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine; [0304] 4-(2-((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-yl)amino)ethyl)phenol; [0305] 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7-isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile; [0306] N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine; [0307] N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; [0308] N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; [0309] N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine; [0310] 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)

[0311] In some embodiments provided herein, the compound of Formula (I) can have the structure of Formula (I-A):

##STR00056##

including pharmaceutically acceptable salts thereof, wherein: R.sup.J can be NR.sup.aR.sup.b; R.sup.a can be hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.b can be R.sup.c or (C.sub.1-C.sub.4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: hydrogen, unsubstituted C.sub.1-6 alkyl; NH(C.sub.1-4 alkyl); N(C.sub.1-4 alkyl).sub.2, unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.K 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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4haloalkyl); Y and Z can each be C; X can be N or CH; W can be O or S; and R.sup.c can be hydrogen or C.sub.1-C.sub.4 alkyl.

[0312] In some embodiments, R.sup.a can be hydrogen. In other embodiments, R.sup.a can be C.sub.1-C.sub.4 alkyl.

[0313] In some embodiments, R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c. For example, R.sup.b can be CH.sub.2R.sup.c, CH.sub.2CH.sub.2R.sup.c, CH.sub.2CH.sub.2CH.sub.2R.sup.c, or CH.sub.2CH.sub.2CH.sub.2CH.sub.2R.sup.c.

[0314] In some embodiments, R.sup.c can be OH. In some embodiments, R.sup.c can be O(C.sub.1-C.sub.4 alkyl). In some embodiments, R.sup.c can be O(C.sub.1-C.sub.4 haloalkyl). In some embodiments, R.sup.c can be C(O)NH.sub.2. In some embodiments, R.sup.c can be unsubstituted C.sub.6-10 aryl. In some embodiments, R.sup.c can be substituted C.sub.6-10 aryl. In some embodiments, R.sup.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.sup.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.sup.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.sub.1-C.sub.4 alkyl. In some embodiments, E can be C.sub.1-C.sub.4 haloalkyl. In some embodiments, E can be O(C.sub.1-C.sub.4 alkyl). In some embodiments, E can be O(C.sub.1-C.sub.4 haloalkyl). In some embodiments R.sup.c can be phenyl. In other embodiments, R.sup.c can be hydroxyphenyl. In still other embodiments, R.sup.c can be indolyl.

[0315] In some embodiments, R.sup.K 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.sup.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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4 haloalkyl). In some embodiments, R.sup.K can be pyridinyl. In other embodiments, R.sup.K can be pyridinyl substituted with one or more substituents Q. For example, R.sup.K can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl.

[0316] In some embodiments, R.sup.c can be hydrogen. In some embodiments, R.sup.c can be C.sub.1-C.sub.4 alkyl. For example, R.sup.c can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl.

[0317] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of unsubstituted C.sub.6-10 aryl; substituted C.sub.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 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, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.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 is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of OH, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4haloalkyl); and R.sup.c can be C.sub.1-C.sub.4 alkyl.

[0318] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (CH.sub.2CH.sub.2)R.sup.c; R.sup.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.sup.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.sub.1-4 alkyl, halo, and cyano; and R.sup.c can be isopropyl.

[0319] In some embodiments, when W is O, R.sup.J can be NR.sup.aR.sup.b; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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 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, C.sub.1-C.sub.4 alkyl, and O(C.sub.1-C.sub.4 alkyl); R.sup.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 a R.sup.K moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of:C.sub.1-4 alkyl, halo, cyano, and O(C.sub.1-4 alkyl); Y and Z can each be C; X can be N or CH; and R.sup.c can be hydrogen or C.sub.1-C.sub.4 alkyl.

[0320] In some embodiments, when W is S, R.sup.J can be NR.sup.aR.sup.b; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, and O(C.sub.1-C.sub.4 alkyl); R.sup.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 a R.sup.K moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of:C.sub.1-4 alkyl, halo, cyano, and O(C.sub.1-4 alkyl); Y and Z can each be C; X can be N or CH; and R.sup.c can be hydrogen or C.sub.1-C.sub.4 alkyl.

[0321] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is C.sub.1-C.sub.4 alkyl; W can be S; R.sup.c can be C.sub.1-C.sub.4 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.

[0322] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; W can be S; R.sup.c can be C.sub.1-C.sub.4 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.

[0323] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.Q can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; W can be S; R.sup.c can be C.sub.1-C.sub.4 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.

[0324] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2RC, R.sup.Q can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E can be OH; R.sup.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.sup.c can be C.sub.1-C.sub.4 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.

[0325] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.Q can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is halo; W can be 0; R.sup.c 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.

[0326] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.Q can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is C.sub.1-C.sub.4 alkyl; W can be 0; R.sup.c 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.

[0327] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G is NR.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.K moiety indicated as substituted is substituted with one or more Q, wherein Q is cyano; W can be 0; R.sup.c 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.

[0328] In some embodiments, the compound of Formula (I-A), or a pharmaceutically acceptable salt thereof, can selected from the group consisting of: [0329] N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine; [0330] 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; [0331] 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol; [0332] N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; [0333] N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; and [0334] 5-(4-((2-(1H-indol-3-yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile.

Formula (I-B)

[0335] In other embodiments provided herein, the compound of Formula (I) can have the structure of Formula (I-B):

##STR00057##

including pharmaceutically acceptable salts thereof, wherein: R.sup.a can be hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.b can be R.sup.Q or (C.sub.1-4 alkyl)-R.sup.c; R.sup.Q can be selected from the group consisting of: OH, O(C.sub.1-C.sub.4 alkyl), O(C.sub.1-C.sub.4 haloalkyl); C(O)NH.sub.2; unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: hydrogen, unsubstituted C.sub.1-6 alkyl; substituted C.sub.1-6 alkyl; NH(C.sub.1-4 alkyl); N(C.sub.1-4 alkyl).sub.2, unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.K 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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4haloalkyl); R.sup.G can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, and (C.sub.1-4 alkyl)-C(O)NH.sub.2; R.sup.f can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, unsubstituted C.sub.6-C.sub.10 aryl, and C.sub.6-C.sub.10 aryl substituted with 1-5 halo atoms; U can be N or CR.sup.U; V can be S or NR.sup.V; R.sup.U can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, halo, and cyano; R.sup.V can be hydrogen or C.sub.1-C.sub.4 alkyl; wherein when U is CR.sup.U and V is NR.sup.V, R.sup.U is selected from the group consisting of C.sub.1-4 alkyl, halo, and cyano; Y and Z can each be C; and X can be N or CH.

[0336] In some embodiments, R.sup.a can be hydrogen. In other embodiments, R.sup.a can be C.sub.1-C.sub.4 alkyl.

[0337] In some embodiments, R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c. For example, R.sup.b can be CH.sub.2R.sup.c, CH.sub.2CH.sub.2R.sup.c, CH.sub.2CH.sub.2CH.sub.2R.sup.c, or CH.sub.2CH.sub.2CH.sub.2CH.sub.2R.sup.c. In certain embodiments, R.sup.b can be (CH.sub.2CH.sub.2)R.sup.c. In certain embodiments, R.sup.b can be (CH.sub.2CH.sub.2)C(O)NH.sub.2. In certain embodiments, R.sup.b can be (CH.sub.2CH.sub.2)-(indolyl). In certain embodiments, R.sup.b can be (CH.sub.2CH.sub.2)-(hydroxyphenyl).

[0338] In some embodiments, R.sup.c can be OH. In some embodiments, R.sup.c can be O(C.sub.1-C.sub.4 alkyl). In some embodiments, R.sup.c can be O(C.sub.1-C.sub.4 haloalkyl). In some embodiments, R.sup.c can be C(O)NH.sub.2. In some embodiments, R.sup.c can be unsubstituted C.sub.6-10 aryl. In some embodiments, R.sup.c can be substituted C.sub.6-10 aryl. In some embodiments, R.sup.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.sup.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.sup.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.sub.1-C.sub.4 alkyl. In some embodiments, E can be C.sub.1-C.sub.4 haloalkyl. In some embodiments, E can be O(C.sub.1-C.sub.4 alkyl). In some embodiments, E can be O(C.sub.1-C.sub.4 haloalkyl).

[0339] In some embodiments, R.sup.K can be hydrogen. In other embodiments, R.sup.K can be C.sub.1-C.sub.4 alkyl. For example, R.sup.K can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In some embodiments, R.sup.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.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4haloalkyl). In certain embodiments, R.sup.K can be benzothiophenyl. In other embodiments, R.sup.K can be pyridinyl substituted with one or more substituents Q. For example, R.sup.K can be methylpyridinyl, ethylpyridinyl cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl.

[0340] In some embodiments, R.sup.G can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, and (C.sub.1-4 alkyl)-C(O)NH.sub.2. In certain embodiments, R.sup.G can be (CH.sub.2CH.sub.2)C(O)NH.sub.2.

[0341] In some embodiments, R.sup.f can be hydrogen. In other embodiments, R.sup.f can be C.sub.1-4 alkyl. For example, R.sup.f can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In some embodiments, R.sup.f can be unsubstituted C.sub.6-C.sub.10 aryl. In other embodiments, R.sup.f can be C.sub.6-C.sub.10 aryl substituted with 1-5 halo atoms. In certain embodiments, R.sup.f can be phenyl substituted with 1-5 halo atoms. In certain embodiments, R.sup.f can be fluorophenyl.

[0342] In some embodiments, U can be N. In other embodiments, U can be CR.sup.U.

[0343] In some embodiments, V can be S. In other embodiments, V can be NR.sup.V.

[0344] In some embodiments, R.sup.U can be hydrogen. In some embodiments, R.sup.U can be C.sub.1-4 alkyl. In other embodiments R.sup.U can be halo. For example, R.sup.U can be fluoro, chloro, bromo, or iodo. In still other embodiments, R.sup.U can be cyano.

[0345] In some embodiments, R.sup.V can be hydrogen. In other embodiments, R.sup.V can be C.sub.1-4 alkyl. For example, R.sup.V 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.

[0346] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (C.sub.1-4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of C(O)NH.sub.2, unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.c 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, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.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 is substituted with one or more substituents Q, wherein each Q can be independently selected from the group consisting of OH, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4haloalkyl); R.sup.G is C.sub.1-4 alkyl or (C.sub.1-4 alkyl)-C(O)NH.sub.2; R.sup.f 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.

[0347] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (CH.sub.2CH.sub.2)R.sup.c; R.sup.c can be selected from the group consisting of C(O)NH.sub.2, substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E can be OH; R.sup.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.sub.1-4 alkyl, halo, and cyano; R.sup.G can be (CH.sub.2CH.sub.2)C(O)NH.sub.2; R.sup.f can be selected from the group consisting of hydrogen, phenyl, and fluorophenyl; Y and Z each can be C; and X can be CH.

[0348] In some embodiments, when V is S, R.sup.a can be hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.b can be R.sup.c or (CH.sub.2CH.sub.2)R.sup.c; R.sup.c can be selected from the group consisting of: C(O)NH.sub.2; unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, and O(C.sub.1-C.sub.4 alkyl); R.sup.K can be selected from the group consisting of: hydrogen, unsubstituted C.sub.1-6 alkyl; substituted C.sub.1-6 alkyl; NH(C.sub.1-4 alkyl); and N(C.sub.1-4 alkyl).sub.2; wherein a R.sup.K 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, C.sub.1-4 alkyl, halo, cyano, and O(C.sub.1-4 alkyl; R.sup.G can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, and (C.sub.1-4 alkyl)-C(O)NH.sub.2; R.sup.f can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, unsubstituted C.sub.6-C.sub.10 aryl, and C.sub.6-C.sub.10 aryl substituted with 1-5 halo atoms; U can be CR.sup.U; R.sup.U can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, halo, and cyano; Y and Z can each be C; and X can be N.

[0349] In some embodiments, when V is NR.sup.V, R.sup.a can be hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.b can be R.sup.c or (CH.sub.2CH.sub.2)R.sup.c; R.sup.c can be selected from the group consisting of: C(O)NH.sub.2; unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4, and O(C.sub.1-C.sub.4 alkyl); R.sup.K can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.K 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, C.sub.1-4 alkyl, halo, cyano, and O(C.sub.1-4 alkyl); R.sup.G can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, and (C.sub.1-4 alkyl)-C(O)NH.sub.2; R.sup.f can be hydrogen; U can be N or CR.sup.U; R.sup.U can be selected from the group consisting of C.sub.1-4 alkyl, halo, and cyano; R.sup.V can be hydrogen or C.sub.1-C.sub.4 alkyl; Y and Z can each be C; and X can be N or CH.

[0350] In some embodiments, when R.sup.J is OR.sup.b; G can be N; joining G and J can be a double bond; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be C(O)NH.sub.2; R.sup.K 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 NR.sup.V; R.sup.V can be C.sub.1-C.sub.4 alkyl; Rican 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.

[0351] In some embodiments, when R.sup.J is O; G can be N substituted with R.sup.G; joining G and J can be a single bond; R.sup.G can be (C.sub.1-4 alkyl)-C(O)NH.sub.2; R.sup.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.sup.V; R.sup.V can be C.sub.1-C.sub.4 alkyl; R.sup.f 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.

[0352] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.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.sup.u; R.sup.u can be cyano; V can be NR.sup.V; R.sup.V can be C.sub.1-C.sub.4 alkyl; Rican 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.

[0353] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.K can be unsubstituted C.sub.1-6 alkyl; U can be CR.sup.u; R.sup.u can be hydrogen; V can be S; Rican 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.

[0354] In some embodiments, when R.sup.J can be NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.K can be hydrogen; U can be CR.sup.u; R.sup.u can be hydrogen; V can be S; Rican 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.

[0355] In some embodiments, the compound of Formula (I-B), or a pharmaceutically acceptable salt thereof, can selected from the group consisting of: [0356] 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-yl)oxy)propanamide; [0357] 3-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide; [0358] 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile; [0359] N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4-amine; and [0360] N-(2-(1H-indol-3-yl)ethyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine.

Formula (I-C)

[0361] In still other embodiments provided herein, the compound of Formula (I) can have the structure of Formula (I-C):

##STR00058##

including pharmaceutically acceptable salts thereof, wherein: R.sup.J can be NR.sup.aR.sup.b; R.sup.a can be hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.b can be R.sup.c or (C.sub.1-C.sub.4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: hydrogen, unsubstituted C.sub.1-6 alkyl; NH(C.sub.1-4 alkyl); N(C.sub.1-4 alkyl).sub.2, unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.K 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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4haloalkyl); A can be N or CH; B can be N or CH; R.sup.g can be selected from the group consisting of hydrogen, C.sub.1-4 alkyl, and N(C.sub.1-4 alkyl).sub.2; Y and Z can each be C; and X can be N or CH.

[0362] In some embodiments, R.sup.K can be NH(C.sub.1-4 alkyl). For example, in some embodiments, R.sup.K can be NH(CH.sub.3), NH(CH.sub.2CH.sub.3), NH(isopropyl), or NH(sec-butyl). In some embodiments, R.sup.K can be unsubstituted benzothiophenyl. In other embodiments, R.sup.K can be substituted pyridinyl. For example, R.sup.K can be methylpyridinyl, ethylpyridinyl, cyanopyridinyl, chloropyridinyl, fluoropyridinyl, or bromopyridinyl.

[0363] 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.

