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1. WO2020113182 - MULTIPLICATION DE CELLULES TUEUSES NATURELLES ET DE CELLULES ILC3 AVEC DE NOUVEAUX COMPOSÉS AROMATIQUES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

WHAT IS CLAIMED IS:

1. A method of producing a cell population comprising natural killer cells, comprising the steps of:

(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 a stem cell mobilizing agent and low molecular weight heparin (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 80% of the natural killer cells are viable.

2. The method of claim 1, wherein said third population of cells comprises natural killer cells that are CD94+ or CD16+.

3. The method of claim 1, wherein said third population of cells comprises natural killer cells that are CD94- or CD16-.

4. The method of claim 1, wherein said third population of cells comprises natural killer cells that are CD94+ and CD16+.

5. The method of claim 1, wherein said third population of cells comprises natural killer cells that are CD94- and CD16-.

6. A method of producing a cell population comprising natural killer cells, comprising the steps of:

(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+.

7. The method of claim 6, wherein the third medium lacks stem cell factor (SCF).

8. The method of claim 7, wherein the third medium lacks LMWH.

9. The method of claim 7, comprising a further step of (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+.

10. The method of any one of claim 6-9, wherein said natural killer cells express perforin and EOMES.

11. The method of any one of claim 6-10, wherein said natural killer cells do not express either RORgt or IL1R1.

12. A method of producing a cell population comprising ILC3 cells, comprising the steps of:

(a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells;

(b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and

(c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking LMWH, to produce a third population of cells;

wherein the third population of cells comprises ILC3 cells that are CD56+, CD3-, and CD11a-.

13. The method of claim 12, wherein the third medium comprises a stem cell mobilizing agent.

14. The method of claim 12, wherein the third medium comprises SCF.

15. The method of claim 12, wherein the third medium comprises a stem cell mobilizing agent and SCF.

16. A method of producing a cell population comprising ILC3 cells, comprising the steps of:

(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 ILC3 cells that are CD56+, CD3-, and CD11a-.

17. The method of any one of claim 12-16, wherein said ILC3 cells express RORgt and IL1R1.

18. The method of any one of claim 12-17, wherein said ILC3 cells do not express either perforin or EOMES.

19. The method of any one of claims 1-18, wherein said third medium lacks desulphated glycosaminoglycans.

20. The method of any one of claim 1-19, wherein said hematopoietic stem or progenitor cells are mammalian cells.

21. The method of claim 20, wherein said hematopoietic stem or progenitor cells are human cells.

22. The method of claim 20, wherein said hematopoietic stem or progenitor cells are primate cells.

23. The method of claim 20, wherein said hematopoietic stem or progenitor cells are canine cells.

24. The method of claim 20, wherein said hematopoietic stem or progenitor cells are rodent cells.

25. The method of claim 20, wherein said hematopoietic stem or progenitor cells are cells from a mammal other than a human, primate, canine or rodent.

26. The method of any one of claims 1-25, wherein said hematopoietic stem or progenitor cells are CD34+ hematopoietic stem cells.

27. The method of any one of claims 1-26, wherein said hematopoietic stem or progenitor cells are placental cells.

28. The method of claim 27, wherein said placental cells are obtained from, or obtainable from, human placental perfusate.

29. The method of claim 27, wherein said placental cells are obtained from, or obtainable from, nucleated cells isolated from human placental perfusate.

30. The method of any one of claims 1-26, wherein said hematopoietic stem or progenitor cells are obtained from, or obtainable from, umbilical cord blood.

31. The method of any one of claims 1-26, wherein said hematopoietic stem or progenitor cells are fetal liver cells.

32. The method of any one of claims 1-26, wherein said hematopoietic stem or progenitor cells are mobilized peripheral blood cells.

33. The method of any one of claims 1-26, wherein said hematopoietic stem or progenitor cells are bone marrow cells.

34. The method of any one of claims 1-33, wherein said Tpo is present in the first medium at a concentration of from 1 ng/mL to 50 ng/mL.

35. The method of claim 34, wherein said Tpo is present in the first medium at a concentration of from 20 ng/mL to 30 ng/mL.

36. The method of claim 34, wherein said Tpo is present in the first medium at a concentration of about 25 ng/mL.

37. The method of any one of claims 1-36, wherein said IL-15 is present in said second medium at a concentration of from 1 ng/mL to 50 ng/mL.

