METHODS AND COMPOSITIONS HIGH SCALE THERAPEUTIC PRODUCTION OF MEMORY NK CELLS

• US 20190314412A1
• Filed: 05/24/2019
• Published: 10/17/2019
• Est. Priority Date: 11/08/2016
• Status: Active Application

• Current Assignee: University of Central Florida Research Foundation Inc. (State University System of Florida)
• Original Assignee: University of Central Florida Research Foundation Inc. (State University System of Florida)
• Inventors: Copik, Alicja J., Igarashi, Robert Y., Oyer, Jeremiah L., Altomare, Deborah
• Application Number:
  US16/422,003
• Publication Number:
  US 20190314412A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 35/33 Fibroblasts
• CPC Class Codes: A61K 38/20 Interleukins [IL]
• CPC Class Codes: A61K 38/208 IL-12
• CPC Class Codes: A61K 38/2086 IL-13 to IL-16
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C12N 2501/20 Cytokines; Chemokines
• CPC Class Codes: C12N 2501/2302 Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/2312 Interleukin-12 (IL-12)
• CPC Class Codes: C12N 2501/2315 Interleukin-15 (IL-15)
• CPC Class Codes: C12N 2501/2318 Interleukin-18 (IL-18)
• CPC Class Codes: C12N 2501/2321 Interleukin-21 (IL-21)
• CPC Class Codes: C12N 5/0646 Natural killers cells [NK], NKT cells

Abstract

Disclosed are compositions and methods relating to the expansion of memory NK cells.

Claims

1-20. -20. (canceled)

21. A method for increasing the number of memory NK cells comprising

a) preactivating NK cells by contacting at least one NK cell with at least one stimulatory cytokine;

and

b) expanding the preactivated NK cells of step a), by contacting the preactivated NK cells with plasma membrane (PM) particles with surface bound interleukin (IL)-21 (PM21) particles, exosomes (EX) with surface bound IL-21 (EX21) exosomes, or feeder cells (FC) with surface bound IL-21 (FC21) feeder cells, or any combination thereof.

22. The method of claim 21, further comprising maintaining the contact in (b) for at least about 15 days, wherein a 10,000 fold increase in NK cell number is achieved.

23. The method of claim 21, further comprising a washing step after the preactivation in step (a) and/or after the expansion in step (b).

24. The method of claim 21, wherein the NK cells are obtained from an unselected population of peripheral blood mononuclear cells, cord blood or a stem cell population.

25. The method of claim 21, wherein preactivating the NK cells comprises contacting the NK cells with at least one stimulatory cytokine selected from the group consisting of Interleukin-12 (IL-12), Interleukin-15 (IL-15), and Interleukin-18 (IL-18).

26. The method of claim 21, wherein preactivating the NK cells comprises contacting the NK cells with any combination of two stimulatory cytokines selected from the group consisting of IL-12, IL-15, and IL-18.

27. The method of claim 21, wherein preactivating the NK cells comprises contacting the NK cells with IL-12, IL-15, and IL-18.

28. The method of claim 24, further comprising contacting the NK cells with an one or more NK cell effector agents selected from the group consisting of 4-1BB ligand (4-1BBL), Interleukin-2 (IL-2), Major histocompatibility complex class I-related chain AB (MICAS), UL16 Binding Protein 2 (ULBP2), Intracellular adhesion molecule 1(ICAM-1), 2B4, /signaling lymphocyte activation molecule (SLAM) family member 2 (F2) (BCM1/SLAMF2), cluster of differentiation 155 CD155), cluster of differentiation 112 (CD112), C-C chemokine receptor type 7 (CCR7), DnaX activation protein of 12 kDa (DAP12), and DnaX activation protein of 10 kDa (DAP10), and any combination thereof.

29. The method of claim 21, wherein preactivating the NK cells comprises contacting the NK cells with the at least one stimulatory cytokine in vitro, in vivo, or ex vivo.

30. The method of claim 21, wherein preactivating the NK cells comprises contacting the NK cells with at least one stimulatory cytokine for a period of about 6 to about 24 hours.

31. The method of claim 21, wherein the PM21 particles, EX21 exosomes, or FC21 feeder cells comprise one or more stimulatory peptides coupled to a membrane-inserting peptide.