[0364] In some embodiments, R.sup.g can be hydrogen. In other embodiments, R.sup.g can be N(C.sub.1-4 alkyl).sub.2. In certain embodiments, R.sup.g can be N(CH.sub.3).sub.2.

[0365] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: NH(C.sub.1-4 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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4 haloalkyl); and R.sup.g can be hydrogen or N(C.sub.1-4 alkyl).sub.2.

[0366] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c; R.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: NH(C.sub.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: OH, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4 haloalkyl); and R.sup.g can be hydrogen or N(C.sub.1-4 alkyl).sub.2.

[0367] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (CH.sub.2CH.sub.2)R.sup.c; R.sup.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.sup.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.sub.1-4 alkyl, halo, and cyano; and R.sup.g can be hydrogen or N(CH.sub.3).sub.2.

[0368] In some embodiments, when A is C and B is C, R.sup.J can be NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be substituted C.sub.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.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.g can be hydrogen; J can be C; X can be N; Y can be C; and Z is C.

[0369] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.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.sup.g can be N(C.sub.1-4 alkyl).sub.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.

[0370] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.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.sup.g 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.

[0371] In some embodiments, when R.sup.1 is NR.sup.aR.sup.b; G is N; joining G and J can be a double bond; R.sup.a can be hydrogen R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.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.sup.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.sup.g 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.

[0372] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.K can be NH(C.sub.1-4 alkyl); A can be CH; B can be CH; R.sup.g 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.sup.4-(2-(1H-indol-3-yl)ethyl)-N.sup.2-(sec-butyl)quinazoline-2,4-diamine.

[0373] In some embodiments, the compound of Formula (I-C), or a pharmaceutically acceptable salt thereof, can selected from the group consisting of: [0374] 4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4-yl)amino)ethyl)phenol; [0375] N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine; [0376] 5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile; and [0377] N.sup.4-(2-(1H-indol-3-yl)ethyl)-N.sup.2-(sec-butyl)quinazoline-2,4-diamine.

Formula (I-D)

[0378] In yet still other embodiments provided herein, the compound of Formula (I) can have the structure of Formula (I-D):

##STR00059##

including pharmaceutically acceptable salts thereof, wherein: R.sup.J can be NR.sup.aR.sup.b; R.sup.a can be hydrogen or C.sub.1-C.sub.4 alkyl; R.sup.b can be R.sup.c or (C.sub.1-4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.K 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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4haloalkyl); R.sup.h can be hydrogen or C.sub.1-4 alkyl; D can be N or CH; Y can be N; Z can be C; and X can be N or CH.

[0379] In some embodiments, R.sup.h can be hydrogen. In other embodiments, R.sup.h can be C.sub.1-4 alkyl. For example, R.sup.h can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl.

[0380] In some embodiments, D can be N. In other embodiments, D can be CH.

[0381] 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.

[0382] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (C.sub.1-4 alkyl)-R.sup.c; R.sup.c can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be selected from the group consisting of: unsubstituted C.sub.6-10 aryl; substituted C.sub.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.sup.K 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, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano, O(C.sub.1-4 alkyl), and O(C.sub.1-4haloalkyl); and R.sup.h can be hydrogen or C.sub.1-4 alkyl.

[0383] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (C.sub.1-C.sub.4 alkyl)-R.sup.c; R.sup.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.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, O(C.sub.1-C.sub.4 alkyl), and O(C.sub.1-C.sub.4 haloalkyl); R.sup.K can be unsubstituted benzothiophenyl; and R.sup.h can be hydrogen or C.sub.1-4 alkyl.

[0384] In some embodiments, R.sup.a can be hydrogen; R.sup.b can be (CH.sub.2CH.sub.2)R.sup.c; R.sup.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.sup.K can be unsubstituted benzothiophenyl; and R.sup.h can be hydrogen or C.sub.1-4 alkyl.

[0385] In some embodiments, when D is N; R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.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.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.K can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; R.sup.b can be C.sub.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.

[0386] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.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.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.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.sup.b can be C.sub.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. 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.

[0387] In some embodiments, when R.sup.J is NR.sup.aR.sup.b; G can be N; joining G and J can be a double bond; R.sup.a can be hydrogen; R.sup.b can be CH.sub.2CH.sub.2R.sup.c; R.sup.c can be substituted C.sub.6-10 aryl, substituted with one or more E, wherein E is OH; R.sup.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.sup.b can be C.sub.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. 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.

[0388] 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.

[0389] 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

[0390] 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.sup.+CD3.sup. cells. In certain embodiments, the NK cells are enriched for CD56.sup.+CD3.sup. 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.sup.+ and CD16.sup. cells, and/or CD94.sup.+ and CD94.sup.. In certain embodiments, cells, e.g., cells produced by the three-step methods described herein, are sorted to separate CDTTa+ and CDTTa cells. In specific embodiments, CDTTa.sup.+ cells are isolated. In certain embodiments, the cells are enriched for CD11a.sup.+ 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.

[0391] 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.sup.+ and CD117.sup. 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.sup.+CD3.sup.CD94.sup.+CD11a.sup.+ 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.sup.+CD3.sup.CD94.sup.+CD11a.sup.+ cells in comparison with total cells produced using the three-stage method, described herein.

[0392] In one embodiment, ILC3 cells are isolated or enriched by selecting for CD56.sup.+CD3.sup.CD11a cells. In certain embodiments, the ILC3 cells are enriched for CD56.sup.+CD3.sup.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.sup.+CD3.sup.CD11a CD117+ cells. In certain embodiments, the ILC3 cells are enriched for CD56.sup.+CD3.sup.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.sup.+CD3.sup.CD11a.sup.CD117.sup.+CDIL1R1.sup.+ cells. In certain embodiments, the ILC3 cells are enriched for CD56.sup.+CD3.sup.CD11a.sup.CD117.sup.+CDIL1R1.sup.+ cells in comparison with total cells produced using the three-stage method, described herein.

[0393] In one embodiment, NK cells are isolated or enriched by selecting for CD56.sup.+CD3.sup.CD94.sup.+CD11a+ cells. In certain embodiments, the NK cells are enriched for CD56.sup.+CD3.sup.CD94.sup.+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.sup.+CD3.sup.CD94.sup.+CD11a.sup.+CD117.sup. cells. In certain embodiments, the NK cells are enriched for CD56.sup.+CD3.sup.CD94.sup.+CD11a.sup.+CD117.sup. cells in comparison with total cells produced using the three-stage method, described herein.

[0394] 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 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 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

[0395] 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. Pat. 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

[0396] 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.

[0397] 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.

[0398] 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 bromide, perfluorodecyl bromide, etc.).

[0399] 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.

[0400] 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.

[0401] 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

[0402] 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.

[0403] Prior to recovery of perfusate, the umbilical cord blood and placental blood are removed. In certain embodiments, after delivery, the cord blood in the placenta is recovered. The placenta can be subjected to a conventional cord blood recovery process. Typically a needle or cannula is used, with the aid of gravity, to exsanguinate the placenta (see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No. 5,415,665). The needle or cannula is usually placed in the umbilical vein and the placenta can be gently massaged to aid in draining cord blood from the placenta. Such cord blood recovery may be performed commercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell. In one embodiment, the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.

[0404] 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. Pat. 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.

[0405] 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

[0406] Methods of perfusing mammalian placentae and obtaining placental perfusate are disclosed, e.g., in Hariri, U.S. Pat. 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.

[0407] 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.

[0408] 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).

[0409] 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.

[0410] 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.

[0411] 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.

[0412] 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.

[0413] 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.

[0414] 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.

[0415] 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.

[0416] The placenta can be perfused a plurality of times over the course of several hours or several days. Where the placenta is to be perfused a plurality of times, it may be maintained or cultured under aseptic conditions in a container or other suitable vessel, and perfused with a cell collection composition, or a standard perfusion solution (e.g., a normal saline solution such as phosphate buffered saline (PBS) with or without an anticoagulant (e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g., -mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 g/ml), penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 0.5 g/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

[0417] 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.sup.+ cells, e.g., hematopoietic stem or progenitor cells. Such cells can, in a more specific embodiment, comprise CD34.sup.+CD45.sup. stem or progenitor cells, CD34.sup.+CD45.sup.+ 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

[0418] 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.

[0419] 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 CD3CD56+ 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% CD3CD56+ cells. In another specific embodiment, said NK cell population comprises no less than 80%, 85%, 90%, 95%, 98%, or 99% CD3CD56+ 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.sup.CD56+ cells.

[0420] In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally NKp46.sup.+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD16. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD16.sup.+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD94.sup.. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD94.sup.+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD11a.sup.+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally NKp30.sup.+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD161.sup.+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally DNAM-1.sup.+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said NK cell population comprises CD3.sup.CD56.sup.+ cells that are additionally T-bet.sup.+.

[0421] 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 perform. 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

[0422] 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.

[0423] 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 CD3CD56+ 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% CD3CD56+ cells. In another specific embodiment, said ILC3 cell population comprises no less than 80%, 85%, 90%, 95%, 98%, or 99% CD3CD56+ cells. In another specific embodiment, said ILC3 cell population comprises between 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-99% CD3CD56+ cells.

[0424] In certain embodiments, said CD3.sup.CD56.sup.+ cells in said ILC3 cell population comprises CD3.sup.CD56.sup.+ cells that are additionally NKp46.sup.. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said ILC3 cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD16.sup.. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said ILC3 cell population comprises CD3.sup.CD56.sup.+ cells that are additionally IL1R1.sup.+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said ILC3 cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD94.sup.. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said ILC3 cell population comprises CD3.sup.CD56.sup.+ cells that are additionally RORt+. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said ILC3 cell population comprises CD3.sup.CD56.sup.+ cells that are additionally CD11a. In certain embodiments, said CD3.sup.CD56.sup.+ cells in said ILC3 cell population comprises CD3.sup.CD56.sup.+ cells that are additionally T-bet+.

[0425] In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are CD117.sup.+. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are NKG2D.sup.. In one embodiment, an ILC3 cell population produced by a three-stage method described herein comprises cells which are NKp30.sup.. 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.

[0426] 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.

[0427] 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.

[0428] 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.

[0429] 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

[0430] 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.sup.CD56.sup.+ 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.sup.CD56.sup.+ cells. In certain embodiments, said CD3.sup.CD56.sup.+ cells are CD16.

[0431] 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.

[0432] 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.

[0433] 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.

[0434] 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.

[0435] 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.

[0436] 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., 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, 110.sup.6, 510.sup.6, 110.sup.7, 510.sup.7, 110.sup.8, 510.sup.8 or more cells per milliliter, or 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, 110.sup.6, 510.sup.6, 110.sup.7, 510.sup.7, 110.sup.8, 510.sup.8, 110.sup.9, 510.sup.9, 110.sup.10, 510.sup.10, 110.sup.11 or more cells per unit. In specific embodiments, the units can comprise about, at least about, or at most about 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, 110.sup.6, 510.sup.6 or more NK cells and/or ILC3 cells per milliliter, or 110.sup.4, 510.sup.4, 110.sup.5, 510.sup.5, 110.sup.6, 510.sup.6, 110.sup.7, 510.sup.7, 110.sup.8, 510.sup.8, 110.sup.9, 510.sup.9, 110.sup.10, 510.sup.10, 110.sup.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. In specific embodiments, the NK cells and ILC3 cells are present in ratios provided herein.

[0437] 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. Pat. 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-(4aminoisolindoline-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

##STR00060##

wherein one of X and Y is CO, the other of X and Y is CO or CH.sub.2, and R.sup.2 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

##STR00061## [0438] wherein one of X and Y is CO and the other is CH.sub.2 or CO; [0439] R.sup.1 is H, (C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.7)cycloalkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, benzyl, aryl, (C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl, (C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl, C(O)R.sup.3, C(S)R.sup.3, C(O)OR.sup.4, (C.sub.1-C.sub.8)alkyl-N(R.sup.6).sub.2, (C.sub.1-C.sub.8)alkyl-OR.sup.5, (C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5, C(O)NHR.sup.3, C(S)NHR.sup.3, C(O)NR.sup.3R3, C(S)NR.sup.3R3 or (C.sub.1-C.sub.8)alkyl-O(CO)R.sup.5; [0440] R.sup.2 is H, F, benzyl, (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, or (C.sub.2-C.sub.8)alkynyl; [0441] R.sup.3 and R.sup.3 are independently (C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.7)cycloalkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, benzyl, aryl, (C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl, (C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl, (C.sub.0-C.sub.8)alkyl-N(R.sup.6).sub.2, (C.sub.1-C.sub.8)alkyl-OR.sup.5, (C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5, (C.sub.1-C.sub.8)alkyl-O(CO)R.sup.5, or C(O)OR.sup.5; [0442] R.sup.4 is (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, (C.sub.1-C.sub.4)alkyl-OR.sup.5, benzyl, aryl, (C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl, or (C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl; [0443] R.sup.5 is (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, benzyl, aryl, or (C.sub.2-C.sub.5)heteroaryl; [0444] each occurrence of R.sup.6 is independently H, (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, benzyl, aryl, (C.sub.2-C.sub.5)heteroaryl, or (C.sub.0-C.sub.8)alkyl-C(O)OR.sup.5 or the R groups can join to form a heterocycloalkyl group; [0445] n is 0 or 1; and [0446] * represents a chiral-carbon center; [0447] 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

##STR00062## [0448] wherein: [0449] one of X and Y is CO and the other is CH.sub.2 or CO; [0450] R is H or CH.sub.2OCOR; [0451] (i) each of R.sup.1, R.sup.2, R.sup.3, or R.sup.4, independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R.sup.1, R.sup.2, R.sup.3, or R.sup.4 is nitro or NHR.sup.5 and the remaining of R.sup.1, R.sup.2, R.sup.3, or Rare hydrogen; [0452] R.sup.5 is hydrogen or alkyl of 1 to 8 carbons [0453] R.sup.6 hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro; [0454] R is R.sup.7CHR.sup.10N(R.sup.8R.sup.9); R.sup.7 is m-phenylene or p-phenylene or (C.sub.nH.sub.2n) in which n has a value of 0 to 4; [0455] each of R.sup.8 and R.sup.9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R.sup.8 and R.sup.9 taken together are tetramethylene, pentamethylene, hexamethylene, or CH.sub.2CH.sub.2X.sub.1CH.sub.2CH.sub.2 in which X.sub.1 is O, S, or NH; [0456] R.sup.10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and [0457] * represents a chiral-carbon center;
or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

[0458] In another specific embodiment, the composition additionally comprises one or more anticancer compounds, e.g., one or more of the anticancer compounds described below.

[0459] 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.

[0460] 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.

[0461] 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.

6. KITS

[0462] 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.

[0463] 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

[0464] CD34.sup.+ 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.

[0465] 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-1) or other stem cell mobilizing agent.

[0466] 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.

[0467] Stage 3 medium: 90% STEMMACS, 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.

[0468] Cells are seeded at Day 0 at 310.sup.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 110.sup.5 cells/mL with Stage 1 medium. At Day 7 cells are counted and seeded to a concentration of 110.sup.5 cells/mL with Stage 1 medium.

[0469] At Day 10, cells are counted and seeded to a concentration of 110.sup.5 cells/mL in Stage 2 medium. At Day 12, cells are counted and seeded to a concentration of 310.sup.5 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.