38. The method of claim 37, wherein said IL-15 is present in said second medium at a concentration of from 10 ng/mL to 30 ng/mL.

39. The method of claim 37, wherein said IL-15 is present in said second medium at a concentration of about 20 ng/mL.

40. The method of any one of claims 1-39, wherein 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.

41. The method of any one of claims 1-39, wherein 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.

42. The method of any one of claims 1-39, wherein 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.

43. The method of any of claims 1-42, wherein said Tpo, IL-2, and IL-15 are not comprised within an undefined component of the first medium, second medium or third medium.

44. The method of any of claims 1-42, wherein said Tpo, IL-2, and IL-15 are not comprised within serum.

45. The method of any of claims 1-44, wherein said stem cell mobilizing agent is a compound of Formula (I) has the following structure:


including pharmaceutically acceptable salts thereof, wherein:

each independently represents a single bond or a double bond;

RJ is selected from the group consisting of–NRaRb, -ORb, and =O; wherein if RJ is =O, then joining G and J represents a single bond and G is N and the N is substituted with RG; otherwise joining G and J represents a double bond and G is N;

Ra is hydrogen or C1-C4 alkyl;

Rb is Rc or -(C1-C4 alkyl)-Rc;

Rc is selected from the group consisting of: -OH, -O(C1-C4 alkyl), -O(C1-C4 haloalkyl); -C(=O)NH2; unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; substituted C1-6 alkyl; -NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4

atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl);

RG is selected from the group consisting of hydrogen, C1-4 alkyl, and -(C1-4 alkyl)-C(=O)NH2;

RY and RZ are each independently absent or selected from the group consisting of: hydrogen, halo, C1-6 alkyl, -OH, -O-(C1-4 alkyl), -NH(C1-4 alkyl), and -N(C1-4 alkyl)2;

or RY and RZ taken together with the atoms to which they are attached are joined together to form a ring selected from:

substituted with one, two, or three groups independently selected from C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -OH, -O-(C1-4 alkyl), -N(C1-4 alkyl)2, unsubstituted C6-C10 aryl, C6-C10 aryl substituted with 1-5 halo atoms, and -O-(C1-4 haloalkyl); and wherein if RY and RZ taken

Rd is hydrogen or C1-C4 alkyl;

Rm is selected from the group consisting of C1-4 alkyl, halo, and cyano;

J is C; and

X, Y, and Z are each independently N or C, wherein the valency of any carbon atom is filled as needed with hydrogen atoms.

46. The method of Claim 45, wherein:

Ra is hydrogen;

Rb is -(C1-C4 alkyl)-Rc;

Rc is selected from the group consisting of: -C(=O)NH2; unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1- C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; -NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl);


y

RY and RZ are each independently absent or selected from the group consisting of: hydrogen, C1-6 alkyl, and -NH(C1-4 alkyl);

or RY and RZ taken together with the atoms to which they are attached are joined together to form a ring selected from:

substituted with one, two, or three groups independently selected from C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -OH, -O-(C1-4 alkyl), -N(C1-4 alkyl)2, unsubstituted C6-C10 aryl, C6-C10 aryl substituted with 1-5 halo atoms, and -O-(C1-4 haloalkyl);

Rd is C1-C4 alkyl;

Rm is cyano; and

X, Y, and Z are each independently N or C, wherein the valency of any carbon atom is filled as needed with hydrogen atoms.

47. The method of Claim 45, wherein, wherein:

Ra is hydrogen;

Rb is -CH2CH2-Rc;

Rc is selected from the group consisting of: unsubstituted phenyl, substituted phenyl, indolyl, and -C(=O)NH2;

RK is selected from the group consisting of: hydrogen, methyl, substituted pyridinyl, unsubstituted benzothiophenyl, and -NH(C1-C4 alkyl);

RG is -CH2CH2-C(=O)NH2;

RY is -NH(C1-C4 alkyl);

RZ is absent or hydrogen;

or RY and RZ taken together with the atoms to which they are attached are joined together to form a ring selected from:

substituted with one, two, or three groups independently selected from C1-C4 alkyl, -N(C1-C4 alkyl)2, cyano, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms;

Rd is C1-C4 alkyl;

Rm is cyano; and

X is N or CH.

