JPWO2021021907A5 - - Google Patents

Claims (30)

A method for enhancing proliferation of natural killer (NK) cells comprising:
co-culturing the NK cell population with a feeder cell population in a culture medium, wherein the feeder cell population was engineered to express 4-1BBL and membrane-bound interleukin-15 (mbIL15) comprising a cell;
adding interleukin 2 (IL2) to the culture medium; and adding at least one soluble stimulating agent to the culture medium, wherein the soluble stimulating agent is interleukin 12 (IL12), interleukin 18 (IL18), interleukin 21 (IL21), and combinations thereof;
method including. 2. The method of claim 1, wherein the concentration of at least one soluble stimulant is between about 0.01 ng/mL and about 50 ng/mL within 24 hours of said co-cultivation. 3. The method of claim 1 or 2 , wherein the at least one soluble stimulant comprises a combination of IL- 12 and IL -18. 4. The method of any one of claims 1-3, wherein at least one stimulating agent comprises soluble IL12 at a concentration of less than about 10 ng/mL within 24 hours of said co-culture. 5. The method of any one of claims 1-4, wherein at least one stimulating agent comprises soluble IL18 at a concentration of less than about 50 ng/mL within 24 hours of said co-culture. The at least one stimulant comprises (i) soluble IL12 at a concentration between about 0.01 ng/mL and about 8 ng/mL and (ii) soluble IL12 at a concentration between about 0.01 ng/mL and about 30 ng/mL. The method of any one of claims 1-5 , comprising IL18. The at least one stimulant comprises (i) soluble IL12 at a concentration between about 0.01 ng/mL and about 8 ng/mL and (ii) soluble IL12 at a concentration between about 0.01 ng/mL and about 30 ng/mL. IL18, wherein the culture medium is supplemented with IL2 a second time at a higher concentration compared to the first addition of IL2 to the culture medium, wherein said concentration is within 120 hours of said co-culture 7. The method of any one of claims 1-6 existing at a point in time. 8. The method of claim 7, wherein the second addition to the culture medium is made between 12 hours and 120 hours after the first addition to the culture medium. 9. The method of any one of claims 1-8 , wherein the feeder cell population comprises K562 cells. 10. The method of any one of claims 1-9 , further comprising contacting the NK cell with a vector encoding a chimeric antigen receptor (CAR). 11. The method of claim 10 , wherein the CAR targets one or more of CD19, CD123, CD70, BCMA, or natural killer receptor group D (NKG2D) ligands. The method is effective in reducing the persistence and/or cytotoxicity of NK cells compared to the persistence and/or cytotoxicity exhibited by NK cells co-cultured with feeder cells in the absence of at least one soluble stimulant. 12. The method of any one of claims 1-11 , further enhancing one or more. 13. The method of any one of claims 7-12 , wherein the medium is supplemented with IL2 a second time to a concentration of less than about 500 IU/mL. 14. The method of any one of claims 7-13 , wherein the medium is supplemented with IL2 the first time to a concentration of less than about 50 IU/mL. A method for enhancing natural killer (NK) cell cytotoxicity comprising:
co-culturing the NK cell population with a feeder cell population in a culture medium, wherein the feeder cell population is engineered to express 4-1BBL and membrane-bound interleukin-15 (mbIL15). comprising the cells;
adding interleukin 2 (IL2) to the culture medium;
adding at least one soluble stimulant to the culture medium, wherein the soluble stimulant is interleukin 12 (IL12) , interleukin 18 (IL18) , interleukin 21 (IL21) , and combinations thereof wherein the concentration of said at least one soluble stimulant is between about 0.01 ng/mL and about 50 ng/mL within 120 hours of said co-cultivation;
contacting an NK cell with a nucleic acid encoding a chimeric antigen receptor (CAR) to cause the NK cell to express the CAR;
including
wherein the addition of said at least one soluble stimulating agent to the medium enhances the cytotoxicity of NK cells expressing said CAR to NK cells co-cultured with feeder cells in the absence of said at least one soluble stimulating agent enhanced, compared to
