METHOD OF CULTURING NK CELLS AND KITS CONTAINING MEDIUM ADDTIONS THEREFORE

• US 20180355317A1
• Filed: 08/15/2018
• Published: 12/13/2018
• Est. Priority Date: 02/15/2016
• Status: Active Grant

• Current Assignee: Dong Hyuk Shin, NKCL Bio Group Incorporated
• Original Assignee: Dong Hyuk Shin
• Inventors: SHIN, Dong Hyuk
• Granted Patent: US 11,613,730 B2
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/2302 Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/2318 Interleukin-18 (IL-18)
• CPC Class Codes: C12N 2501/515 CD3, T-cell receptor complex
• CPC Class Codes: C12N 2501/599 with CD designations not provided for elsewhere
• CPC Class Codes: C12N 5/0646 Natural killers cells [NK], NKT cells

Abstract

The present invention relates to a method for culturing natural killer cells (NK cells) applied to immune therapy and, more particularly, to a medium addition kin for culturing NK cells with which lymphocytes derived from human peripheral blood can be cultured to effectively amplify and activate NK cells, which have an outstanding therapeutic effect on malignant tumors, while remarkably increasing the share of NK cells, and an NK cell culturing method using the kit.

Claims

1. A method of culturing NK cells comprising following steps:

a) adding an antibody 1 solution to a separated lymphocyte followed by adding autologous plasma;

b) adding an antibody-cytokine mixed solution to the medium collected from step a);

c) adding a cytokine 1 solution to the medium collected from step b) followed by adding autologous plasma;

d) adding an antibody 2 solution to the medium collected from step c) followed by adding autologous plasma, FBS or the like;

e) adding a cytokine 2 to the medium collected from the step d) followed by adding autologous plasma, FBS or the like;

f) adding an antibody 3 solution and autologous plasma, FBS or the like to the medium collected from step e); and

g) adding a basic solution and autologous plasma, FBS or the like to the medium collected from step f).

2. The method of claim 1, wherein the basic solution comprises IL-2, L-glutamine and a cell culture medium, wherein 100 ng/mL-300 ng/mL of IL-2 and 5 mM of L-glutamine are dissolved in the cell culture medium.

3. The method of claim 1, wherein the cytokine 1 solution comprises IL-12, IL-18 and the basic solution, wherein 0.5-5 ng/mL of IL-2 and 2-50 nm/mL of IL-18 are dissolved in the basic solution.

4. The method of claim 1, wherein the cytokine 2 solution comprises IL-15 and the basic solution, wherein 10-50 ng/mL of IL-15 is dissolved in the basic solution.

5. The method of claim 1, wherein the antibody 1 solution comprises an anti-CD16 antibody and an anti-CD56 antibody dissolved in the basic solution, wherein a concentration of anti-CD16 and anti-CD56 is about 0.1-15 μ

g/mL respectively.

6. The method of claim 1, wherein the antibody 2 solution comprises the antibody 1 solution and the basic solution in a volume ratio of 1:

6-10.

7. The method of claim 1, wherein the antibody 3 solution uses a part of antibody 2 solution.

8. The method of claim 1, wherein the antibody-cytokine mixed solution comprises anti-CD3 and the cytokine 1 solution, wherein 1-12 μ

g/mL of anti-CD3 is dissolved in the cytokine 1 solution.

9. The method of claim 1, wherein autotrophic plasma contains 40 usp units or more of heparin per 1 mL of blood.

10. The method of claim 1, wherein the content of the added autologous plasma or FBS is less than 10% by volume of the total medium.

11. The method of claim 1, wherein the NK cells are cultured for 24 hours or more before the step b) is performed after the step a).

18. A NK cell culture medium prepared using the method of claim 1.

20. The culture medium of claim 18, wherein the NK cell culture medium is obtained by culturing NK cells for 8 days or less, wherein the number of NK cells contained in the medium is 5×

10⁷ or more.

22. The culture medium of claim 18, wherein the number of NK cells contained in the medium is 2×

10⁹ or more, wherein the ratio of NK cells is 80% or more.

12. A culture medium addition kit for culturing NK cells comprising:

a B unit comprising a basic solution comprising IL-2, L-glutamine dissolved in a cell culture medium;

a C1 unit comprising of a cytokine 1 solution comprising IL-12 and IL-18 dissolved in the basic solution;

a C2 unit comprising of a cytokine 2 solution comprising IL-15 dissolved in the basic solution;

an A1 unit comprising of an antibody 1 solution comprising an anti-CD16 antibody and an anti-CD56 antibody dissolved in the basic solution;

an A2 unit comprising of an antibody 2 solution comprising the antibody 1 solution and the basic solution in a volume ratio of 1;

6-10; and

a D unit comprising of an antibody-cytokine mixed solution comprising an anti-CD3 antibody dissolved in the cytokine 1 solution.

13. The kit of claim 12, wherein the A1 unit is added to the lymphocyte culture medium prior to the D unit.

14. The kit of claim 12, wherein the anti-CD16 antibody, the anti-CD56 antibody and the basic solution of the A1 unit are separately packaged and mixed during the medium addition step.

15. The kit of claim 12, wherein the anti-CD16 antibody, the anti-CD56 antibody and the basic solution of the A2 unit are separately packaged and mixed during the medium addition step.

16. The kit of claim 12, wherein anti-CD3 antibody and the cytokine 1 solution of the D unit are separately packaged and mixed during the medium addition step.

17. The kit of claim 12, wherein the units are configured to be added to a lymphocyte culture medium in the order of the A1 unit, the D unit, the C1 unit, the A2 unit, the C2 unit, the A2 unit and the B unit.

19. A NK cell culture medium prepared using the medium addition kit of claim 12.

21. The culture medium of claim 19, wherein the NK cell culture medium is obtained by culturing NK cells for 8 days or less, wherein the number of NK cells contained in the medium is 5×

10⁷ or more.

23. The culture medium of claim 19, wherein the number of NK cells contained in the medium is 2×

10⁹ or more, wherein the ratio of NK cells is 80% or more.