# US 12,006,512 B2 · Method for producing CD8alpha+beta+ cytotoxic t cells

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- Number: US 12,006,512 B2
- Document ID: US12006512B2
- Status: Granted
- Kind: B2
- Application number: 16/479,575
- Filed: 2018-01-19
- Published: 2019-12-05
- Granted: 2024-06-11
- Earliest claimed: 2017-01-20
- Assignees: Kyoto University
- Applicants: Kyoto University
- Inventors: Shin Kaneko; Yohei Kawai
- Web page: https://patentgrep.com/patent/US12006512B2

## CPC

- A61P 37/02: Immunomodulators
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- 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)
- A61K 39/12: Viral antigens
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/421: {Immunoglobulin superfamily}
- A61K 40/46: Viral antigens
- A61P 31/18: for HIV
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- A61K 2039/572: {cytotoxic response}
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C12N 2500/38: Vitamins
- C12N 2501/2307: Interleukin-7 (IL-7)
- C12N 2501/2312: Interleukin-12 (IL-12)
- C12N 2501/2315: Interleukin-15 (IL-15)
- C12N 2501/2318: Interleukin-18 (IL-18)
- C12N 2501/2321: Interleukin-21 (IL-21)
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2501/42: Notch; Delta; Jagged; Serrate
- C12N 2501/48: Regulators of apoptosis
- C12N 2501/50: Cell markers; Cell surface determinants
- C12N 2501/515: CD3, T-cell receptor complex
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2502/30: tumour cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2533/50: Proteins
- C12N 2533/52: Fibronectin; Laminin
- C12N 2740/16334: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

A method efficiently produces cytotoxic T lymphocytes having intrinsic properties of lymphocytes of the acquired immune system suitable for cellular immunotherapy. The method includes culturing CD4/CD8 double-positive T cells in a medium containing IL-7 and a T-cell receptor activator, to induce CD8α+β+ cytotoxic T lymphocytes.

## Claims

### Claim 1

A method of producing CD8α+β+ cytotoxic T lymphocytes, the method comprising:
(a) culturing CD4/CD8 double-positive T cells in the presence of IL (interleukin)-7 and a T-cell receptor activator, wherein the T-cell receptor activator is an anti-CD3 antibody, thereby producing CD8α+β+ cytotoxic T lymphocytes, and
(b) culturing the cells obtained in step (a) in the presence of IL-7, but in the absence of a T-cell receptor activator, wherein the culture (b) is carried out using a culture vessel containing a fibronectin fragment and/or a Notch ligand.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the medium further contains IL-21 and Flt3L (Flt3 ligand).

### Claim 3 (depends on 1)

The method according to claim 1, wherein the fibronectin fragment is RetroNectin, and the Notch ligand is Delta-like 4 (DLL4).

### Claim 4 (depends on 1)

The method according to claim 1, further comprising the following step (c):
(c) culturing the cells obtained in step (b) in a medium containing IL-7, IL-21, and Flt3L using a culture vessel containing neither a fibronectin fragment nor a Notch ligand.

### Claim 5 (depends on 4)

The method according to claim 4, comprising the following steps (a1), (b1), and (c1):
(a1) culturing CD4/CD8 double-positive T cells in a medium containing IL-7, Flt3L, IL-21, and an anti-CD3 antibody;
(b1) culturing the cells obtained in step (a1) in a medium containing IL-7, Flt3L, and IL-21, but not containing an anti-CD3 antibody, using a culture vessel containing a fibronectin fragment; and
(c1) culturing the cells obtained in step (b I) in a medium containing IL-7, IL-21, and Flt3L using a culture vessel containing neither a fibronectin fragment nor a Notch ligand.

### Claim 6 (depends on 4)

The method according to claim 4, comprising the following steps (a2), (b2), and (c2):
(a2) culturing CD4/CD8 double-positive T cells in a medium containing IL-7, Flt3L, and an anti-CD3 antibody;
(b2) culturing the cells obtained in step (a2) in a medium containing IL-7 and Flt3L, but not containing an anti-CD3 antibody, using a culture vessel containing a fibronectin fragment and a Notch ligand; and
(c2) culturing the cells obtained in step (b2) in a medium containing IL-7, IL-21, and Flt3L using a culture vessel containing neither a fibronectin fragment nor a Notch ligand.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the culture is carried out without using feeder cells.

### Claim 8 (depends on 1)

The method according to claim 1, further comprising the step of sorting the CD8α+β+ cytotoxic T lymphocytes obtained.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the sorting step is carried out using as an index/indexes, wherein the index/indexes is one or more of CD8α+β+ positivity, CD5 positivity, CD336 negativity, and CD1 a negativity.

### Claim 10 (depends on 1)

The method according to claim 1, further comprising the step of performing expansion culture of CD8α+β+ cytotoxic T lymphocytes in a medium containing IL-7, IL-15, and IL-21.

### Claim 11 (depends on 1)

The method according to claim 1, further comprising the step of performing expansion culture of CD8α+β+ cytotoxic T lymphocytes in a medium containing IL-7 and IL-15, and one or more of IL-21, IL-18, IL-12, and TL1A.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the CD4/CD8 double-positive T cells are induced from pluripotent stem cells.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells.

### Claim 14 (depends on 12)

The method according to claim 12, wherein the induction of the CD4/CD8 double-positive T cells from the pluripotent stem cells comprises the following steps (a) and (b):
(a) culturing the pluripotent stem cells in a medium supplemented with vitamin C, to induce hematopoietic progenitor cells; and
(b) culturing the hematopoietic progenitor cells obtained in step (a) in a medium containing vitamin C, FLT3L, and IL-7, to induce CD4/CD8 double-positive T cells.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the CD8α+β+ cytotoxic T lymphocytes produced do not show natural killer (NK) activity.