Medium system and method for ex vivo expansion of NK cells

• US 10,450,547 B1
• Filed: 03/06/2019
• Issued: 10/22/2019
• Est. Priority Date: 10/25/2018
• Status: Expired due to Fees

• Current Assignee: Purecell Biomedical Technology Company Limited
• Original Assignee: Purecell Biomedical Technology Company Limited
• Inventors: Sun, Zhenhua, Cao, Hui
• Primary Examiner(s): Fan, Lynn Y
• Application Number:
  US16/293,710
• Time in Patent Office: 230 Days
• CPC Class Codes: A61K 39/4613 Natural-killer cells [NK or NK-T]
• CPC Class Codes: A61K 39/4644 Cancer antigens
• CPC Class Codes: C12N 2500/72 from bacteria
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/2302 Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/2304 Interleukin-4 (IL-4)
• CPC Class Codes: C12N 2501/2315 Interleukin-15 (IL-15)
• CPC Class Codes: C12N 2501/2321 Interleukin-21 (IL-21)
• CPC Class Codes: C12N 2501/599 with CD designations not provided for elsewhere
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0639 Dendritic cells, e.g. Langherhans cells in the epidermis
• CPC Class Codes: C12N 5/0646 Natural killers cells [NK], NKT cells

Abstract

This invention relates to a medium system and a method for ex vivo expansion of natural killer (NK) cells. This invention directly cultures Ficoll-separated PBMC by using immobilized anti-CD137 and RetroNectin, and uses OK-432 as a biological effector under the co-existence of GM-CSF, IL-4, IL-2, IL-15, and IL-21 for ex vivo activation and proliferation of NK cells, creating an efficient method for ex vivo expansion of NK cells. The expression rate of NK cells CD3-CD16+/CD56+ prepared by the method is as high as 92.5% or more. After 14 days of culture, NK cells can be expanded 1000 to 2000 times and have strong in vitro cytotoxic activity.

Claims

1. A method for ex vivo expansion of natural killer (NK) cells, comprising:

1) culturing a population of peripheral blood mononuclear cells (PBMC) in an induction medium comprising a basic culture medium and a group of induction factors comprising OK-432, IL-2, IL-15, and IL-21; and

2) culturing the cells obtained from step (1) in a proliferation medium comprising a basic culture medium and a group of proliferation factors comprising rhGM-CSF and rhlL-4, wherein a population of NK cells is obtained.

2. A method for ex vivo expansion of natural killer (NK) cells, comprising the steps of:

a) culturing peripheral blood mononuclear cells (PBMC) using an induction medium in a first cell culture flask pre-coated with anti-CD137, wherein said induction medium comprises a basic culture medium and a group of induction factors comprising OK-432, IL-2, IL-15, and IL-21;

b) transferring suspension cells from the first cell culture flask to a second cell culture flask pre-coated with RetroNectin;

c) culturing adherent dendritic cells in the first cell culture flask using a proliferation medium, wherein said proliferation medium comprises a basic culture medium and a group of proliferation factors comprising rhGM-CSF and rhlL-4; and

d) collecting and transferring suspended non-adherent dendritic cells in the first cell culture flask to the second cell culture flask and culturing cells in said second culture flask using said proliferation medium for a period of time, thereby obtaining the population of NK cells.

3. A method for ex vivo expansion of natural killer (NK) cells, comprising the steps of:

a) providing peripheral blood mononuclear cells (PBMC) to a first cell culture flask pre-coated with anti-CD137 and culturing said PBMC using an induction medium, wherein said induction medium comprises a basic culture medium and a group of induction factors comprising OK-432, IL-2, IL-15, and IL-21;

b) transferring suspension cells from said first cell culture flask to a second cell culture flask pre-coated with RetroNectin;

c) culturing adherent dendritic cells in said first cell culture flask using a proliferation medium, wherein said proliferation medium comprises a basic culture medium and a group of proliferation factors comprising rhGM-CSF and rhlL-4;

d) collecting non-adherent dendritic cells from the first cell culture flask after step c) and transferring the collected non-adherent dendritic cells to the second cell culture flask;

e) culturing cells in said second cell culture flask using the proliferation medium for a first period of time; and

f) adding IL-2, IL-15 and IL-21 to said second cell culture flask and culturing the cells for a second period of time, thereby obtaining the population of NK cells.

4. The method of claim 3, wherein the first cell culture flask is pre-coated with anti-CD137 at a concentration of 5 μ

g/mL.

5. The method of claim 3, wherein the second cell culture flask is pre-coated with RetroNectin at a concentration of 5 μ

g/mL.

6. The method of claim 3, wherein the first period of time is four to six days.

7. The method of claim 3, wherein the second period of time is three days or more.

8. The method of claim 3, wherein the population of NK cells is obtained at least 14 days after the start of step e).

9. The method of claim 3, wherein the population of NK cells is obtained at least 21 days after the start of step e).

10. The method of claim 7, producing a population of NK cells wherein at least 90% of the NK cells are CD3-CD16+/CD56+.

11. The method of claim 3, wherein the induction medium contains OK-432 at a concentration of 1 μ

g/mL, IL-2 at a concentration of 500 IU/mL, IL-15 at a concentration of 20 ng/mL, and IL-21 at a concentration of 50 ng/mL.

12. The method of claim 3, wherein the proliferation medium contains rhGM-CSF at a concentration of 1000 U/mL and rhlL-4 at a concentration of 500 U/mL.

13. The method of claim 3, wherein the induction factors in step f) comprises IL-2 at a concentration of 500 IU/mL, IL-15 at a concentration of 20 ng/mL, and IL-21 at a concentration of 50 ng/mL in a basic culture medium.

14. The method of claim 13, wherein the number of NK cells in said population of NK cells is at least 2500×

10⁶.

15. The method of claim 8, wherein the number of NK cells in said population of NK cells is 1000 to 2000 times higher than the number of NK cells in the PBMC provided in step a).