32. The method of claim 31, wherein the stimulatory peptide coupled to a membrane-inserting peptide comprises a fused peptide that is capable of membrane insertion with affinity for a lipid bilayer, wherein the fused peptide comprises a segment of IG4, CD4, or a combination thereof.

33. The method of claim 31, wherein the one or more stimulatory peptides coupled to a membrane-inserting peptide is a fusion protein encoded by recombinant DNA.

34. The method of claim 31, wherein the membrane-inserting peptide comprises human Fc, GPI, trans-membrane T-cell receptor, or pHLIP.

35. The method of claim 31, wherein the one or more stimulatory peptides are selected from the group consisting of 4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA/B, ULBP2, ICAM-1, 2B4,BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.

36. The method of claim 21, wherein the NK cells are contacted with the PM21 particles, EX21 exosomes, or FC21 feeder cells in vitro, in vivo, or ex vivo.

37. The method of claim 21, wherein the NK cells of step (a) are contacted with PM21 particles, EX21 exosomes, or FC21 feeder cells for a period of 7 days to 60 days.

38. The method of claim 21, further comprising resting the expanded memory NK cells following step (b).

39. The method of claim 38, wherein the cells are memory NK cells are rested for at least 1, 2, 3, 4 or 5 days.

40. The method of claim 38, wherein the memory NK cells are rested for no more than 5 days.

41. The method of claim 21, wherein the PM21 particles, EX21 exosomes, or FC21 feeder cells comprise 4-1BBL and IL-21.

42. The method of claim 41, wherein the 4-1BBL and IL-21 are coupled to a membrane-inserting peptide.

43. An activated NK cell composition comprising NK cells following contact with at least one stimulatory cytokine selected from the group consisting of IL-12, IL-15, and IL-18 for at least about 6 hours.

44. The cell composition of claim 43, in contact with PM21 particles, EX21 exosomes, FC21 feeder cells, or any combination thereof.

45. The composition of claim 44, further comprising an additional one or more cytokines selected from the group consisting of 4-1BBL, IL-2, IL-21, MICA/B, ULBP2, ICAM-1, 2B4BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, DAP10, and any combination thereof.

46. The composition of claim 44, in vitro or ex vivo.

47. The composition of claim 44, wherein the PM21 particles, EX21 exosomes, or FC21 feeder cells comprise one or more stimulatory peptides coupled to a membrane-inserting peptide.

48. The composition of claim 47, wherein each of the one or more stimulatory peptides coupled to a membrane-inserting peptide comprises a fused peptide that is capable of membrane insertion with affinity for a lipid bilayer, wherein the fused peptide comprises a segment of IG4, CD4, or a combination thereof.

49. The composition of claim 47, wherein the one or more stimulatory cytokines coupled to a membrane-inserting peptide is a fusion protein encoded by recombinant DNA.

50. The composition of claim 47, wherein the membrane-inserting peptide comprises human Fc, GPI, trans-membrane T-cell receptor, or pHLIP.

51. The composition of claim 47, maintained in contact with the PM21 particles, EX21 exosomes, FC21 feeder cells or combination thereof for a period of 7 days to 60 days.

52. The composition of claim 47, wherein the PM21 particles, EX21 exosomes, or FC21 feeder cells comprise IL-21 and further comprise 4-1BBL.

53. The method of claim 51, wherein the 4-1BBL and IL-21 are coupled to a membrane-inserting peptide.

54. A kit for increasing the number of memory NK cells comprising amounts of one or more stimulatory cytokines and amounts of one or more vesicles comprising IL-21, and optionally amounts of one or more NK cell effector agents.

55. The kit of claim 54, wherein the one or more stimulatory cytokines are selected from the group consisting of IL-12, IL-15, IL-18 and any combination thereof.

56. The kit of claim 54, wherein the one or more vesicles comprising IL-21 are selected from a PM21 particle, an EX21 exosome, a FC21 feeder cell and any combination thereof.

57. The kit of claim 54, wherein the one or more NK cell effector agents are selected from the group consisting of 41BBL, IL-2, IL-15, IL-21, MICA/B, ULBP2, ICAM-1, 2B4, BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, DAP10 and any combination thereof.

58. The kit of claim 54, further comprising an amount of NK cells or an NK cell line.