[0470] Alternatively, the following protocol is used through Day 14: Cells seeded at Day 0 at 7.510.sup.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 310.sup.5 cells/mL with Stage 1 medium. At Day 9 cells are counted and seeded to a concentration of 310.sup.5 cells/mL with Stage 2 medium. At Day 12, cells are counted and seeded to a concentration of 310.sup.5 cells/mL in Stage 2 medium. At Day 14, cells are counted and seeded to a concentration of 310.sup.5 cells/mL in Stage 2 medium.

[0471] 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.

[0472] For harvest, cells are spun at 400g for seven minutes, followed by suspension of the pellet in an equal volume of Plasmalyte A. The suspension is spun at 400g 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 m 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

[0473] The following compounds were investigated for their ability to promote the expansion of NK cell populations in vitro: [0474] 4-(2-((2-(benzo[b]thiophen-3-yl)-6-(isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol) (CRL1)

##STR00063## [0475] 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-yl)amino)ethyl)phenol)) (CRL2)

##STR00064## [0476] 4-(2-((2-(benzo[b]thiophen-3-yl)-7-isopropyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)ethyl)phenol (CRL3)

##STR00065## [0477] 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (CRL4)

##STR00066## [0478] 3-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-yl)oxy)propanamide (CRL5)

##STR00067## [0479] 4-(2-((2-(benzo[b]thiophen-3-yl)-8-(dimethylamino)pyrimido[5,4-d]pyrimidin-4-yl)amino)ethyl)phenol (CRL6)

##STR00068## [0480] 5-(2-((2-(1H-indol-3-yl)ethyl)amino)-6-(sec-butylamino)pyrimidin-4-yl)nicotinonitrile (CRL7)

##STR00069## [0481] N-(2-(1H-indol-3-yl)ethyl)-2-methyl-6-phenylthieno[2,3-d]pyrimidin-4-amine (CRL8)

##STR00070## [0482] N-(2-(1H-indol-3-yl)ethyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-amine (CRL9)

##STR00071## [0483] 3-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-6-oxo-6,9-dihydro-1H-purin-1-yl)propanamide (CRL10)

##STR00072## [0484] N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine (CRL11)

##STR00073## [0485] 5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile (CRL12)

##STR00074## [0486] N.sup.4-(2-(1H-indol-3-yl)ethyl)-N.sup.2-(sec-butyl)quinazoline-2,4-diamine (CRL13)

##STR00075## [0487] 2-(benzo[b]thiophen-3-yl)-4-((4-hydroxyphenethyl)amino)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (CRL14)

##STR00076## [0488] N-(2-(1H-indol-3-yl)ethyl)-6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine (CRL15)

##STR00077## [0489] 4-(2-((6-(benzo[b]thiophen-3-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-yl)amino)ethyl)phenol (CRL16)

##STR00078## [0490] 5-(4-((2-(1H-indol-3-yl)ethyl)amino)-7-isopropylthieno[3,2-d]pyrimidin-2-yl)nicotinonitrile (CRL17)

##STR00079## [0491] N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-7-isopropylthieno[3,2-d]pyrimidin-4-amine (CRL18)

##STR00080## [0492] N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)furo[3,2-d]pyrimidin-4-amine (CRL19)

##STR00081## [0493] N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine (CRL20)

##STR00082## [0494] N-(2-(1H-indol-3-yl)ethyl)-7-isopropyl-2-(5-methylpyridin-3-yl)thieno[3,2-d]pyrimidin-4-amine (CRL21)

##STR00083## and [0495] 5-(4-((2-(1H-indol-3-yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile (CRL22)

##STR00084##

7.3. Example 3: Characterization of Three-Stage NK Cells

Methods

[0496] 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.

[0497] 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 M concentration. SR1 (at 10 M) 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

[0498] Cell expansion data showed that 20 out of the 22 compounds supported NK expansion at 10 M concentration. Except for CRL7 and CRL13, the rest of the compounds all resulted in a NK expansion of 2,00015,000 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).

[0499] Cytotoxicity assay was run using compound cultured cells against K562 tumor cells at 10:1 effector to target ratio (FIGS. 5A-5B) to evaluate cell functions. The results showed that the cells cultured with compounds killed 3060% 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:

[0500] In summary, we found that all the compounds except CRL7 and CRL13 supported PNK-007 expansion and differentiation. Expansion with the compounds ranged from 2,00015,000 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

[0501] 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.

[0502] Magnetic-activated cell sorting: PNK cells were stained with PE Mouse Anti-Human CD 11a (BD) and CD11a+ PNK cells concentrated using anti-PE MicroBeads according to manufacturer's instructions (Miltenyi Biotec).

[0503] Single cell RNA sequencing: CYNK cells were combined with PB-NK at 1:1 ratio and gene expression analyzed on single cell level using 10 Genomics Chromium platform and Illumina sequencing. Bioinformatics analysis utilized 10 Genomics Cell Ranger analysis pipeline.

[0504] 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).

[0505] 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 Ct method.

Results

[0506] 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).

[0507] FGFBP2, encoding fibroblast growth factor-binding protein, is known to be secreted by cytotoxic lymphocytes.

[0508] 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.

[0509] CXCR4 regulates NK cell homing to bone marrow.

[0510] 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.

[0511] Transcription factor KLF2 that regulates both NK cell proliferation and survival.

[0512] NK cell-derived IFN- (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.

TABLE-US-00003 TABLE 1 Top 50 upregulated genes per PB-NK cluster. Feature CYNK PB-NK PB-NK Log2 PB-NK P- Feature ID Name Average Average Fold Change Value 1 ENSG00000137441 FGFBP2 0.099352 2.935962 4.88363 4.09E78 2 ENSG00000100450 GZMH 0.136708 2.484828 4.182845 2.49E49 3 ENSG00000276085 CCL3L3 0.072152 1.251852 4.115143 2.13E49 4 ENSG00000197540 GZMM 0.134235 1.982728 3.883559 1.40E50 5 ENSG00000121966 CXCR4 0.403236 5.935725 3.879087 9.19E51 6 ENSG00000169554 ZEB2 0.127877 1.860789 3.861967 7.03E50 7 ENSG00000127528 KLF2 0.172475 1.92761 3.481483 1.86E40 8 ENSG00000189067 LITAF 0.297791 3.231559 3.439184 1.06E39 9 ENSG00000069667 RORA 0.101913 1.055542 3.371425 3.26E37 10 ENSG00000145220 LYAR 0.142448 1.306592 3.196402 2.39E33 11 ENSG00000125107 CNOT1 0.208595 1.809824 3.116348 3.39E32 12 ENSG00000111537 IFNG 0.193317 1.639941 3.083863 1.11E29 13 ENSG00000158050 DUSP2 0.40774 3.322164 3.025836 4.12E30 14 ENSG00000110046 ATG2A 0.190226 1.508942 2.987028 3.39E29 15 ENSG00000173762 CD7 0.492697 3.641922 2.885402 1.77E27 16 ENSG00000141682 PMAIP1 0.252398 1.820017 2.849558 6.51E26 17 ENSG00000078304 PPP2R5C 0.381864 2.591665 2.762207 6.15E25 18 ENSG00000153234 NR4A2 0.399174 2.622622 2.715393 5.59E24 19 ENSG00000152518 ZFP36L2 0.856899 5.585388 2.703993 4.72E24 20 ENSG00000145675 PIK3R1 0.325168 2.078618 2.675822 2.70E23 21 ENSG00000150045 KLRF1 0.191285 1.177103 2.620822 4.78E22 22 ENSG00000255198 SNHG9 0.516983 2.951818 2.512937 1.34E20 23 ENSG00000125148 MT2A 0.51504 2.913311 2.499426 9.06E20 24 ENSG00000116741 RGS2 0.203737 1.147279 2.492865 1.51E19 25 ENSG00000153922 CHD1 0.252574 1.350762 2.418474 9.42E19 26 ENSG00000120129 DUSP1 2.078529 9.865317 2.24638 2.58E16 27 ENSG00000143924 EML4 0.256284 1.150299 2.165756 7.80E15 28 ENSG00000128016 ZFP36 2.22866 9.777355 2.132849 1.32E14 29 ENSG00000163874 ZC3H12A 0.261759 1.120475 2.097382 7.47E14 30 ENSG00000105993 DNAJB6 0.6506 2.667169 2.035058 2.98E13 31 ENSG00000126524 SBDS 0.534822 2.185078 2.030148 3.57E13 32 ENSG00000125347 IRF1 1.450448 5.812277 2.002193 7.32E13 33 ENSG00000157514 TSC22D3 1.103379 4.30409 1.963373 2.57E12 34 ENSG00000184205 TSPYL2 0.592137 2.247746 1.924086 1.14E11 35 ENSG00000146278 PNRC1 1.362312 5.156149 1.919832 7.77E12 36 ENSG00000135070 ISCA1 0.27898 1.043084 1.90227 2.06E11 37 ENSG00000171223 JUNB 4.09462 15.11622 1.883884 2.20E11 38 ENSG00000156232 WHAMM 0.316425 1.146147 1.856513 7.14E11 39 ENSG00000164327 RICTOR 0.318279 1.101977 1.791406 3.85E10 40 ENSG00000118503 TNFAIP3 0.550807 1.902316 1.787777 3.93E10 41 ENSG00000120616 EPC1 0.562199 1.846066 1.714953 2.17E09 42 ENSG00000167508 MVD 0.309448 1.00722 1.702322 4.11E09 43 ENSG00000013441 CLK1 0.690164 2.216412 1.682859 4.62E09 44 ENSG00000188042 ARL4C 0.437325 1.388136 1.666056 8.18E09 45 ENSG00000162924 REL 0.553809 1.736208 1.648145 1.14E08 46 ENSG00000005483 KMT2E 0.79402 2.460289 1.631225 1.47E08 47 ENSG00000119801 YPEL5 0.966141 2.98202 1.625617 1.70E08 48 ENSG00000123505 AMD1 0.558578 1.664102 1.574595 6.03E08 49 ENSG00000159388 BTG2 0.751541 2.22132 1.563151 7.55E08 50 ENSG00000010404 IDS 0.723193 2.128073 1.556757 8.48E08

[0513] 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-US-00004 TABLE 2 Top 50 upregulated genes per CYNK cluster. CYNK Log2 Feature PBNK CYNK Fold CYNK P- Feature ID Name Average Average Change Value 1 ENSG00000102471 NDFIP2 0.077391 1.45981 4.230949 1.69E22 2 ENSG00000242258 LINC00996 0.063046 1.183921 4.222944 5.04E22 3 ENSG00000172005 MAL 0.057005 1.03529 4.173813 1.35E21 4 ENSG00000108702 CCL1 0.078524 1.334494 4.080611 5.11E09 5 ENSG00000198125 MB 0.10193 1.683947 4.041355 1.45E20 6 ENSG00000128040 SPINK2 0.087962 1.233641 3.804242 7.88E19 7 ENSG00000166920 C15orf48 0.078901 1.018246 3.683547 6.40E18 8 ENSG00000134072 CAMK1 0.151762 1.932724 3.667647 2.13E18 9 ENSG00000134545 KLRC1 0.509273 4.740451 3.217889 9.47E16 10 ENSG00000121858 TNFSF10 0.295975 2.682764 3.178801 6.44E15 11 ENSG00000186891 TNFRSF18 1.182011 10.09017 3.093605 6.96E15 12 ENSG00000008517 IL32 4.345617 37.08234 3.093395 6.60E15 13 ENSG00000042493 CAPG 0.369213 3.112494 3.074529 9.91E15 14 ENSG00000235576 AC092580.4 0.44736 3.660475 3.031759 2.23E14 15 ENSG00000163191 S100A11 0.41527 3.364804 3.017543 2.42E14 16 ENSG00000186827 TNFRSF4 0.135529 1.097816 3.01448 1.91E13 17 ENSG00000074800 ENO1 2.166202 16.05066 2.889567 1.86E13 18 ENSG00000158869 FCER1G 0.734274 5.393877 2.876632 2.43E13 19 ENSG00000118971 CCND2 0.457175 3.324621 2.861636 3.21E13 20 ENSG00000205426 KRT81 0.169883 1.187806 2.803005 3.69E12 21 ENSG00000243927 MRPS6 0.358643 2.29304 2.675597 6.10E12 22 ENSG00000182718 ANXA2 0.206125 1.282389 2.635118 3.48E11 23 ENSG00000125384 PTGER2 0.175546 1.08713 2.628037 4.29E11 24 ENSG00000124767 GLO1 0.214053 1.289543 2.588793 6.50E11 25 ENSG00000135077 HAVCR2 0.175924 1.031051 2.548543 1.51E10 26 ENSG00000103490 PYCARD 0.183097 1.070527 2.545209 1.34E10 27 ENSG00000086730 LAT2 0.178566 1.04156 2.541707 1.53E10 28 ENSG00000141526 SLC16A3 0.282006 1.622835 2.523282 1.73E10 29 ENSG00000103187 COTL1 0.894342 5.013779 2.486834 1.45E10 30 ENSG00000067225 PKM 1.099712 6.145949 2.482453 1.11E10 31 ENSG00000177156 TALDO1 0.196687 1.084745 2.46115 4.23E10 32 ENSG00000153283 CD96 0.368458 2.029162 2.460314 1.66E10 33 ENSG00000204475 NCR3 0.640272 3.472457 2.438804 2.31E10 34 ENSG00000170442 KRT86 0.257845 1.372733 2.410873 1.02E09 35 ENSG00000117632 STMN1 0.468878 2.413499 2.36315 1.22E09 36 ENSG00000227507 LTB 3.831437 19.41653 2.341609 1.09E09 37 ENSG00000130429 ARPC1B 0.570053 2.846585 2.31957 1.27E09 38 ENSG00000162704 ARPC5 0.347317 1.717418 2.30484 1.66E09 39 ENSG00000088832 FKBP1A 0.40017 1.978205 2.304629 1.60E09 40 ENSG00000102265 TIMP1 0.385447 1.902345 2.302248 1.96E09 41 ENSG00000113088 GZMK 0.290312 1.403201 2.27168 1.37E08 42 ENSG00000085063 CD59 0.215186 1.035997 2.265377 7.12E09 43 ENSG00000102144 PGK1 1.405879 6.735348 2.260328 2.92E09 44 ENSG00000148908 RGS10 0.217451 1.014713 2.220352 1.33E08 45 ENSG00000196405 EVL 1.186164 5.50471 2.214345 5.41E09 46 ENSG00000128340 RAC2 1.063092 4.917253 2.209516 5.72E09 47 ENSG00000100097 LGALS1 4.427539 20.46621 2.208968 6.05E09 48 ENSG00000139626 ITGB7 0.50059 2.285445 2.19016 8.54E09 49 ENSG00000196230 TUBB 1.062715 4.838214 2.186651 1.22E08 50 ENSG00000171314 PGAM1 0.670096 3.046436 2.18433 8.56E09

[0514] 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 perform (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.