48. The method of Claim 45, wherein, wherein:

Ra is hydrogen;

Rb is -CH2CH2-Rc;

Rc is selected from the group consisting of: unsubstituted phenyl, substituted phenyl, indolyl, and -C(=O)NH2; wherein the substituted phenyl is substituted with one substituent E, wherein E is -OH;

RK is selected from the group consisting of: hydrogen, methyl, substituted pyridinyl, unsubstituted benzothiophenyl, and -NH(sec-butyl); wherein the substituted pyridinyl moiety is substituted with one substituent Q, wherein Q is selected from the group consisting of: C1-4 alkyl, halo, and cyano;

RG is -CH2CH2-C(=O)NH2;

RY is -NH(isopropyl) or -NH(sec-butyl);

RZ is absent or hydrogen;

or RY and RZ taken together with the atoms to which they are attached are joined together to form a ring selected from:


substituted with one, two, or three groups independently selected from C1-C4 alkyl, cyano, unsubstituted phenyl, and 4-fluorophenyl;

Rd is isopropyl;

Rm is cyano; and

X is N or CH.

49. The method of Claim 45, wherein the compound of Formula (I) has the structure of Formula (I-A):


including pharmaceutically acceptable salts thereof, wherein:

RJ is–NRaRb;

Ra is hydrogen or C1-C4 alkyl;

Rb is Rc or -(C1-C4 alkyl)-Rc;

Rc is selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; -NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl);

Y and Z are each C;

X is N or CH;

W is O or S; and

Re is hydrogen or C1-C4 alkyl.

50. The method of Claim 49, wherein:

Ra is hydrogen;

Rb is -(C1-C4 alkyl)-Rc;

Rc is selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is 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 is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); and

Re is C1-C4 alkyl.

51. The method of Claim 49, wherein:

Ra is hydrogen;

Rb is -(CH2-CH2)-Rc;

Rc is selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E is -OH;

RK is selected from the group consisting of: unsubstituted benzothiophenyl and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one substituent Q, wherein Q is selected from the group consisting of: C1-4 alkyl, halo, and cyano; and

Re is isopropyl.

52. The method of Claim 49, wherein the compound is selected from the group consisting of, or a pharmaceutically acceptable salt of:

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

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

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

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

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

N-(2-(1H-indol-3-yl)ethyl)-2-(5-methylpyridin-3-yl)furo[3,2-d]pyrimidin-4-amine; and

5-(4-((2-(1H-indol-3-yl)ethyl)amino)furo[3,2-d]pyrimidin-2-yl)nicotinonitrile.

53. The method of Claim 45, wherein the compound of Formula (I) has the structure of Formula (I-B):


including pharmaceutically acceptable salts thereof, wherein:

Ra is hydrogen or C1-C4 alkyl;

Rb is Rc or -(C1-4 alkyl)-Rc;

Rc is selected from the group consisting of: -OH, -O(C1-C4 alkyl), -O(C1-C4 haloalkyl); -C(=O)NH2; unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; substituted C1-6 alkyl; -NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6- 10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl);

RG is selected from the group consisting of hydrogen, C1-4 alkyl, and -(C1-4 alkyl)-C(=O)NH2;

Rf is selected from the group consisting of hydrogen, C1-4 alkyl, unsubstituted C6-C10 aryl, and C6-C10 aryl substituted with 1-5 halo atoms;

U is N or CRU;

V is S or NRV;

RU is selected from the group consisting of hydrogen, C1-4 alkyl, halo, and cyano; RV is hydrogen or C1-C4 alkyl;

wherein when U is CRU and V is NRV, RU is selected from the group consisting of C1-4 alkyl, halo, and cyano;

Y and Z are each C; and

X is N or CH.

54. The method of Claim 53, wherein:

Ra is hydrogen;

Rb is -(C1-4 alkyl)-Rc;

Rc is selected from the group consisting of: -C(=O)NH2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is 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 is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl);

RG is C1-4 alkyl or -(C1-4 alkyl)-C(=O)NH2;

Rf is selected from the group consisting of hydrogen, unsubstituted phenyl, and phenyl substituted with 1-5 halo atoms;

Y and Z are each C; and

X is CH.

