IP Library › Granted Patent US 12,351,824
Granted Patent B2
US 12,351,824 · App. 17/186,877 · Granted Jul 8, 2025

Method for producing natural killer cells from pluripotent stem cells

Inventors: Yu Qian (Cambridge, MA); Hui-Hsin Chang (Cambridge, MA); Xi Shi (Cambridge, MA); Jianxin Hu (Cambridge, MA); Lan Cao (Cambridge, MA)
Assignee: Takeda Pharmaceutical Company Limited
C12N5/0646A61K40/15A61K40/31A61K40/35A61K40/4211C07K14/5443C07K14/705C07K14/7051C07K14/70517C07K14/70578C07K16/2803A61K2239/31A61K2239/38A61K2239/48C07K2319/03C12N2500/02C12N2501/115C12N2501/125C12N2501/155C12N2501/165C12N2501/2302C12N2501/2307C12N2501/26C12N2501/515C12N2501/998C12N2501/999C12N2506/45
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Quick Facts
Patent No.
US 12,351,824
App. No.
17/186,877
Granted
Jul 8, 2025
Kind
B2
Abstract

The present disclosure provides, among other things, a method for efficiently producing natural killer cells from induced pluripotent cells. The method includes the steps of: (I) culturing pluripotent stem cells in a culture medium to produce CD56+/CD3− immune cells.

Claims (22)

1. A method of producing pluripotent stem cell-derived natural killer (NK) cells comprising:

(A) providing a bulk cell population in a bulk cell medium, comprising hematopoietic progenitor cells (HPC) (HP cell bulk) derived from pluripotent stem cells;

(B) culturing HP cell bulk in a CD4/CD8 induction medium comprising a p38 inhibitor and SDF-1 to obtain a heterogeneous population of CD4+/− cells and CD8+/− cells (DP cell bulk);

(C) culturing the DP cell bulk in NK induction medium comprising IL-2 or IL-7 to obtain NK cells; wherein step (C) does not include a step of isolating CD4+/CD8+ cells from the DP cell bulk and wherein >50% of the cells produced from step (C) are NK cells.

2. The method of claim 1 , wherein the method does not include a cell isolation step in step (B).

3. The method of claim 1 , wherein the HP cell bulk in step (A) comprises CD34+ cells.

4. The method of claim 1 , wherein the DP cell bulk of step (B) comprises an intermediate heterogeneous cell population comprising CD4−/CD8− cells, CD4−/CD8+ cells, CD4+/CD8− cells, and CD4+/CD8+ cells.

5. The method of claim 1 , wherein step (B) does not include a step of isolating CD4+/CD8+ cells.

6. The method of claim 1 , wherein step (A) comprises culturing pluripotent stem cells in HPC induction media to produce the HP cell bulk.

7. The method of claim 1 , wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC).

8. The method of claim 4 , wherein the CD4/CD8 induction media further comprises a compound selected from the group consisting of ascorbic acid, stem cell factor (SCF), IL-7, Flt3L, thrombopoietin (TPO), and combinations thereof.

9. The method of claim 4 , wherein the NK induction media of step (C) further comprises a CD3 activator.

10. The method of claim 1 , wherein each of the culturing steps is performed at 3-6% oxygen.

11. The method of claim 6 , wherein culturing pluripotent stem cells in the HPC induction medium to obtain HP cell bulk lasts for greater than 6 days.

12. The method of claim 7 , wherein the iPSC is obtained from peripheral blood mononuclear cells.

13. The method of claim 1 , wherein at least 70% or more of CD56+/CD3− cells are produced without a step of enrichment.

14. The method of claim 1 , wherein the NK cells are genetically modified to express one or more chimeric antigen receptors (CAR).

15. A method of producing induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells comprising the following steps:

(1) culturing iPSC in HPC induction media comprising at least one compound selected from vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and ascorbic acid to obtain a heterogeneous population of cells comprising hematopoietic progenitor cells (HPC) (HP cell bulk);

(2) culturing the HP cell bulk obtained in (1) in CD4/CD8 induction media comprising ascorbic acid, p38 inhibitor and SDF-1 to obtain an intermediate heterogeneous population of CD4+/− and CD8+/− cells (DP cell bulk); and

(3) culturing the DP cell bulk from (2) in NK induction media comprising a compound selected from the group consisting of a CD3 activator, IL-2 and IL-7 to obtain NK cells; and wherein step (3) does not include a step of isolating CD4+/CD8+ cells from the DP cell bulk and wherein >50% of the cells produced from step (3) are NK cells.

16. The method of claim 1 , wherein at least 60% of the cells produced from step (C) are NK cells.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 7, 2025
From: MILLENNIUM PHARMACEUTICALS, INC.
To: TAKEDA PHARMACEUTICAL COMPANY LIMITED
Reel/Frame 071053/0759 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 16, 2021
From: QIAN, YU; CHANG, HUI-HSIN; SHI, XI; HU, JIANXIN; CAO, LAN
To: MILLENNIUM PHARMACEUTICALS, INC.
Reel/Frame 056567/0157 →
Continuity (2)
Provisional Application 62983511 · Feb 28, 2020
Related Publication 20210292713A1 · Sep 23, 2021
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