TABLE-US-00005 TABLE 3 Top differentially expressed genes encoding factors regulating NK cell cytolytic function. Genes that had <1 count per cell across the entire cluster were excluded. Log2 Feature CYNK PBNK Fold CYNK P- Feature ID Name Alias Average Average Change Value 1 ENSG00000134545 KLRC1 NKG2A, 4.740451 0.509273 3.217889 9.47E16 CD159a 2 ENSG00000121858 TNFSF10 TRAIL 2.682764 0.295975 3.178801 6.44E15 3 ENSG00000186891 TNFRSF18 GITR 10.09017 1.182011 3.093605 6.96E15 4 ENSG00000186827 TNFRSF4 CD134, 1.097816 0.135529 3.014481 1.91E13 OX40 5 ENSG00000135077 HAVCR2 TIM-3 1.031051 0.175924 2.548543 1.51E10 6 ENSG00000153283 CD96 Tactile 2.029162 0.368458 2.460314 1.66E10 7 ENSG00000204475 NCR3 CD337, 3.472457 0.640272 2.438804 2.31E10 NKp30 8 ENSG00000085063 CD59 MAC-IP, 1.035997 0.215186 MIRL, protectin 2.265377 7.12E09 9 ENSG00000139626 ITGB7 2.285445 0.50059 2.19016 8.54E09 10 ENSG00000180644 PRF1 3.589295 0.887169 2.016259 8.95E08 11 ENSG00000100453 GZMB 11.6194 3.515453 1.725026 4.27E06 12 ENSG00000100385 IL2RB 2.568753 0.956632 1.424929 0.000126 13 ENSG00000205809 KLRC2 NKG2C, 1.419451 0.784861 0.854636 0.026587 CD159c 14 ENSG00000111796 KLRB1 CD161 18.74844 10.45953 0.842324 0.027995 15 ENSG00000150045 KLRF1 NKp80 0.191285 1.177103 2.62082 4.78E22

[0515] 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 naive 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.

[0516] 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.

[0517] 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: Cleavage Resistant CD16 Expressing NK Cells

[0518] Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the shelf, ex-vivo expanded and allogenic Natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified with the 158 Val/Val form has shown to have higher IgG binding affinity comparing with the 158Phe/Phe form. The high IgG binding allele are found in about 10-20% of the normal population. In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells. Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158 Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity. Methods:

[0519] Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3106) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism.

[0520] Cell culture: Human placental CD34.sup.+ cells were isolated and cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate NK cells.

[0521] Cell Expansion and Characterization: Cell expansion was recorded during the culture process. On day 35, CD16VP cells were evaluated for NK surface markers CD56+/CD3, and CD16, using flow cytometry.

[0522] CD16VP Shedding Assay: Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage followed by immunostaining with CD16 antibody and analyzed using flow cytometry.

[0523] In vitro ADCC Assay: ADCC activity of CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E/T) ratios. IgG was used as ADCC control. In sustained ADCC assay, CD16VP cells were treated with PMA/ionomycin and then evaluated for ADCC activity as described above.

[0524] Animal Study: In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3106) were intravenously (IV) administered at day 0, followed by CD16VP cells (10106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI).

[0525] Statistical Analysis: Statistical analysis was performed using Prism/Excel program. Data are presented as meanstandard deviation. Paired or unpaired two-tailed Student's test were used for comparing two groups.

Results:

[0526] Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34.sup.+donors. These cells expanded [70952998 folds (n=8)] and differentiated into PNK cells (>90% CD56.sup.+CD3) at day 35. PNK-CD16VP expressed 64.610.3% (n=8) of CD16, while the NT expressed 12.13.3% (n=8) CD16. PMA/ionomycin induced >89% shedding of CD16 in NT cells, while significantly less (<11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 4713% against Daratumumab opsonized Daudi cells versus 255% (n=5; p<0.05); 3013% against Daratumumab opsonized HS-Sultan cells versus 2114% (n=3; p<0.05); 307% against Daratumumab opsonized Sudh16 cells versus 1610% (n=3; p<0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji, and Sudh14 with Daratumumab and Rituximab antibodies.

[0527] In this study, we genetically modified placental CD34.sup.+ cells to over-express CD16VP and evaluated the phenotype and functions of CD16VP cells regarding enhancement of ADCC and cleavage resistance.

[0528] 7000-fold expansion and >90% NK purity were achieved by the process.

[0529] 65% of CD16VP expression was maintained during the culture.

[0530] CD16VP expression was shown to be resistant to shedding after activation.

[0531] CD16VP cells demonstrated enhanced ADCC in vitro against lymphoma cell lines in combination with Daratumumab or Rituximab.

[0532] CD16VP resistance to activation induced shedding supported sustained killing in vitro.

[0533] CD16VP cells showed in vivo anti-tumor activities at early time points in an ADCC lymphoma model.

[0534] CD16VP provides a promising approach to augment the anti-tumor activities in combination with monoclonal antibodies. Further investigation is perused to support [0535] development of CD16VP in combination with therapeutic antibodies for various hematological malignancies and solid tumors.

[0536] PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal (>50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity.

Conclusions:

[0537] In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted.

7.6 Example 6: Cleavage Resistant CD16 Expressing CYNK Cells

[0538] CYNK cells were transduced with a CD16VP lentivirus and expanded as set forth above followed by analysis of cell surface marker expression, CD16 expression and CD16 shedding. See, FIG. 15A, FIG. 15B, FIG. 16, and FIG. 17.

TABLE-US-00006 TABLE 4 NK Surface Marker Expression Under CD56+/CD3 Donor Viability CD56+/CD3 CD16+ CD226+ NKG2D+ CD94+ CD11a+ NKp30+ NKp44+ NKp46+ PL 6 VP 91.5 97 78.8 57.4 49.6 54 71.9 49.8 95.3 9.77 PL 7 VP 92.6 95.2 65.9 43.9 33.7 42.9 49.5 30 92 24.9 DQ 1 VP 92.2 96.5 71.1 62.1 67.5 66.1 97.6 68.5 96.2 17.8 DQ 2 VP 83.9 90.7 73 50.3 36.9 53.7 62.8 29 89.7 11.2 DQ 3 VP 90.9 94.3 83.3 50.9 38.9 51.3 55.7 27.5 90.2 15.9 DQ 4 VP 87.8 95.2 73.4 50.7 33.5 41.4 53.5 38.1 97.1 14 DQ 5 VP 85 94.1 73.4 31.8 24.4 33.2 37.9 18.7 95.9 16.2 Average: 89.1 94.7 74.1 49.6 40.6 48.9 61.3 37.4 93.8 15.7 Stdev: 3.6 2.1 5.6 9.7 14.0 10.7 19.2 16.8 3.1 5.0

[0539] The results of this analysis showed the following: Average of 94.7% 2.1% of CD56+/CD3. Less than 1% CD3+ and CD19+. Average of 74.1%5.6% of CD16

[0540] Post thaw viability: 89.1%3.6%. Expressing activating receptors such as CD226, NKG2D, CD11a, NKp30, NKp44 and NKp46, and maturation marker CD94.

[0541] Shedding assays demonstrated shedding resistance of CYNK-101 (n=7 donors) induced by PMAi at both post thaw and after 2 days recovery.

[0542] CYNK-101 showed greater than 90% CD56.sup.+CD3, less than 1% CD3 or CD19, greater than 65% CD16, and expression of NK surface markers such as CD226, NKG2D, CD11a, NKp30, NKp44, NKp46, and CD94.

[0543] CYNK-101 was resistant to CD16 shedding following PMAi stimulation.

[0544] CYNK-101 displayed cytotoxicity against K562 cells with a dose dependent manner. In the mixed targets culture system of K562 plus normal PBMCs, CYNK-101 can specifically kill K562 while sparing normal PBMCs even at the E:T ratio up to 100:1.

[0545] Increased GM-CSF, IFN-, and TNF-a secretion was observed from CYNK-101 in the presence of K562 compared to that of CYNK-101 alone.

[0546] Increased intracellular cytokine production such as GM-CSF, TNF-a, IFN-g was shown from CYNK-101 in the presence of K562, or stimulated with PMAi, or IL-12+IL-18 compared to that of CYNK-101 alone.

7.7 Example 7: Cleavage Resistant CD16 Expressing CYNK Cells Are Effective Against HER2+ Cancers

[0547] CYNK-101 is a human placental hematopoietic stem cell derived natural killer (NK) cell product, that is genetically modified to express a variant of CD16, Fc gamma receptor III (FcgRIII), via lentiviral vector transduction. CD16 plays a central role in antibody dependent cell mediated cytotoxicity (ADCC) in NK cells through binding to the Fc portion of IgG antibodies. FIG. 18 shows the rationale of ADCC by engineering CD16 on NK cells. CYNK-101 was designed to express a high-affinity proteolytic cleavage-resistant CD16 variant with a Valine at amino acid position 158 and Proline at position 197 as demonstrated in the Construct Design in FIG. 19 [Wu, 1997; Sugita, 1999; Koene, 1997; Jing, 2015].

[0548] To assess the anti-tumor ADCC activity of CYNK-101, extensive in vitro, ex vivo and in vivo studies have been conducted using CYNK-101 in combination with trastuzumab (Herceptin) against HER2.sup.+ Gastric/Gastroesophageal Junction (G/GEJ) cancer cell lines, and HER2.sup.+ breast cancer cell lines. The in vitro study examined CYNK-101 multi-parameter phenotypic characterization, CD16 shedding resistance evaluation of CYNK-101 followed by PMAi stimulation, cytotoxicity evaluation of CYNK-101 in combination with Herceptin against G/GEJ cancer cell lines as NCI-N87 and OE19; and breast cancer cell lines as AU565, BT-474, HCC-1954, SKBR-3, ZR-75-30; and assessment of cytokine secretion such as IFN-, TNF-, and GM-CSF from CYNK-101 in combination with Herceptin in the presence of the above tumor cell lines. An ex vivo study was conducted to further evaluate ADCC activity of CYNK-101 administered to NSG mice. CYNK-101 cells were isolated from mouse liver thirteen days post injection and evaluated for phenotypic characterization, CD16 shedding resistance and anti-tumor ADCC activity. An in vivo study was further performed to evaluate the anti-tumor activity of CYNK-101 in combination with Trastuzumab in a subcutaneous gastric tumor mouse model. In addition, the safety of CYNK-101 in combination with Trastuzumab was also evaluated in this study. The results summarized here provide a rationale to support clinical development of CYNK-101 in combination with trastuzumab against HER2 overexpressing G/GEJ cancer.

Methods and Materials

xCELLigence Real Time Cell Analysis-Based Antibody Dependent Cellular Cytotoxicity Assay Against Tumor Cell Lines

[0549] CYNK-101 cells (donor IDs: 2000112643, 2000113629, 2000113366, 2000113511, 2000113472, 2000113880, 2000114102) with purity of >85% CD56.sup.+CD3 were served as effector cells in this assay. The following HER2 overexpressing tumor cell lines were used as target cells: NCI-N87 (ATCC, Cat #CRL-5822), OE19 (Sigma, Cat #96071721), HCC-1954 (ATCC, Cat #CRL-2338), SKBR-3 (ATCC, Cat #HTB-30), BT-474 (ATCC, Cat #CRL-3247), AU565 (ATCC, Cat #CRL-2351), and ZR-75-30 (ATCC, Cat #CRL-1504). The anti-HER2 antibody Herceptin (Blue Door Pharma, Rockville, MD, Cat #50242-132-01) or the Ultra-LEAF purified human IgG.sub.1 isotype control recombinant antibody (Biolegend, Cat #403501) was used at 1 g/mL. The xCELLigence real time cell analysis (RTCA) system (ACEA Biosciences, San Diego, CA) was used for the determination of ADCC activities mediated by the combination of effector cells and antibody. 810.sup.4/well NCI-N87, 610.sup.4/well OE19, 110.sup.4/well HCC-1954, 210.sup.4/well SKBR-3, 210.sup.4/well BT-474, 210.sup.4/well AU565, or 210.sup.4/well ZR-75-30 target cells were seeded in the E-plates in 100 L of assay buffer (RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin) for 24 hours at 37 C. in 5% CO.sub.2. IgG.sub.1 or Herceptin was then added 30 minutes prior to the addition of different amounts of CYNK-101 cells to achieve various E:T ratios (10:1, 5:1, 2.5:1, 1:1 and 0.6:1) for each donor in duplicate. Target cells were incubated with CYNK-101 cells in the presence of the antibodies in 200 L final volume. The xCELLigence software was then used to determine the percent cytotoxicity at 4 h and 24 h after effector cells added. The results from different experiments were reported as mean of donorsstandard deviation of the mean (SD).

Flow Cytometry-Based Cytotoxicity Assay with Tumor Cell Lines

[0550] CYNK-101 from seven different donors (donor IDs: 2000112643, 2000113629, 2000113366, 2000113511, 2000113472, 2000113880, 2000114102) used in all the assays were >85% CD56.sup.+CD3.sup. cells, >70% viability by 7-AAD.sup.. Cytotoxicity assays were performed by using CYNK-101 cells as effector cells and the tumor cell lines, such as K562 (ATCC, Cat #CCL-243) as the target cells. Target cell number was fixed at 110.sup.4 while CYNK-101 cells were used in different amounts to achieve various E:T ratios (10:1, 5:1, 2.5:1, and 1.25:1). To distinguish target cells from CYNK-101 cells on the flow cytometer, target cells were labeled with 7.5 M PKH26 fluorescent dye (Sigma-Aldrich, St Louis, MO, Cat #PKH26-GL). Target cells were incubated with NK cells in 96-well U-bottom tissue culture plates in 200 L of assay buffer for 4 hours at 37 C. in 5% CO.sub.2. After incubation, cells were harvested and TO-PRO-3 (Invitrogen, Carlsbad, CA Cat #T3605), a membrane-impermeable DNA stain, was added to the cultures at 1 M final concentration in order to identify dead cells (TO-PRO-3.sup.+). To determine spontaneous target cell death, PKH26-labeled target cells were cultured alone for the duration of the assay. As a positive control for dead cells, 110.sup.5 labeled target cells were permeabilized with 350 L of Cytofix/Cytoperm buffer (BD Biosciences, Cat #554722) for 20 minutes at 4 C. The data were acquired on a FACSCanto X (BD Biosciences, San Jose, CA) and analyzed by FlowJo (Tree Star, Ashland, OR).

[0551] The percentage of dead target cells in each sample was calculated as follows: % TO-PRO-3.sup.+PKH26.sup.+ cells/(% TO-PRO-3.sup.+PKH26*+% TO-PRO-3-PKH26.sup.+) * 100%. Percent cytotoxicity reported was calculated by subtracting the percent of dead target cells in cultures of target cells alone from the percent of dead target cells in co-cultures of effector and target cells. The results are reported as the mean of the donorsstandard deviation of the mean (SD).

CD16 Shedding Resistance Assay

[0552] To evaluate the CD16 shedding resistance of CYNK-101 followed by cell stimulation, 510.sup.5 CYNK-101 cells (donor IDs: 2000112643, 2000113629, 2000113366, 2000113511, 2000113472, 2000113880, 2000114102) were incubated for 2 h with either Phorbol 12-myristate 13-acetate plus ionomycin (PMAi) (eBioscience, Cat #00-4970-93) or ethanol vehicle control in RPMI-1640 media with 10% FBS. NK cells without CD16 transduction were served as assay control. Cells from each condition were then incubated at room temperature for 10 minutes in FACS buffer (PBS with 0.5 mM EDTA and 2% FBS) containing human Fc block (BD Biosciences, Cat #564220). The cells were stained with anti-CD16 PE (BD Biosciences, Cat #556619), anti-CD56 APC (BD Biosciences, Cat #555518), and anti-CD11a FITC (BD Biosciences, Cat #555383) for 30 minutes at 4 C. After washing, 7-AAD (BD Biosciences, Cat #559925) was added to distinguish live and dead cells, the data were acquired on FACSCanto X (BD Biosciences, San Jose, CA) and analyzed by FlowJo (Tree Star, Ashland, OR). The data were reported as % CD56.sup.+CD16 cells gated under 7-AAD.sup. cells. Setting of the % positive gate was done using isotype stained samples as controls.