55. The method of Claim 53, wherein:

Ra is hydrogen;

Rb is -(CH2-CH2)-Rc;

Rc is selected from the group consisting of: -C(=O)NH2, substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E is -OH;

RK is selected from the group consisting of: unsubstituted benzothiohenyl and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one substituent Q, wherein Q is selected from the group consisting of: C1-4 alkyl, halo, and cyano;

RG is -(CH2CH2)-C(=O)NH2;

Rf is selected from the group consisting of hydrogen, phenyl, and fluorophenyl; Y and Z are each C; and

X is CH.

56. The method of Claim 53, wherein the compound is selected from the group consisting of, or a pharmaceutically acceptable salt of:

57. The method of Claim 45, wherein the compound of Formula (I) has the structure of Formula (I-C):


including pharmaceutically acceptable salts thereof, wherein:

RJ is–NRaRb;

Ra is hydrogen or C1-C4 alkyl;

Rb is Rc or -(C1-C4 alkyl)-Rc;

Rc is selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: hydrogen, unsubstituted C1-6 alkyl; -NH(C1-4 alkyl); -N(C1-4 alkyl)2, unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl);

A is N or CH;

B is N or CH;

Rg is selected from the group consisting of hydrogen, C1-4 alkyl, and -N(C1-4 alkyl)2;

Y and Z are each C; and

X is N or CH.

58. The method of Claim 57, wherein:

Ra is hydrogen;

Rb is -(C1-C4 alkyl)-Rc;

Rc is selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: -NH(C1-4 alkyl); unsubstituted five-to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein the substituted heteroaryl is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); and

Rg is hydrogen or -N(C1-4 alkyl)2.

59. The method of Claim 57, wherein:

Ra is hydrogen;

Rb is -(C1-C4 alkyl)-Rc;

Rc is 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 is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: -NH(C1-4 alkyl); unsubstituted benzothiophenyl; and substituted pyridinyl; wherein the substituted pyridinyl is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); and

Rg is hydrogen or -N(C1-4 alkyl)2.

60. The method of Claim 57, wherein:

Ra is hydrogen;

Rb is -(CH2CH2)-Rc;

Rc is selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E is -OH;

RK is 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 is independently selected from the group consisting of: C1-4 alkyl, halo, and cyano; and

Rg is hydrogen or -N(CH3)2.

61. The method of Claim 57, wherein the compound is selected from the group consisting of, or a pharmaceutically acceptable salt of:

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

N-(2-(1H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)quinazolin-4-amine;

5-(4-((2-(1H-indol-3-yl)ethyl)amino)quinazolin-2-yl)nicotinonitrile; and N4-(2-(1H-indol-3-yl)ethyl)-N2-(sec-butyl)quinazoline-2,4-diamine.

62. The method of Claim 45, wherein the compound of Formula (I) has the structure of Formula (I-D):


including pharmaceutically acceptable salts thereof, wherein:

RJ is–NRaRb,

Ra is hydrogen or C1-C4 alkyl;

Rb is Rc or -(C1-4 alkyl)-Rc;

Rc is selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl);

Rh is hydrogen or C1-4 alkyl;

D is N or CH;

Y is N;

Z is C; and

X is N or CH.

63. The method of Claim 62, wherein:

Ra is hydrogen;

Rb is -(C1-4 alkyl)-Rc;

Rc is selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a Rc moiety indicated as substituted is substituted with one or more substituents E, wherein each E is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is selected from the group consisting of: unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a RK moiety indicated as substituted is substituted with one or more substituents Q, wherein each Q is independently selected from the group consisting of: -OH, C1-4 alkyl, C1-4 haloalkyl, halo, cyano, -O-(C1-4 alkyl), and -O-(C1-4 haloalkyl); and

Rh is hydrogen or C1-4 alkyl.

64. The method of Claim 62, wherein:

Ra is hydrogen;

Rb is -(C1-C4 alkyl)-Rc;

Rc is 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 is independently selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, -O(C1-C4 alkyl), and -O(C1-C4 haloalkyl);

RK is unsubstituted benzothiophenyl; and

Rh is hydrogen or C1-4 alkyl.

65. The method of Claim 62, wherein:

Ra is hydrogen;

Rb is -(CH2-CH2)-Rc;

Rc is selected from the group consisting of: substituted phenyl and unsubstituted indolyl; wherein the substituted phenyl is substituted with one substituent E, wherein E is -OH;

RK is unsubstituted benzothiophenyl; and

Rh is hydrogen or C1-4 alkyl.