Method. 16. The method of claim 15 , wherein the at least one soluble stimulant comprises a combination of IL -12 and IL -18. 17. The method of claim 15 or 16 , wherein at least one stimulating agent comprises soluble IL12 at a concentration of less than about 10 ng/mL within 120 hours of said co-culture. 18. The method of any one of claims 15-17 , wherein at least one stimulating agent comprises soluble IL18 at a concentration of less than about 50 ng/mL within 120 hours of said co-culture. at least one stimulating agent comprising (i) soluble IL12 at a concentration between about 0.01 ng/mL and about 8 ng/mL at a time point within 24 hours of said co-culture; and (ii) within 24 hours of said co-culture. 19. The method of any one of claims 15-18 , comprising soluble IL18 at a concentration between about 0.01 ng/mL and about 30 ng/mL at the time point of . The at least one stimulant comprises (i) soluble IL12 at a concentration between about 0.01 ng/mL and about 8 ng/mL and (ii) soluble IL12 at a concentration between about 0.01 ng/mL and about 30 ng/mL. IL18, wherein the culture medium is supplemented with IL2 a second time at a higher concentration compared to the first addition of IL2 to the culture medium, wherein each concentration is 120 hours from said co-culture 20. The method of any one of claims 15-19 , wherein the time point is within 21. Any one of claims 15-20 , wherein the CAR is configured to target one or more of CD19, CD123, CD70, BCMA, or Natural Killer Receptor Group D (NKG2D) ligands. Method. 22. The method of claim 20 or 21 , wherein the medium is supplemented with IL2 a second time within 120 hours of said co-cultivation to a concentration of less than about 500 IU/mL. 23. The method of any one of claims 15-22 , wherein the medium is supplemented with IL2 a first time within 24 hours of said co-cultivation to a concentration of less than about 50 IU/mL. A pharmaceutical composition comprising NK cells proliferated by the method of any one of claims 1-23 for use in treating cancer. A natural killer (NK) cell population expanded by the method of any one of claims 1-23. an engineered chimeric receptor configured to bind to a marker on a target cancer cell, and which upon binding induces natural killer (NK) cells to exert a cytotoxic effect on the target cancer cell An engineered NK cell population comprising
wherein said NK cells are grown in medium in the presence of at least one soluble stimulant;
wherein said soluble stimulating agent is selected from interleukin 12 (IL12) , interleukin 18 (IL18) , interleukin 21 (IL21) , and combinations thereof; and wherein said engineered NK cell population comprises at least exhibit a memory cell-like phenotype characterized in part by (i) increased expression of NKG2C by NK cells and/or (ii) decreased or equivalent expression of CD62 ligand by NK cells , where expression in both (i) and (ii) is by comparison to NK cells cultured under identical conditions but without one or more soluble stimulatory molecules,
Engineered NK cell populations. A sequence wherein the engineered chimeric receptor is at least 95% identical to the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27 is coded by ; or
amino acids that the engineered chimeric receptor is at least 95% identical to the sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 27. The NK cell population of claim 26 , having a sequence . A method for enhancing proliferation of natural killer (NK) cells comprising:
co-culturing the NK cell population with a feeder cell population in a culture medium, wherein the feeder cell population is fertilized with cells engineered to express 4-1BBL and membrane-bound interleukin-15; a step comprising;
adding interleukin 2 (IL2) to the culture medium;
At a first time point, adding at least one soluble stimulant to the culture medium, wherein the soluble stimulant is interleukin 12 (IL12), interleukin 18 (IL18), interleukin 21 ( IL21), and combinations thereof, wherein the concentration of said at least one soluble stimulant is between about 0.01 ng/mL and about 100 ng/mL within 120 hours of said co-cultivation. ;
adding an additional amount of at least one of said soluble stimulants to the culture medium at a second time point, wherein the first and second time points are greater than 12 hours and less than 120 hours apart. and
co-culturing the NK cells with the feeder cells for a second period of time ;
wherein addition of said at least one soluble stimulating agent to the medium reduces NK cell proliferation compared to co-culturing NK cells with feeder cells in the absence of said at least one soluble stimulating agent bring about enhancement,
Method. A culture medium for cell growth, comprising:
Interleukin 2 provided at a concentration of less than about 500 IU/mL;
interleukin 12 provided at a concentration of less than about 10 ng/mL; and interleukin 18 provided at a concentration of about 30 ng/mL.
culture medium. A combination culture medium for cell growth, the combination comprising:
Interleukin 2 provided at a concentration of less than about 500 IU/mL;
Interleukin 12 provided at a concentration of less than about 10 ng/mL;
Interleukin 18, provided at a concentration of about 30 ng/mL; and Interleukin 15 (mbIL15), which binds to the cell membrane surface.
Combination culture medium.