Flow Cytometric Detection of NK Surface Receptors

[0553] 110.sup.6 CYNK-101 cells from each donor (donor IDs: 2000112643, 2000113629, 2000113366, 2000113511, 2000113472, 2000113880, 2000114102) were first labeled with a viability stain (LIVE/DEAD Aqua, Invitrogen, Cat #L34957) according to the manufacturer's protocol (Invitrogen). Cells were then stained with anti-CD56 Alexa Fluor 700 (BD Biosciences, Cat #557919), anti-CD3 APC-H7 (BD Biosciences, Cat #560176), and anti-CD16-PE (BD Biosciences, Cat #556619) antibodies, as well as with an additional seven antibodies against NK receptors: NKG2D (BD Biosciences, Cat #562364), NKp46 (BD Biosciences, Cat #563230), NKp44 (BD Biosciences, Cat #744305), NKp30 (BD Biosciences, Cat #563385), CD94 (R & D Systems, Cat #FAB1058A), CD11a (BD Biosciences, Cat #561387), and DNAM-1 (BD Biosciences, Cat #559788). Samples were washed and immediately analyzed on BDLSR Fortessa X-20. Data were analyzed using the FlowJo software. The data were expressed as % positive cells gated under CD56.sup.+CD3.sup. Aqua single cells. Setting of the % positive gate was done using unstained and isotype stained samples as controls.

Intracellular Cytokine Staining (ICCS) Assay

[0554] CYNK-101 cells (donor IDs: 2000112643, 2000113629, 2000113366, 2000113511, 2000113472, 2000113880, 2000114102) were plated in 96-well plates at 210.sup.5 cells/well in 200 L of RPMI 1640 medium with 10% FBS and antibiotics. Stimulating agents were added to the corresponding wells as followings: 1 Cell Stimulation Cocktail containing PMAi (eBiosciences, Cat #00-4970-03), or IL-12 (20 ng/mL) (R&D Systems, Minneapolis, MN, Cat #219-IL-025) plus IL-18 (100 ng/mL) (R&D Systems, Cat #B003-2). Additionally, 110.sup.5 K562 tumor cells were added with the CYNK-101 cells at E:T ratio of 2:1. Control wells were left unstimulated for the duration of stimulation. After 1 hour, the protein transport inhibitor Brefeldin A (BD FastImmune, San Diego, CA, Cat #347688) was added to all the wells at 10 g/mL final concentration. Stimulation was performed for an additional 4 hours, after which cells were labeled with a viability stain, LIVE/DEAD Aqua, according to the manufacturer's protocol (Invitrogen, Cat #L34957). Cells were then stained with anti-CD56 PE-Cy7 (Biolegend, Cat #362510), anti-CD3 APC-H7 (BD Biosciences, Cat #560176), and anti-CD11a BV711 (BD Biosciences, Cat #563935) antibodies for 15 min at 4 C. in brilliant stain buffer (BD Biosciences, Cat #563794), fixed and permeabilized using Cytofix/Cytoperm kit according to the manufacturer's protocol (BD Biosciences, Cat #554722), and then washed with perm wash buffer (BD Biosciences, Cat #554723). Permeabilized cells were stained with anti-IFN- APC (BD Biosciences, Cat #554702), anti-TNF- BV650 (BD Biosciences, Cat #563418), anti-GM-CSF PE (BD Biosciences, Cat #554507), anti-Perforin BV421 (Biolegend, Cat #308122), and anti-Granzyme B AF700 (BD Biosciences, Cat #560213) antibodies in brilliant stain buffer. Data were acquired on BD LSRFortessa X-20 and analyzed using the FlowJo software. Data were expressed as % positive cells gated under CD56.sup.+CD3-Aqua single cells. The gate that delineated cells positive for a given cytokine was set by utilizing unstimulated samples, isotypes, and/or separation of stained populations. The data were analyzed and presented using GraphPad Prism (GraphPad Software, La Jolla, CA) and depicted as mean of the donorsSD.

Cytokine Secretion Assay

[0555] CYNK-101 cells (donor IDs: 2000112643, 2000113629, 2000113366, 2000113511, 2000113472, 2000113880, 2000114102) were incubated with tumor cell lines, or tumor cell lines with either the IgG.sub.1 or Herceptin antibodies at 1 g/mL in 96-well U-bottom tissue culture plates at an E:T ratio of 1:1 (110.sup.5 cells each) in 200 L of RPMI 1640 supplemented with 10% FBS and antibiotics. After 24 hours incubation at 37 C. and 5% CO.sub.2, the supernatant was collected and cytokine concentrations were determined by Luminex analysis using MILLIPLEX MAP magnetic bead kits (EMD Millipore, Burlington, MA, Cat #HCD8MAG-15K-07 for granulocyte-macrophage colony-stimulating factor (GM-CSF), perforin, TNF-, IL-10, granzyme A, granzyme B and IFN-) according to the protocol provided by the manufacturer. The data were analyzed using Milliplex Xponent and Analyst software (EMD Millipore). The results from different experiments were depicted as mean of donorsSD.

Ex Vivo Characterization of CYNK-101

[0556] 210.sup.7 CYNK-101 cells were intravenous (IV) injected to busulfan pretreated NOD-scid IL2Rgamma.sup.null immunodeficient (NSG) mice at Day0. Recombinant human IL-15 was intraperitoneally injected at Days 0, 2, 4, 6, 8, 10 and 12 to support NK cell proliferation and survival. At thirteen days post IV injection, CYNK-101 cells were isolated from mouse liver and evaluated for phenotype characterization, CD16 shedding resistance followed by PMAi stimulation and ADCC activity against NCI-N87. The phenotyping panel included the following: AQUA, mCD45, hCD45, CD3, CD56, CD16, CD11a, CD94, CD158a, CD158e1/e2, CD158b1/b2, NKG2D, NKp46, and NKp44.

In Vivo Efficacy and Safety Study of CYNK-101

[0557] The anti-tumor activity and safety of CYNK-101 in combination with Trastuzumab were evaluated in a subcutaneous gastric tumor model using the NSG mice with transgenic expression of human IL-15 (NSG-Tg-hIL15).

[0558] A total of eighty female NSG-Tg-hIL15 mice were preconditioned with busulfan (30 mg/kg) to deplete myeloid cells on Day 3. As shown in FIG. 20, NCI-N87 gastric cancer cell line was subcutaneously inoculated at the dose of 310.sup.6 cells/mouse on Day 0. One week later (Day 7), seventy-three mice with palpable tumors were randomized to the following four groups: Vehicle control (n=19), Trastuzumab (n=17), CYNK-101 (n=19), CYNK-101+Trastuzumab (n=18). Trastuzumab (10 mg/kg) was administered intraperitonially (IP) once on Day 7, and CYNK-101 at the dose of 1010.sup.6 cells/mouse was administered intravenously (IV) three times on Days 7, 14 and 21.

In Vitro Anti-Tumor ADCC Activity of CYNK-101 in Combination with Herceptin Against Her2.sup.+Solid Tumor Cancer Cells

Enhanced in Vitro ADCC Activities of CYNK-101 Against G/GEJ Tumor Cell Lines

[0559] To assess the in vitro ADCC activity of CYNK-101 in combination with the anti-HER2 antibody Herceptin against HER2 overexpressing G/GEJ cancer cell lines, NCI-N.sub.87 and OE-19 were used as targets in a xCELLigence real time cell analysis-based assay. To assess NK cell dose dependent effects, varying E:T ratios from 10:1 to 0.6:1 for 7 different CYNK-101 donors were used in the assay. The Herceptin alone or IgG1 control alone against the targets were included as controls. In addition, low HER2 expression normal human dermal fibroblasts cells (NHDF) were used as target cells to determine whether cytotoxic effects of CYNK-101 plus Herceptin were specific for HER2.sup.+ tumor cells.

[0560] The results showed that CYNK-101, when combined with Herceptin, displayed enhanced cytotoxic activity against both NCI-N87 and OE19 G/GEJ tumor cell lines at an E:T ratio as low as 0.6:1 compared to when the IgG antibody was added. This activity increased with increasing E:T ratios in a dose-dependent manner at the early timepoint of 4 h for both tumor lines and at 24 h for OE19 (FIGS. 21A-21B). By 24 h at an E:T ratio of 0.6:1, CYNK-101 cells (n=7 donors) lysed 91.3%15.0% of NCI-N87 cells in the presence of Herceptin compared to 7.5%9.3% of NCI-N87 cells in the presence of IgG control, and 44.6%20.1% of OE19 cells in the presence of Herceptin compared to 14.7% 14.8% in the presence of IgGcontrol (FIGS. 21A-21B Error! Reference source not found).

[0561] FIGS. 21A-21B shows the average cytotoxic activity of CYNK-101 cells in combination with Herceptin or a control IgG against the indicated gastric tumor cell lines, NCI-N87 and OE19. The error bars represent the SD from the mean calculated from seven different CYNK-101 donors. Cytotoxicity from Herceptin or IgG alone is included for reference. * indicate significantly higher activity of CYNK-101 in combination with Herceptin compared to that of IgG (***p<0.001 and *p<0.05).

[0562] Furthermore, when using mixed target of NCI-N87 with Low HER2 expression NHDF (FIG. 22A), CYNK-101 cells in combination with Herceptin specifically kill NCI-N87, while spare NHDF cells at any of the E:T ratios tested up to 100:1 (FIG. 22B), indicating that CYNK-101 cells in combination with Herceptin were capable not only of lysing HER2.sup.+ tumor cells but also of discriminating between HER2.sup.+ normal and tumor targets.

Secretion of IFN-Gamma, TNF-Alpha and GM-CSF Cytokines in Response to Gastric Cancer Tumor Cell Lines

[0563] Because NK cell cytokine secretion is implicated in anti-tumor immune responses [Marcus, 2014], CYNK-101 cytokine secretion was tested following co-culture with gastric cancer tumor cell lines. After 24 hours of co-culture, supernatant was analyzed for the presence of IFN-, TNF-, and GM-CSF. The results are summarized in Table 5.

[0564] IFN- was significantly induced from CYNK-101 in the presence of both NCI-N87 and OE19 when Herceptin was added (Table 5). Specifically, 110.sup.6 CYNK-101 cells secreted 803559 g IFN- with NCI-N87 and 8872 g with OE19 when Herceptin was present compared to 1520 g and 00 g when IgG.sub.1 was present, respectively; the secretion in the presence of IgG was not significantly different from that of the baseline for CYNK-101 cells of 1316 g. In addition, TNF- and GM-CSF were significantly induced in the presence of both gastric cancer tumor cell lines when Herceptin was added in combination (Table 5).

[0565] In summary, CYNK-101 cells secreted relevant immunomodulatory cytokines in the presence of Herceptin and gastric cancer tumor cell lines. Thus, CYNK-101 cells in combination with Herceptin were not only capable of directly lysing gastric cancer tumor cells but also capable of indirectly eliciting anti-tumor responses through their ability to secrete IFN-, TNF-, and GM-CSF.

TABLE-US-00007 TABLE 5 Summary of cytokine secretion from CYNK-101 in response to gastric cancer tumor cells Tumor IFN- (pg/10.sup.6 cells) TNF- (pg/10.sup.6 cells) GM-CSF (pg/10.sup.6 cells) cells .sup.a)N .sup.b)Ave SD Min Max Ave SD Min Max Ave SD Min Max None 6 13 16 0 40 87 47 20 140 167 114 60 340 NCI-N87 6 10 17 0 40 103 46 20 160 303 167 160 540 NCI-N87 + 6 15 20 0 50 110 55 40 200 307 169 140 560 IgG.sub.1 NCI-N87 + 6 *803 559 200 1520 *610 212 320 960 *3417 1276 1380 4840 Herceptin OE19 5 0 0 0 0 88 23 60 120 396 157 180 580 OE19 + 5 0 0 0 0 76 30 40 120 412 247 200 840 IgG.sub.1 OE19 + 5 *88 72 20 180 *248 106 140 420 *2112 585 1360 2960 Herceptin .sup.a)Number of CYNK-101 donors .sup.b)Average *P < 0.05 (Student's T-test, in the presence of tumor cells vs. none)

CYNK-101 is Resistant to CD16 Shedding Post PMAi Stimulation

[0566] High expression of CD16 was detected from CYNK-101 as seen in FIG. 23, the average of CD16 expression of CYNK-101 (n=7 donors) is 74.1%5.6%. To assess if CYNK-101 is resistant to CD16 shedding, CYNK-101 was stimulated with PMAi for 2 h, followed by surface staining of CD16, non-transduced NK cells were served as control. As shown in FIGS. 24A-24B, followed by PMAi stimulation, the percentage of CD16 expression on CYNK-101 maintained compared to that of control, while around 70% CD16 expression decreased from non-transduced NK cells. In summary, CYNK-101 demonstrates CD16 shedding resistant post PMAi stimulation.

Expression of Cytotoxic Mediators, Perforin and Granzyme B, and NK Activating Receptors in CYNK-101

[0567] To confirm that CYNK-101 cells express molecules required for cytotoxic activity, we analyzed intracellular expression of perform, granzyme B, and cell surface expression of activating receptors, such as CD16, DNAM-1 (CD226), NKG2D, CD94, NKp30, NKp44, and NKp46, which are known to be important for cytotoxic effects of NK cells [Marcus, 2014] by flow cytometry. Data for CYNK-101 from seven donors were summarized in Table 6.

[0568] Results indicated that CYNK-101 cells expressed all of the factors required for cytotoxic activity tested: perform (25.8%23.0% cells), granzyme B (30.4%30.4% cells), CD16 (74.1%5.6% cells), CD226 (49.6%9.7% cells), NKG2D (40.6%14.0% cells), CD94 (48.9%10.7% cells), NKp30 (37.4%16.8% cells), NKp44 (93.8%3.1% cells), and NKp46 (15.7%5.0% cells). Results suggested that CYNK-101 cells exerted cytotoxic effects onto their targets by utilizing pathways that have been well described for NK cells involving recognition of tumor targets via activating receptor engagement and secretion of perforin and granzyme B-containing granules.

TABLE-US-00008 TABLE 6 Assessment of expression of perforin, Granzyme B, and activation receptors of CYNK-101 by Flow Cytometry Donor Perforin+ Granzyme CD16+ CD226+ NKG2D+ CD94+ NKp30+ NKp44+ NKp46+ ID % B+ % % % % % % % % Gated under CD56+CD3 live cells PL6 13.1 17.7 78.8 57.4 49.6 54.0 49.8 95.3 9.8 PL7 21.3 19.3 65.9 43.9 33.7 42.9 30.0 92.0 24.9 DQ1 73.7 99.1 71.1 62.1 67.5 66.1 68.5 96.2 17.8 DQ2 20.6 21.2 73.0 50.3 36.9 53.7 29.0 89.7 11.2 DQ3 11.7 18.2 83.3 50.9 38.9 51.3 27.5 90.2 15.9 DQ4 6.0 16.4 73.4 50.7 33.5 41.4 38.1 97.1 14.0 DQ5 34.3 20.6 73.4 31.8 24.4 33.2 18.7 95.9 16.2 Average 25.8 30.4 74.1 49.6 40.6 48.9 37.4 93.8 15.7 SD 23.0 30.4 5.6 9.7 14.0 10.7 16.8 3.1 5.0 Min 6.0 16.4 65.9 31.8 24.4 33.2 18.7 89.7 9.8 Max 73.7 99.1 83.3 62.1 67.5 66.1 68.5 97.1 24.9

Enhanced in Vitro ADCC Activities of CYNK-101 Against HER2.SUP.+ Breast Cancer Cell Lines

[0569] To assess the in vitro ADCC activity of CYNK-101 in combination with Herceptin against HER2.sup.+ breast cancer cell lines, AU565, BT-474, HCC-1954, SKBR-3, ZR-75-30 were used as targets in a xCELLigence real time cell analysis-based assay. To assess NK cell dose dependent effects, varying E:T ratios from 10:1 to 0.6:1 for 7 different CYNK-101 donors (unless specified) were used in the assay.

