METHOD FOR PRODUCING HELPER T CELLS FROM PLURIPOTENT STEM CELLS

• US 20200129551A1
• Filed: 03/13/2018
• Published: 04/30/2020
• Est. Priority Date: 03/14/2017
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: KANEKO, Shin, UEDA, Norihiro, UEMURA, Yasushi, FUKUDA, Kyoko
• Granted Patent: US 11,559,548 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/48 Blood cells, e.g. leukemia or lymphoma
• 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: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61K 35/76 Viruses; Subviral particles; Bacteriophages
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/464453 Wilms tumor 1 [WT1]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/2302 Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/2315 Interleukin-15 (IL-15)
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/998 : Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 : Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/1394 : Bone marrow stromal cells; whole marrow
• CPC Class Codes: C12N 2502/45 : Artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2506/45 : from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

A method of producing helper T cells, comprising:

• (i) culturing T cells, which have been induced from pluripotent stem cells and into which a CD4 gene or a gene product thereof has been introduced, in a medium containing IL-2 and IL-15; and (ii) isolating CD40L-highly expressing T cells from cells obtained in step (i).

Claims

1. A method of producing helper T cells comprising:

(i) culturing T cells, which have been induced from pluripotent stem cells and into which a CD4 gene or a gene product thereof has been introduced, in a medium containing IL-2 and IL-15; and

(ii) isolating CD40L-highly expressing T cells from cells obtained in Step (i).

2. The method according to claim 1, wherein the concentration of the IL-2 is 10 to 500 IU/ml, and the concentration of the IL-15 is 1 to 50 ng/ml.

3. The method according to claim 1, wherein the T cells have been induced from the pluripotent stem cells by a method comprising:

(1) inducing CD34-positive hematopoietic progenitor cells from pluripotent stem cells; and

(2) culturing CD34-positive hematopoietic progenitor cells obtained in Step (1), in the presence of FLT3L and IL-7.

4. The method according to claim 3, wherein Step (1) comprises co-culturing pluripotent stem cells with C3H10T1/2, followed by co-culturing with C3H10T1/2 in the presence of VEGF, FLT3L, and SCF.

5. The method according to claim 3, wherein Step (2) comprises co-culturing the CD34-positive hematopoietic progenitor cells with stromal cells.

6. The method according to claim 3, wherein said method of inducing the T cells from the pluripotent stem cells further comprises:

(3) co-culturing cells obtained in Step (2), with peripheral blood mononuclear cells in the presence of IL-7 and IL-15.

7. The method according to claim 3, wherein said method of inducing T cells from the pluripotent stem cells further comprises:

bringing cells obtained in Step (2) into contact with mitogen, and/or

bringing cells obtained in Step (3) into contact with mitogen.

8. The method according to claim 1, wherein the CD4 gene has been introduced using a retrovirus vector.

9. The method according to claim 1, wherein the pluripotent stem cells are pluripotent stem cells having a rearranged TCR sequence of interest.

10. The method according to claim 9, wherein the pluripotent stem cells are human iPS cells induced from lymphocytes that recognize a desired antigen(s).

11. The method according to claim 10, wherein the lymphocytes that recognize a desired antigen(s) are lymphocytes that recognize BCR/ABL.

12. A method of activating dendritic cells comprising:

bringing helper T cells containing CD4-positive CD40L-highly expressing T cells produced by the method according to claim 1 into contact with isolated dendritic cells in vitro in the presence of an antigen(s).

13. Helper T cells induced from pluripotent stem cells, the helper T cells comprising CD4-positive CD40L-highly expressing T cells.

14. A pharmaceutical comprising the helper T cells according to claim 13.

15. The pharmaceutical according to claim 14, further comprising dendritic cells.

16. The pharmaceutical according to claim 14, further comprising an antigen.

17. The pharmaceutical according to claim 16, wherein the antigen is a BCR/ABL fragment.

18. The pharmaceutical according to claim 14, which is a therapeutic agent for cancer.