66. The method of Claim 62, wherein the compound is selected from the group consisting of, or a pharmaceutically acceptable salt 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.

67. The method of Claim 65, wherein the compound is selected from the group consisting of, or a pharmaceutically acceptable salt of:

5-(2-((2-(1H-indol-3-yl)ethyl)amino)-6-(sec-butylamino)pyrimidin-4-yl)nicotinonitrile;

4-(2-((2-(benzo[b]thiophen-3-yl)-6-(isopropylamino)pyrimidin-4-yl)amino)ethyl)phenol;

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

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

68. The method of any of claims 1-67, wherein said first medium does not comprise LMWH.

69. The method of any of claims 1-68, wherein said first medium does not comprise a desulphated glycosaminoglycan.

70. The method of any of claims 1-69, wherein said first medium comprises each of Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF.

71. A population of natural killer cells produced by the method of any of claims 1-70.

72. A population of ILC3 cells produced by the method of any of claims 1-70.

73. A method of suppressing the proliferation of tumor cells comprising contacting the tumor cells with a plurality of natural killer cells and / or ILC3 cells, wherein the natural killer cells are produced by the method of any one of claims 70.

74. The method of claim 73, wherein said contacting takes place in vitro.

75. The method of claim 74, wherein said contacting takes place in vivo.

76. The method of any of claims 73-75, wherein said tumor cells are acute myeloid leukemia (AML) cells.

77. The method of any of claims 73-75, wherein said tumor cells are breast cancer cells, head and neck cancer cells, or sarcoma cells.

78. The method of any of claims 73-75, wherein said tumor cells are primary ductal carcinoma cells, leukemia cells, acute T cell leukemia cells, chronic myeloid lymphoma (CML) cells, chronic myelogenous leukemia (CML) cells, lung carcinoma cells, colon adenocarcinoma cells, histiocytic lymphoma cells, colorectal carcinoma cells, colorectal adenocarcinoma cells, or retinoblastoma cells.

79. The method of any of claims 73-75, wherein said tumor cells are solid tumor cells.

80. The method of any of claims 73-75, wherein said tumor cells are liver tumor cells.

81. The method of any of claims 73-75, wherein said tumor cells are lung tumor cells.

82. The method of any of claims 73-75, wherein said tumor cells are pancreatic tumor cells.

83. The method of any of claims 73-75, wherein said tumor cells are renal tumor cells.

84. The method of any of claims 73-75, wherein said tumor cells are glioblastoma multiforme (GBM) cells.

85. The method of any of claims 73-84, wherein said natural killer cells have been cryopreserved prior to said contacting or said administering.

86. The method of any of claims 73-84, wherein said natural killer cells have not been cryopreserved prior to said contacting or said administering.

87. A natural killer cell 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.

88. The natural killer cell of claim 87, 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.

89. The natural killer cell of claim 87 or claim 88, 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.

90. The natural killer cell of claim 87, 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.

91. The natural killer cell of any one of claims 87– 90, wherein expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers selected from the group consisting of NDFIP2, LINC00996, MAL, CCL1, MB, SPINK2, C15orf48, CAMK1, KLRC1, TNFSF10,

TNFRSF18, IL32, CAPG, AC092580.4, S100A11, TNFRSF4, ENO1, FCER1G, CCND2, KRT81, MRPS6, ANXA2, PTGER2, GLO1, HAVCR2, PYCARD, LAT2, SLC16A3, COTL1, PKM, TALDO1, CD96, NCR3, KRT86, STMN1, LTB, ARPC1B, ARPC5,

FKBP1A, TIMP1, GZMK, CD59, PGK1, RGS10, EVL, RAC2, LGALS1, ITGB7, TUBB, PGAM1, PRF1, GZMB, IL2RB, KLRC2, and KLRB1 is higher than expression of said markers in peripheral blood natural killer cells.

92. The natural killer cell of any one of claims 87– 91, wherein the natural killer cells are hematopoietic stem cell-derived natural killer cells.

93. The natural killer cell of any one of claims 87– 92, wherein the natural killer cells are CD34+ hematopoietic stem cell-derived natural killer cells.

94. The natural killer cell of claim 92 or claim 93, wherein the hematopoietic stem cells are placenta-derived hematopoietic stem cells.

95. The natural killer cell of any one of claims 87– 94, wherein the natural killer cells are prepared by the method of any one of claims 1– 70.