[0570] As shown in FIGS. 25A-25B, enhanced anti-tumor ADCC activity of CYNK-101 in combination with Herceptin against the HER2.sup.+ breast cancer cell lines was demonstrated at both 4 h and 24 h with the indicated E:T ratios compared to that of CYNK-101 plus IgG control.

Secretion of IFN-Gamma, TNF-Alpha and GM-CSF Cytokines in Response to Breast Cancer Cell Lines

[0571] CYNK-101 cytokine secretion was tested following co-culture with breast cancer cell lines in the presence of Herceptin or IgG control. After 24 hours of co-culture, supernatant was analyzed for the production of IFN-, TNF-, and GM-CSF.

[0572] As seen in FIG. 26, IFN- was significantly induced from CYNK-101 in the presence of the breast cancer cell lines as indicated compared to that of IgG control. In addition, TNF- and GM-CSF were significantly induced in the presence of the breast cancer cell lines when Herceptin was added in combination.

[0573] In summary, CYNK-101 cells secreted relevant immunomodulatory cytokines in the presence of Herceptin and breast cancer cell lines. Thus, CYNK-101 cells in combination with Herceptin were not only capable of directly lysing breast cancer cells but also capable of indirectly eliciting anti-tumor responses through their ability to secrete IFN-, TNF-, and GM-CSF.

Ex Vivo Anti-Tumor ADCC Activity of CYNK-101 in Combination with Herceptin Against Her2+ Gastric Cancer Cells

[0574] An ex vivo model was developed to further evaluate ADCC activity of CYNK-101 administered to NSG mice. In brief, CYNK-101 was IV-injected at the dose of 210.sup.7 cells/animal into busulfan-pretreated NSG mice at Day 0. Recombinant human IL-15 was intraperitoneally injected at Days 0, 2, 4, 6, 8, 10 and 12 to support CYNK-101 in vivo expansion and persistence. At Day 13, CYNK-101 cells were isolated from mouse livers (ex vivo-CYNK-101) and assessed for phenotype and ADCC activity. FIG. 27 shows the percentage of CYNK-101 cells from the livers pre and post mouse cell depletion in the ex vivo study. In this study, among the cells from post mouse cell depletion, 69.3% of which were live hCD45Y, and of those 97.6% were CD56.sup.+CD3.sup. cells. Surface marker expression profiling revealed that the ex vivo isolated cells have increased CD94 and CD158 expression, retained their CD16 expression, and decreased NKp44 expression compared to the corresponding prior infusion CYNK-101 donor (FIG. 28). Thus, the increased expression of CD94 and CD158 indicated the maturation of CYNK-101 cells in NSG mice after 13 days IV administration.

[0575] After stimulated by PMAi for 2 h, both ex vivo and in vitro CYNK-101 demonstrated CD16 shedding resistance, while 60% CD16 shedding observed from control sample (FIG. 29). FIG. 30 demonstrated that enhanced ADCC activity of ex vivo-CYNK-101 in combination with Herceptin against NCI-N87 at E:T ratio of 0.5:1 compared to that of IgG, or in vitro CYNK-101 plus Herceptin, in vitro CYNK-101 plus IgG. Increased cytokine secretion such as IFN-, TNF- and GM-CSF was observed from ex vivo CYNK-101 in combination with Herceptin against NCI-N87 at E:T ratio of 0.5:1 (FIG. 31).

[0576] In summary, the study demonstrated that ex vivo-CYNK-101 can be enriched from mouse liver followed by 13 days post injection. Ex vivo-CYNK-101 was CD16 shedding resistance post PMAi stimulation. Ex vivo-CYNK-101 in combination with Herceptin showed enhanced ADCC activity and enhanced cytokine secretion against NCI-N87 compared to that of ex vivo-CYNK-101+IgG, in vitro CYNK-101+Herceptin, and in vitro CYNK-101+IgG.

[0577] Taken together, the in vitro and ex vivo results demonstrated ADCC activity of CYNK-101 in combination with Herceptin against HER2 overexpressing G/GEJ tumor cell lines. The data outlined here support the clinical development of CYNK-101 in combination with trastuzumab for patients with HER2 overexpressing G/GEJ adenocarcinoma.

In Vivo Efficacy of CYNK-101 in Combination with Trastuzumab in a Subcutaneous Gastric Tumor Mouse Model

[0578] In this study, the anti-tumor activity and safety of CYNK-101 in combination with Trastuzumab were evaluated in a subcutaneous gastric tumor model using NSG-Tg-hIL15. As shown in FIG. 32, CYNK-101 alone had no impact on tumor growth, whereas Trastuzumab alone significantly inhibited tumor growth from Day 14 to Day 28 compared with vehicle control. In consistent with our in vitro and ex vivo findings, CYNK-101 in combination with Trastuzumab demonstrated a synergistic anti-tumor activity and significantly suppressed tumor growth compared to Trastuzumab or CYNK-101 alone. More significantly, CYNK-101 plus Trastuzumab treatment continuously reduced tumor volume from Day 7 to Day 28, and approximately 30% tumor volume reduction was achieved on Day 28 compared to Day 7 baseline.

[0579] Three repeated weekly IV administration of CYNK-101 at the dose of 1010.sup.6 cells/mouse was safe and well tolerated with or without Trastuzumab combination therapy. No abnormal clinical symptoms, morbidity and mortality occurred throughout the study. No CYNK-101 or CYNK-101+Trastuzumab related abnormal effects on body weight and gross pathology at necropsy were observed.

[0580] Taken together, this study demonstrated that CYNK-101 in combination with Trastuzumab had a synergistic in vivo anti-tumor activity against gastric cancer, and the combination therapy was safe and well tolerated.

[0581] In summary, our results demonstrated synergistic anti-tumor ADCC activities of CYNK-101 plus Trastuzumab against HER2.sup.+ gastric cancer cells in vitro, ex vivo and in vivo. Further development of CYNK-101 in combination with Trastuzumab for HER2.sup.+ gastric cancer immunotherapy is underway.

REFERENCES

[0582] Jing Y, Ni Z, Wu J, Higgins L, Markowski T W, Kaufman D S, Walcheck B. Identification of an ADAM17 cleavage region in human CD16 (FcRIII) and the engineering of a non-cleavable version of the receptor in NK cells. PLoS One. 2015 Mar. 27; 10(3):e0121788. [0583] Koene H R, Kleijer M, Algra J, Roos D, von dem Borne A E, de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood. 1997 Aug. 1; 90(3):1109-14. [0584] Marcus A, Gowen B G, Thompson T W, Iannello A, Ardolino M, Deng W, et al. Recognition of tumors by the innate immune system and natural killer cells. Adv Immunol. 2014; 122:91-128. [0585] Sugita N, Yamamoto K, Kobayashi T, Van Der Pol W, Horigome T, Yoshie H, Van De Winkel J G, Hara K. Relevance of Fe gamma RIIIa-158V-F polymorphism to recurrence of adult periodontitis in Japanese patients. Clin Exp Immunol. 1999 August; 117(2):350-4. [0586] Wu J, Edberg J C, Redecha P B, Bansal V, Guyre P M, Coleman K, Salmon J E, Kimberly R P. A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to autoimmune disease. J Clin Invest. 1997 Sep. 1; 100(5):1059-70.

7.8 Example 8: Development of Additional Cleavage Resistant CD16 Variants

[0587] CYNK-101 is a human placental hematopoietic stem cell derived natural killer (NK) cell product, that is genetically modified to express a variant of CD16, Fc gamma receptor III (FcgRIII), via lentiviral vector transduction. CD16 plays a central role in antibody dependent cell mediated cytotoxicity (ADCC) in NK cells through binding to the Fc portion of IgG antibodies. FIG. 33 shows the rationale of ADCC by engineering CD16 on NK cells. CYNK-101 was designed to express a high-affinity proteolytic cleavage-resistant CD16 variant with a Valine at amino acid position 158 and Proline at position 197 as demonstrated in the Construct Design in FIG. 34 [Wu, 1997; Sugita, 1999; Koene, 1997; Jing, 2015].

[0588] In order to generate second generation of CYNK-101, we have designed a series of CD16 variant constructs in order to improve expression and or ADCC relative to the S197P mutation currently used in our CD16VP construct (Table 7). Our strategy is to screen the CD16 variant constructs, identify the candidate of CD16 variant lentivector which shows the comparable or better functional activity compared to CD16VP with CD16 shedding resistance for second generation of CYNK-101.

TABLE-US-00009 TABLE7 ListofCD16variantsconstructs indicates data missing or illegible when filed

[0589] Proposal 1 (Table 7, constructs1&2) is to use Cysteine or Glycine to replace Serine at position 197 for potential shedding resistance. The rationale is to use minimum deviation from the wild-type sequences. Serine is an amino acid with polar neutral sidechain. Cysteine is an amino acid with polar neutral sidechain and similar size as Serine. On the other side, Cysteine might drive disulfide bond. Glycine is classified as unique amino acid like Prolineit is structurally simplest and non-reactive in proteins (FIG. 35).

[0590] Proposal 2 (Table 7, constructs 38) is to replace entire membrane proximal stalk sequence with CD8a or CD28 used in CARs, scrambled sequence or CD64 (sequence 189-208) (FIG. 36).

[0591] Proposal 3 (Table 7: constructs 913) is to replace multiple amino acids in the cleavage region AVSTI reported in the literature (Jing, 2015). Amino acids A, V, S and I can be replaced. The followings are proposed replacements: [0592] A (alanine) with V (valine) [0593] V (valine) with A (alanine) [0594] S (serine) with C (cysteine) or G (glycine) [0595] I (Isoleucine) with L (Leucine) [0596] The proposed replacements of A, V and I are to alter ADAM17 recognition domain with amino acids with similar hydrophobic side chains (FIG. 37).

[0597] We are planning to first generate lentivirus vector from the listed CD16 variant constructs, then use Celularity's proprietary placental CD34 derived CYNK-101 process to screen the Lentivectors to identify the candidate/(s). Two selection criteria will be applied for identifying CD16 variant candidate: 1) The comparable or better ADCC activity of CD16-CYNK in combination with Herceptin against NCI-N87 compared to that of current CD16VPCYNK; 2) CD16 shedding resistance followed by PMAi stimulation. After the candidate of CD16 variant is identified, we will use the same approach to validate the Preclinical grade Lentivirus CD16 variant vector, followed by generation of GMP grade CD16 variant lentivector for second generation of CYNK-101. Currently the study is ongoing.

Example 8 References

[0598] Jing Y, Ni Z, Wu J, Higgins L, Markowski T W, Kaufman D S, Walcheck B. Identification of an ADAM17 cleavage region in human CD16 (FcRIII) and the engineering of a non-cleavable version of the receptor in NK cells. PLoS One. 2015 Mar. 27; 10(3):e0121788. [0599] Koene H R, Kleijer M, Algra J, Roos D, von dem Borne A E, de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood. 1997 Aug. 1; 90(3):1109-14. [0600] Sugita N, Yamamoto K, Kobayashi T, Van Der Pol W, Horigome T, Yoshie H, Van De Winkel J G, Hara K. Relevance of Fc gamma RIIIa-158V-F polymorphism to recurrence of adult periodontitis in Japanese patients. Clin Exp Immunol. 1999 August; 117(2):350-4. [0601] Wu J, Edberg J C, Redecha P B, Bansal V, Guyre P M, Coleman K, Salmon J E, Kimberly R P. A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to autoimmune disease. J Clin Invest. 1997 Sep. 1; 100(5):1059-70.

7.9 Example 9: Testing and Validation of Cleavage Resistant CD16 Variants

Summary/Synopsis

[0602] Celularity is developing CYNK-101 as a novel product in combination with trastuzumab and pembrolizumab for the treatment of human epidermal growth factor 2 (HER2) overexpressing gastric/gastroesophageal junction (G/GEJ) adenocarcinoma. CYNK-101 is a human placental hematopoietic stem/progenitor cell derived natural killer (NK) cell product, which is genetically modified to express a variant of CD16, Fc gamma receptor III (FcRIII), via lentiviral vector transduction. CD16 plays a central role in antibody-dependent cell-mediated cytotoxicity (ADCC) in NK cells through binding to the Fc portion of IgG antibodies. CYNK-101 was designed to express a high-affinity proteolytic cleavage-resistant CD16 variant (CD16VP) with a Valine at amino acid position 158 and Proline at position 197.

[0603] Celularity has designed, synthesized and evaluated a number of constructs using placental CD34 derived CYNK culture platform. With total of 16 donors evaluated, CD16VS (construct #5) has been nominated based on its comparable fold expansion, CD16 expression, shedding resistance post PMAi stimulation, phenotype characterization, ADCC activity and cytokine secretion profiling in comparison to that of CD16VP.

Introduction

[0604] Celularity is developing CYNK-101 as a novel product in combination with trastuzumab and pembrolizumab for the treatment of HER2 overexpressing G/GEJ adenocarcinoma. CYNK-101 is a human placental hematopoietic stem/progenitor cell derived NK cell product, which is genetically modified to express a variant of CD16 (FcRIII) via lentiviral vector transduction. CD16 plays a central role in antibody-dependent cell-mediated cytotoxicity (ADCC) in NK cells through binding to the Fc portion of IgG antibodies. CYNK-101 was designed to express a high-affinity proteolytic cleavage-resistant CD16 variant (CD16VP) with a Valine at amino acid position 158 and Proline at position 197 (Koene, 1997; Jing, 2015). CD16VP lentivirus vector (LVV) is generated by Lentigen using its proprietary lentivirus backbone.

[0605] The higher affinity polymorphism variant 158V is referenced in Koene, 1997 is considered safe to retain in the new construct without IP concern.

[0606] Celularity has designed, synthesized and evaluated a number of constructs using placental CD34 derived CYNK culture platform. Day10 transduction efficiency, in process and post-thaw phenotype, shedding assay stimulated by PMAi, cytotoxicity of CYNK cell in combination with trastuzumab against HER2+ gastric cancer cell line NCI-N87 and cytokine secretion profile were performed to compare the activity of CD16 variants vs. CD16VP. The ten constructs were first designed with Myc tag added for Sirion Bio to generate lentivirus vectors (LVV). Followed by the results from in house evaluation, five constructs #1, 2, 5, 9 and 10 without Myc tag were selected for Sirion Bio to generate LVV. Based on the in house data, three constructs: #1, 5 and 10 were selected for Lentigen to generate Research Grade LVVs using the same backbone of CD16VP. With total of 16 donors evaluated, CD16VS (construct #5) has been nominated based on its comparable fold expansion, CD16 expression, shedding resistance post PMAi stimulation, phenotype characterization, ADCC activity and cytokine secretion profiling in comparison to that of CD16VP.

