Method of producing natural killer cells and composition for treating cancer

1. A method of expanding natural killer cells in culture, comprising:

isolating peripheral blood mononuclear cells (PBMCs) from a blood sample;

isolating at least one of CD56+ cells and/or CD3−/CD56+ cells from the PBMCs; and

co-culturing the at least one of CD56+ cells and/or CD3−/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines;

wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells,

wherein the at least two cytokines comprise IL-2 and IL-21.

2. The method of claim 1 , wherein isolating at least one of CD56+ cells and/or CD3−/CD56+ cells from the PBMCs is conducted by using at least one of CD56 microbeads and CD3 microbeads.

3. The method of claim 1 , wherein the at least two cytokines further comprise one or more cytokines selected from a group consisting of IL-15, Flt3-L, SCF, IL-7, IL-18, IL-4, type I interferons, GM-CSF, IGF 1, and combinations thereof.

4. The method of claim 1 , wherein IL-2 is added at a concentration of 50-1000 IU/mL.

5. The method of claim 1 , wherein IL-21 is added at a concentration of 10-100 ng/mL.

6. The method of claim 1 , wherein the ratio of the irradiated Jurkat cells and the irradiated EBV-LCL cells is about 1:0.1-5 or 0.1-5:1.

7. The method of claim 1 , wherein the co-culturing comprises co-culturing for 1-50 days.

8. The method of claim 1 , further comprising:

co-culturing the at least one of CD56+ cells and/or CD3−/CD56+ cells with the combination of feeder cells, in the presence of IL-2 for a first period; and

co-culturing the at least one of CD56+ cells and/or CD3−/CD56+ cells with the combination of feeder cells, in the presence of IL-21 for a second period.

9. The method of claim 8 , wherein IL-21 is added more than once during Day 0-6 of the second period.

10. The method of claim 8 , wherein IL-21 and the combination of feeder cells are added more than once during Day 0-6 of the second period.

11. The method of claim 8 , wherein IL-21 is added more than once during the first six days of every fourteen-day cycle during the second period.

12. The method of claim 1 , wherein the at least one of CD56+ cells and/or CD3−/CD56+ cells and the combination of feeder cells is co-cultured with a ratio of about 1:1-100 of CD56+ cells and/or CD3−/CD56+ cells to feeder cells.

13. A composition made by the method of claim 1 .

14. A method of expanding natural killer cells in culture, comprising:

isolating peripheral blood mononuclear cells (PBMCs) from a blood sample;

isolating at least one of CD56+ cells and/or CD3−/CD56+ cells from the PBMCs; and

co-culturing the at least one of CD56+ cells and/or CD3−/CD56+ cells with a combination of feeder cells in the presence of at least two cytokines;

wherein the combination of feeder cells comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells,

wherein the at least two cytokines comprise IL-2 and IL-21, and

wherein both the feeder cells and the cytokines are supplemented at intervals throughout the expansion in culture.

15. The method of claim 14 , wherein isolating at least one of CD56+ cells and/or CD3−/CD56+ cells from the PBMCs is conducted by using at least one of CD56 microbeads and CD3 microbeads.

16. The method of claim 14 , wherein IL-21 is added at a concentration of 10-100 ng/mL.

17. The method of claim 14 , wherein IL-21 is added once or more during each of the following periods: Day 0-6; Day 14-20; and Day 28-34.

18. The method of claim 14 , wherein the co-culturing comprises co-culturing for 1-50 days.