Purpose/Study Objectives

[0607] The purpose of this study is to design and generate Celularity owned CD16 variants with proteolytic cleavage resistance for NK cells ADCC enhancement and to overcome the IP hurdle of CD16VP for CYNK-101. Lentivirus vectors for CD16 variants constructs were generated by Sirion bio and Lentigen, respectively. Placental CD34 derived CYNK culture system was used for the candidate selection. Day10 transduction efficiency, in process and post-thaw phenotype, shedding assay stimulated by PMAi, cytotoxicity of CYNK cell in combination with trastuzumab against HER2+ gastric cancer cell line NCI-N87 and cytokine secretion profile were performed to compare the activity of CD16 variants vs. CD16VP.

Methods

Cd16 Variants Lentivirus Vector Design and Generation

[0608] Lentivirus vectors for in house designed 10 constructs with Myc tag, construct #1, 2, 5, 9, 10 without Myc tag were generated by Sirion Bio. Construct #1, 5, 10 without Myc tag were generated by Lentigen.

Cynk Cell Generation

[0609] Placental CD34+ cells were acquired from healthy donors under fully informed consent. With donor eligibility documentation, tissues were qualified using a series of tests including serology and bacteriology (Lifebank USA). Blood was isolated from healthy donor tissues and processed by red blood cell depletion using Hetastarch (Hospira). The resulting cells were then magnetically labeled using Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec). CD34+ cells were positively selected using CliniMACS Cell Separator following manufacturer's protocol. Placental CD34+ cells were then cryopreserved in CryoStor CS10 (Biolife Solutions) and stored in liquid nitrogen before use.

[0610] Human placental CD34+ cells were transduced with a lentivirus vector expressing a CD16 variants as indicated and cultured for 35 days in the presence of cytokines, including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, to generate CYNK cells.

Immunophenotypical Characterization

[0611] The phenotype of CYNK cells was analyzed by multicolor flow cytometry. CYNK cells were washed and stained with fluorochrome-conjugated antibodies diluted in staining buffer [2% fetal bovine serum (FBS) in phosphate buffered saline (PBS)] according to the manufacturer's protocol. Dead cells were labelled with the Live/Dead Fixable Aqua Stain (Invitrogen, Cat #L34957) and gated out. Data were acquired on BD Fortessa X20 flow cytometer (BD Biosciences) and were analyzed using FlowJo software. The data were expressed as % positive cells gated under CD56+CD3-Aqua single cells based on the viability staining used. Setting of the % positive gate was done using the corresponding isotype-stained samples as controls.

TABLE-US-00010 TABLE 8 List of Antibodies for NK Phenotypical Characterization Volume of Antibody or Stain Fluorochrome/Antibody Catalog # Clone # per 1 10.sup.5 cells Supplier FITC/CD226 559788 DX11 20 L BD Biosciences PE/CD16 556619 3G8 10 L BD Biosciences PerCP-Cy5.5/NKG2D 562364 1D11 5 L BD Biosciences APC/CD94 FAB-1058A 131412 5 L R & D Systems PE-Cy7/CD11a 561387 HI111 2.5 L BD Biosciences AF700/CD56 557919 B159 2 L BD Biosciences APC-H7/CD3 560176 SK7 2 L BD Biosciences BV421/NKp30 563385 P30-15 5 L BD Biosciences BV650/NKp46 563230 9E2/NKP 10 L BD Biosciences BUV395/NKp44 744305 p44-8 5 L BD Biosciences

Shedding Assay

[0612] To assess the shedding resistance of the genetically modified CD16 variants, 210.sup.5 post-thawed CD16 variants transduced CYNK cells were incubated for 2 hours with either 1 Cell Stimulation Cocktail containing PMAi (eBioscience, Cat #00-4970-9303) or ethanol vehicle control in assay buffer (RPMI-1640 media supplemented with 10% FBS, 1% penicillin, and 1% streptomycin). Non-transduced (NT) CYNK cells were used as a control. After 2-hour stimulation, cells from each treatment condition were then washed twice with FACS buffer (PBS with 0.5 mM EDTA and 2% FBS) and incubated human Fc block (BD Biosciences, Cat #564220) at room temperature for 10 minutes. Staining was then performed with anti-human CD16 PE (BD Biosciences, Cat #556619), anti-human CD56 APC (BD Biosciences, Cat #555518), and anti-human CD3 APC-H7 (BD Biosciences, Cat #560176), or with the corresponding isotype controls for 30 minutes at 4 C. Following washing 2 with FACS buffer the viability stain 7-Aminoactinomycin D (7-AAD) (BD Biosciences, Cat #559925) was added to all stained samples and incubated for ten minutes prior to data acquisition. The data was acquired on a FACS Canto 10 (BD Biosciences, San Jose, CA) and analyzed in FlowJo (Tree Star, Ashland, OR). The data were expressed as % CD56.sup.+CD16+ cells gated under 7-AAD.sup. cells. Setting of the % positive gate was done using isotype-stained samples as controls.

xCELLigence Real Time Cell Analysis ADCC Assay

[0613] The HER2.sup.+ NCI-N87 gastric tumor cell line was used as the target cells. CD16 variant transduced CYNK cells were thawed followed by two days recovery were used as the effector cells. The anti-HER2 antibody trastuzumab (Blue Door Pharma, Rockville, MD, Cat #50242-132-01), the Ultra-LEAF purified Human IgG.sub.1 isotype control recombinant antibody (Biolegend, Cat #403501) were used for the assay. Target cells were seeded at 810.sup.4 cells/well in the 96-well plates in 100 l assay buffer for 24 hours at 37 C. in 5% CO.sub.2. After 24 h, 1 g/mL trastuzumab, or 1 g/mL IgG.sub.1 control was added as indicated. CYNK cells were added post 30 minutes antibodies incubation at 37 C. with 5% CO.sub.2.

[0614] The xCELLigence real time cell analysis (RTCA) system (ACEA Biosciences, San Diego, CA) was used for the determination of the ADCC response mediated by the combination of antibody and effector cells. This system can determine the real time proliferation or cell death in each well of a specialized 96-well plate with electrodes attached to the bottom E-plate by measuring cell adherence as determined by the impedance of the electrical signal that passes through the plate. The more cells that are attached to the bottom of the well the higher the resulting impedance or cell index that is measured. The cell index over time in wells with effector cells, antibody, and target is compared to the cell index in wells with target alone and is converted to a percent cytolysis.

[0615] The xCELLigence system was used for the measurement of the percent cytolysis by each of the antibodies alone, by the effector CYNK cells in the presence of IgG.sub.1 control, and by the effector CYNK cells in the presence of trastuzumab at different E:T ratios as indicated. Target cells were incubated with the effector cells in the presence of the antibodies in 200 l final volume for 24 hours. The xCELLigence software was then used to determine the percent cytotoxicity at 4 and 24 hours of co-culture time.

Cytokine Secretion Assay

[0616] Two-day in vitro recovered CYNK cells were used in this assay as the effector cells. The HER2.sup.+ NCI-N87 gastric tumor cell line was used as the target cell. The effector and target cells were incubated at the E:T ratio of 1:1 (110.sup.5 cells each) in the presence of either 1 g/ml trastuzumab or IgG control antibody, or media control. The supernatant was then collected after 24 h and used for downstream analysis. The cytokine concentrations were determined by Luminex analysis using a customer selected MILLIPLEX MAP magnetic bead kit (EMD Millipore, Billerica, MA, Cat #HCD8MAG-15K-07 for GM-CSF, TNF-, and IFN-) according to the protocol provided by the manufacturer. The data were analyzed using Belysa software (Millipore Sigma).

Results

Design of CD16 Variant Constructs

[0617] As shown in Table 8, total of 10 constructs have been designed for assessing the potential proteolytic cleavage resistance. Myc tag is added for the first run by Sirion Bio to facilitate the expression detection of the designed constructs.

TABLE-US-00011 TABLE9 DesignofCD16variants # AminoAcidSequence 1 176V(also MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP referredto QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI as158V); DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK 197 EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA Glycine TLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVGTISSFFPPGY (G);Myc QVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRK tag DPQDK 2 176V;197 MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP Phenylalanine QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI (F); DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK Myctag EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA TLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVFTISSFFPPGYQ VSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDP QDK 3 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP CD28Stalk QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI (Full DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK Length); EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA Myctag TLKDSGSYFCRGLVGSKNVSSETVNITIEVMYPPPYLDNEKSNG TIIHVKGKHLCPSPLFPGPSKPVSFCLVMVLLFAVDTGLYFSVKT NIRSSTRDWKDHKFKWRKDPQDK 4 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP CD28Stalk QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI (Last DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK 19AA); EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA Myctag TLKDSGSYFCRGLVGSKNVSSETVNITHVKGKHLCPSPLFPGPS KPVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRK DPQDK 5 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP SCRAMBLED QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI original DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK sequence; EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA Myctag TLKDSGSYFCRGLVGSKNVSSETVNITITQGVITALSSSFFPPGYQ VSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDP QDK 6 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP CD8aStalk QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI (Full DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK Length); EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA Myctag TLKDSGSYFCRGLVGSKNVSSETVNITTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDVSFCLVMVLLFAVDTGL YFSVKTNIRSSTRDWKDHKFKWRKDPQDK 7 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP CD8aStalk QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI (Last DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK 19AA); EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA Myctag TLKDSGSYFCRGLVGSKNVSSETVNITCRPAAGGAVHTRGLDF ACDVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKW RKDPQDK 8 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP CD64 QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI Stalk;Myc DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK tag EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA TLKDSGSYFCRGLVGSKNVSSETVNITPELELQVLGLQLPTPVW FHVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRK DPQDK 9 176V(also MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLEP referredto QWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI as158V); DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFK 197Proline EEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKA (P);Myc TLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVPTISSFFPPGYQ tag VSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDP QDK # AminoAcidSequence 1 176V MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE (also PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY referred FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV toas FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI 158 PKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVGTISSFFP V); PGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFK 197 WRKDPQDK Glycine (G) 2 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE 197 PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY phenylalanine FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV (F) FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI PKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVFTISSFFP PGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFK WRKDPQDK 3 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE CD28 PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY Stalk FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV (Full FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI Length) PKATLKDSGSYFCRGLVGSKNVSSETVNITIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGPSKPVSFCLVMVLLFAVDTGLY FSVKTNIRSSTRDWKDHKFKWRKDPQDK 4 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE CD28 PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY Stalk FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV (Last FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI 19AA) PKATLKDSGSYFCRGLVGSKNVSSETVNITHVKGKHLCPSPLFP GPSKPVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFK WRKDPQDK 5 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE SCRAMBLED PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY original FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV sequence FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI PKATLKDSGSYFCRGLVGSKNVSSETVNITITQGVITALSSSFFP PGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFK WRKDPQDK 6 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE CD8a PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY Stalk FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV (Full FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI Length) PKATLKDSGSYFCRGLVGSKNVSSETVNITTTTPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDVSFCLVMVLLFA VDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK 7 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE CD8a PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY Stalk FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV (Last FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI 19AA) PKATLKDSGSYFCRGLVGSKNVSSETVNITCRPAAGGAVHTRG LDFACDVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHK FKWRKDPQDK 8 176V; MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE CD64 PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY Stalk FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI PKATLKDSGSYFCRGLVGSKNVSSETVNITPELELQVLGLQLPT PVWFHVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKF KWRKDPQDK 9 176V MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE (also PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY referred FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV toas FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI 158 PKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVPTISSFFP V); PGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFK 197 WRKDPQDK Proline(P) 10 176V MWQLLLPTALLLLVSADIEQKLISEEDLGMRTEDLPKAVVFLE (also PQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSY referred FIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWV toas FKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYI 158 PKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFP V): PGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFK 197 WRKDPQDK Serine (S)

Selection of Constructs #1, 5 and 10

[0618] In vitro placental CD34.sup.+ derived CYNK cell culture system was used for assessing the designed constructs, in house preclinical grade CD16VP (MOI: 150, titrated before) generated by Lentigen was served as internal control. At Day5, the cultured CD34 cells were transduced with LVV CD16 variants constructs with different MOI as indicated. The expression of CD16 was detected at Day 10. As shown in FIG. 38, with MOI at 200, constructs #1, 2, 5, 9 and 10 showed comparable CD16 expression compared to that of CD16VP. MOI 200 for CD16 variants has been used for the further studies.

[0619] With MOI 200 at Day5 CD16 variants LVV transduction, CD16 expression were tested at Day10, Day21, Day28, Day35 and Day40. As shown in FIGS. 39A-39B, construct #5, 1, 2, 9 and 10 showed comparable CD16 expression at different time points as indicated compared to that of CD16VP.

[0620] To determine whether overexpression of the CD16 variant in CYNK cells would confer protease cleavage resistance, a CD16 shedding assay was conducted. PMAi stimulation was used in this assay to activate NK cells and subsequently resulted in increased secretion of the metalloprotease ADAM17, which cleaves endogenous CD16 on the surface of NK cells (Lajoie, 2014). In CD16VP, the modified version of CD16 is unable to be cleaved due to the lack of amino acid recognition sites that are identified by ADAM17. However, in a NT CYNK donor that expresses only endogenous CD16, the receptor will be cleaved and thus shed from the cell surface. As shown in FIG. 40, with significant CD16 shedding from NT CYNK cells, construct #5 showed comparable shedding resistance in comparison to that of CD16VP. In addition, constructs #1, 2, 9 and 10 showed the trend of shedding resistance compared to that of NT control.

[0621] To evaluate the function of CD16 variants transduced CYNK cells, ADCC assays were performed in combination with trastuzumab against the HER2.sup.+ NCI-N87 gastric tumor cells. As shown FIGS. 41A-41B, Construct #5 transduced CYNK demonstrated comparable ADCC activity in combination with trastuzumab against NCI-N87 compared to that of CD16VP at both 4 h and 24 h with different E:T ratios as indicated. Similar trend was observed on Constructs #1, 2, 9 and 10 transduced CYNK compared to that of CD16VP transduced CYNK cells.

[0622] The effect of CD16 variants on fold expansion of CYNK had been evaluated, as shown in FIG. 42, comparable fold expansion of construct #5, 1, 6, 9 and 10 was observed in comparison with that of CD16VP.

[0623] To evaluate the effect of phenotype by CD16 variants, the listed surface markers have been tested on Day40 from CD16 variants transduced CYNK cells. As shown in Table 10, comparable surface markers expression: CD56.sup.+CD3, CD226, NKG2D, CD94, CD11a, NKp30, NKp44 and NKp46 was observed between construct #5 and CD16VP transduced CYNK cells.

TABLE-US-00012 TABLE 10 Phenotype characterization of CD16 variants transduced CYNK cells at Day40 Under Live Cells Under CD56+/CD3 Donor Condition Viability CD56+/CD3 CD16+ CD226+ NKG2D+ 2000118306 Comstrict 1-200 MCI 71.7% 88.0% 7.25% 9.17% 3.11% (Celularity) Construct 2-200 MCI 62.3% 86.2% 7.37% 11.4% 4.89% Construct 3-200 MCI 73.3% 85.2% 2.48% 9.76% 3.87% Construct 4-200 MCI 70.9% 77.7% 3.26% 14.0% 6.04% Construct 5-200 MCI 74.5% 87.0% 31.9% 11.2% 7.87% Construct 6-200 MCI 66.4% 89.1% 14.5% 11.0% 5.08% Construct 7-200 MCI 66.6% 83.7% 5.68% 9.42% 5.56% Construct 8-200 MCI 66.1% 84.6% 3.07% 11.5% 5.29% Construct 9-200 MCI 66.9% 83.9% 9.10% 12.3% 3.94% Corstret 10-200 MCI 61.4% 79.8% 16.5% 11.2% 5.38% CD16VP-150 MCI 71.1% 84.7% 56.5% 13.4% 9.70% NT 77.9% 87.7% 49.2% 15.1% 8.82% Under CD56+/CD3 Donor CD94+ CD11a+ NKp30+ NKp44+ NKp46+ 2000118306 4.62% 6.01% 4.16% 98.4% 5.92% (Celularity) 5.63% 7.99% 6.40% 98.1% 5.87% 4.24% 6.17% 5.10% 98.9% 5.32% 6.29% 9.44% 8.40% 98.3% 6.55% 4.58% 5.74% 5.76% 98.9% 5.53% 6.39% 8.97% 9.10% 98.5% 7.93% 6.00% 7.53% 8.04% 98.5% 6.22% 6.57% 7.64% 7.81% 97.7% 6.24% 4.90% 6.82% 5.59% 97.8% 6.61% 5.78% 7.47% 6.77% 97.4% 6.24% 8.30% 10.8% 5.36% 97.5% 8.18% 9.00% 13.2% 13.8% 98.6% 9.00%

[0624] In summary, similar trend was observed from the other 5 donors evaluated regarding to CD 16 expression, CD 16 shedding resistance, ADCC activity and phenotype characterization in comparison with that CD16VP transduced CYNK cells. Based on the results, Lentivirus vectors for Construct #1, 2, 5, 9, and 10 without Myc tag were generated by Sirion Bio for further evaluation. Three donors were used for this study, similar results were demonstrated in comparison to the corresponding constructs with Myc tag. Taken together, Construct #1, 5, 10 were selected to generate Research Grade lentivirus by Lentigen using the same lentivirus backbone as that of CD16VP.

Construct #5: CD16VS Nomination

[0625] Constructs #1, 5 and 10 have been selected and used to generate lentivirus vector by Lentigen, the same lentivirus vector backbone as CD16VP. Placental CD34.sup.+ derived CYNK cell culture system had been used for further candidate selection. As shown in FIG. 6, among the 6 donors evaluated, comparable CD16 expression of constructs #1, 5 and 10 were observed at Day10, Day35 and post thaw in comparison with that of CD16VP.

[0626] PMAi stimulated CD16 shedding assay was used for assessing proteolytic cleavage resistance. As shown in FIGS. 44A-44B whereas 74.7%16.3% CD16 shed from the NT CYNK cell control post 2 h PMAi stimulation, the CD16 shedding percentage for construct #1, 5 and 10 transduced CYNK cells (n=6 donors) was 9.4%8.5%, 0.6%0.9%, 1.8%2.2%, respectively, with construct #5 being the most shedding resistant construct. 4.3%3.0% of CD16 shedding was shown from CD16VP transduced CYNK (n=6 donors), which are consistent with historical data of CYNK-101.

[0627] The effects of CD16 variants on the fold expansion also was evaluated, as shown in FIG. 45, comparable fold expansion was observed from constructs #1, 5 and 10 compared to that of CD16VP and NT.

[0628] XCELLigence based ADCC assay was used to evaluate the function of CD16 variants transduced CYNK cells in combination with trastuzumab against the HER2.sup.+ NCI-N.sub.87 gastric tumor cells. As shown in FIG. 46, significant difference of cytotoxicity change [Cytotoxicity(CYNK+Trastuzumab)Cytotoxicity(CYNK+IgG)] was observed from Construct #1, 5, 10 and CD16VP transduced CYNK cells in combination with trastuzumab against NCI-N87 compared to that of NT for both 4 h and 24 h at E:T ratio of 2:1. Furthermore, there is no significant difference of cytotoxicity change between the tested CD16 variants and CD16VP transduced NK cells.

[0629] Cytokine secretion profiling of CD16 variants transduced CYNK cells was evaluated by Luminex. As shown in FIG. 47, significant difference of GM-CSF secretion was observed from Construct #1, 5, 10 and CD16VP transduced NK cell in the presence of trastuzumab and NCI-N87 compared to that of NT. Furthermore, there was no significant difference of cytokine secretion (IFNg, TNFa and GM-CSF) in the presence of trastuzumab and NCI-N87 between Construct #1, 5, 10 transduced NK cells and CD16VP transduced NK cells.

[0630] Extensive immunophenotyping analyses of CD16 variants transduced CYNK (n=6 donors) were conducted, and the data were summarized in Table 11. In comparison with CD16VP transduced CYNK, Construct #5 transduced CYNK showed comparable expression of CD56.sup.+CD3; and under the gating of CD56.sup.+CD3 population with the expression of CD16, CD226, CD94, CDT 1a, NKp30, NKp44 and NKp46.

[0631] In summary, as shown in Table 12, construct #5 (CD16VS) was nominated as replacement of CD16VP for CYNK-101 based on comparable CD16 expression, CD16 shedding resistance post PMAi stimulation, fold expansion, ADCC activity in combination with trastuzumab against NCI-N87, and cytokine secretions (IFN-, TNF- and GM-CSF) in the presence of trastuzumab against NCI-N87 in comparison with that of CD16VP.

TABLE-US-00013 TABLE 11 Assessment of Immunophenotype of CD16 variants transduced CYNK by Flow Cytometry # of Under CD56 + CD3 donors CD56 + CD3 % CD16+ % CD226+ % NKG2D+ % Construct#1 5 Average 59.5 70.5 93.0 24.0 STDEV 5.2 11.3 14.5 11.1 Construct#5 5 Average 58.5 88.2 41.3 57.5 STDEV 5.1 12.2 10.8 11.2 Construct#10 5 Average 39.1 83.1 94.5 91.3 STDEV 5.7 10.9 14.5 13.7 CD15VP 5 Average 90.5 51.9 34.2 25.2 STDEV 5.5 15.4 14.2 12.5 NT 5 Average 91.2 12.2 35.4 14.9 STDEV 4.3 9.4 16.3 9.0 Under CD56 + CD3 CD94+ % CD11a+ % NKp30+ % NKp44+ % NKp46+ % Construct#1 29.7 39.5 9.7 90.4 18.4 17.4 20.7 3.5 5.1 9.2 Construct#5 28.8 41.9 11.5 88.9 15.4 14.7 17.8 7.1 7.0 7.4 Construct#10 29.5 99.3 9.7 99.5 15.9 16.5 19.3 3.0 5.4 7.5 CD15VP 30.9 43.1 13.7 90.5 23.4 17.1 21.7 5.2 5.5 12.5 NT 29.5 43.3 21.3 90.8 25.4 17.2 21.9 14.2 4.3 13.9

TABLE-US-00014 TABLE 12 CD16VS as the nominated candidate for CD16VP replacement # of Construct #5 donors (CD16VS) CD16VP CD16 expression 6 88.2% 12.2% 61.9% 15.4% CD16 shedding resistance post 6 Yes Yes PMAi stimulation Fold expansion 6 3355 2321 3260 1491 Phenotype characterization 6 Comparable phenotype 4 h cytotoxicity against NCI-N87 in 6 64.9% 15.8% 73.1% 20.7% the presence of Trastuzumab at E:T ratio of 2:1 24 h cytotoxicity against NCI-N87 in 6 97.5% 2.3% 94.5% 7.7% the presence of Trastuzumab at E:T ratio of 2:1 IFNg secretion against NCI-N87 in 6 0.9 0.6 1.8 1.6 the presence of Trastuzumab at E:T ratio of 1:1 (ng/1 10.sup.6 cells) TNFa secretion against NCI-N87 in 6 0.6 0.3 0.8 0.3 the presence of Trastuzumab at E:T ratio of 1:1 (ng/1 10.sup.6 cells) GM-CSF secretion against NCI-N87 in 6 3.9 1.1 5.4 2.7 the presence of Trastuzumab at E:T ratio of 1:1 (ng/1 10.sup.6 cells)

Conclusions

[0632] CD16VS is designed and demonstrated for CYNK-101 for further drug development.

References For Example 9

[0633] Jing Y, Ni Z, Wu J, Higgins L, Markowski T W, Kaufman D S, et al. Identification of an ADAM17 cleavage region in human CD16 (FcRIII) and the engineering of a non-cleavable version of the receptor in NK cells. PLoS One. 2015 Mar. 27; 10(3):e0121788. [0634] Koene H R, Kleijer M, Algra J, Roos D, von dem Borne A E, de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood; 1997 90(3):1109-14. [0635] Lajoie L, Congy-Jolivet N, Bolzec A, Gouilleux-Gruart V, Sicard E, Sung H C, et al. ADAM17-mediated shedding of FcRIIIA on human NK cells: identification of the cleavage site and relationship with activation. J Immunol. 2014 Jan. 15; 192(2):741-51.

7.10 Example 10: Testing and Validation of Cleavage Resistant CD16 in Placental T Cells

Pt-CD16VS-TRAC KO Cells with Potent ADCC Against PDL-1+ Cancer Cells Lines

[0636] Celularity has developed methods for expanding and using placental T cells, e.g., as described in PCT International Patent Application Nos. PCT/US2019/064074 and PCT/US2020/063473. Celularity is developing PT-CD16VS as a novel product in combination with approved monoclonal antibodies for the treatment of various cancers. For proof-of-concept studies, we evaluated PT-CD16VS in combination with trastuzumab for the treatment of HER2 overexpressing G/GEJ adenocarcinoma. PT-CD16VS is a human placental-derived T cell product, which is genetically modified via lentiviral vector transduction with a proprietary CD16 construct designed to express a high affinity and cleavage resistant CD16 variant (CD16VS). High affinity to Fc is achieved by presenting a Valine at amino acid position 176, and cleavage resistance is achieved by scrambling six amino acids at positions 194-199. Following transduction, PT-CD16VS cells also undergo CRISPR-Cas9 mediated knockout of the endogenous T cell receptor (TCR) to mitigate any potential for graft-vs-host disease (GvHD) and to ensure the safety of the allogeneic, off-the-shelf cell therapy.

[0637] CD16 (FcRIII) plays a central role in antibody-dependent cell-mediated cytotoxicity (ADCC) in NK cells and macrophages through binding to the Fc portion of IgG antibodies For T cells, gamma delta T cells represent a population of nonconventional T cells that naturally express CD16 and exhibit ADCC against various cancer cells when combined with different monoclonal antibodies (mAbs) (Barros et al., 2021). Conventional alpha beta T cells do not express CD16 but can be genetically modified to express the receptor and mediate ADCC in a similar fashion (Ollier et al., 2017). Some advantages of CD16 T cells include HLA unrestricted cell-mediated anti-tumor activity, the ability to target different tumors or antigens with the same drug product by simply changing the monoclonal antibody used in combination, and the ability to manage off-target toxicities through elimination of mAb administration. For these reasons, Celularity is developing PT-CD16VS as an adoptive cell therapy for the treatment of multiple liquid and solid tumors. Here, we evaluate PT-CD16VS ADCC with Avelumab when targeting PD-L1 cancer cell lines.

Study Design/Experimental Procedures:

[0638] 2 PT NT-KO and CD16VSKO donors (25280 and 30890) were thawed and recovered for 1 day by the CyCART team. They were then used in ADCC assays against 5 PD-L1 expressing tumor cell lines, 5637 (20,000 cells/well seeding density), NCI-H1975 (10,000 cells/well seeding density), MDA-MB-231 (40,000 cells/well seeding density), RT-112 (20,000 cells/wells seeding density) and T-24 (10,000 cells/well seeding density). The ADCC was performed at 5 E:T ratios: 5:1, 2.5:1, 1.25:1, 0.6:1, and 0.3:1. IgG and Avelumab were both at a concentration of 0.1 g/ml to be able to be compared to CYNK-101 data. The experiments ran for at least 24 hours and then data from hour four and hour twenty-four were analyzed (hour four and hour twelve in the case of MDA-MB-231 due to target alone cell index drop after that time point). Additionally, supernatant was collected after 24 hours of 1:1 co-culture of PT-CD16VS cells and tumor cells with IgG or Avelumab for later cytokine secretion analysis.

Results:

[0639] FIG. 48 shows the data from the 2 PT donors of the NT and CD16VS KO samples with IgG and Avelumab against RT-112. The two graphs on the left depict the 24-hour ADCC data of the PT donors against RT-112, which show that the PT-CD16VS cells with Avelumab had significantly higher cytolysis then when with IgG. The low cytolysis for the NT samples show that there's no ADCC activity without the CD16 receptor. The graph on the right shows that PT-CD16VS cells in combination with Avelumab was significantly more effective than the CYNK-101 cells with Avelumab.

[0640] FIG. 49 shows the experimental data with PT-CD16VS cells for the T-24 tumor cell line at 24 hours. T-24 also has a significant increase in ADCC when PT-CD16VS cells are paired with Avelumab, and significantly increased cytolysis with the PT-CD16VS when compared to CYNK-101 cells with Avelumab.

[0641] FIG. 50 shows the data for MDA-MB-231 tumor cells with the 2 PT donors at 12 hours. Once again, there was a significant increase in ADCC when PT-CD16VS were paired with Avelumab compared to the IgG control. The effectiveness of the PT-CD16VS cells and the CYNK-101 cells with Avelumab were similar, however due to the lower baseline cytolysis of the PT-CD16VS cells with IgG, there was a greater increase in ADCC with the PT cells.

[0642] FIG. 51 shows the data for NCI-H1975 tumor cells with the 2 PT donors at 24 hours. There was significant increase in ADCC with the PT-CD16VS cells with Avelumab compared to the IgG control. The PT-CD16VS cells performed slightly worse than the CYNK-101 cells when both in combination with Avelumab, however like MDA-MB-231 there was a more significant increase compared to the control.

[0643] FIG. 52 shows data of the 5637 tumor cells with the 2 PT donors. Like all the other tumor cell lines, there was significant increase in cytolysis with the PT-CD16VS cells in combination with Avelumab compared to IgG control. However, for 5637 the CYNK-101 cells had significantly greater ADCC than the PT-CD16VS cells.

Conclusion

[0644] The results of this study show that PT-CD16VS cells in combination with Avelumab significantly improved killing of tumor cells through ADCC. The 5637, T-24, RT-112, and NCI-H1975 tumor cell lines all displayed 100% cytolysis at 24 hours at the 5:1 E:T ratio while MDA-MB-231 reached 70% cytolysis at 12 hours at the 5:1 E:T ratio. PT-CD16VS cells in combination with Avelumab had much better cytolysis than CYNK-101 cells for lower PD-L1 expressing tumor cell lines and had similar cytolysis at higher PD-L1 expressing tumor cell lines. 5637 was once again an outlier in the data set, most likely attributed to 5637 being susceptible to CYNK-101 cells with Avelumab. Overall, the experiment shows that PT-CD16VS cells can be paired with Avelumab for effective killing.

[0645] 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.

[0646] 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.