# US 2014/0154228 A1 · Antigen-specific central-memory t cell preparations having high cd4+ fraction

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- Number: US 2014/0154228 A1
- Document ID: US20140154228A1
- Status: Published application
- Kind: A1
- Application number: 14/125,101
- Filed: 2012-06-11
- Published: 2014-06-05
- Earliest claimed: 2011-06-11
- Inventors: Hans-dieter Volk; Michael Schmuck; Petra Reinke
- Web page: https://patentgrep.com/patent/US20140154228A1

## CPC

- A61K 35/26: Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/4243: {Wilms tumor 1 [WT1]}
- A61K 40/4245: {Tyrosinase or tyrosinase related proteinases [TRP-1 or TRP-2]}
- A61K 40/4268: {MAGE}
- A61K 40/46: Viral antigens
- C12N 5/0636: {T lymphocytes}
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2501/2307: Interleukin-7 (IL-7)
- C12N 2501/727: Kinases (EC 2.7.)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to a method for generation of T cell preparations that are specific for at least one target antigen, comprising the steps of expanding lymphoid cells in vitro in the presence of a target antigen or peptide fragments thereof in an expansion step, isolating cells that secrete interferon gamma and culturing, the cells in the presence of interleukin 2 and interleukin 7 and either an inhibitor of the mTOR Complex 1, or in the presence of an inhibitor of IL-2/IL-2R interaction. The invention further relates to preparations obtained by the method of the invention.

## Claims

### Claim 1

A method for generation of T cell preparations that are specific for at least one target antigen, comprising the steps of:
expanding lymphoid cells in vitro in the presence of a target antigen or peptide fragments thereof in an expansion step, yielding a first T cell preparation;
isolating responding cells from the first T cell preparation in an isolation step, yielding a second T cell preparation;
culturing, in a culturing step, the cells obtained as second T cell preparation in the presence of a cytokine, preferably interleukin 2 and/or interleukin 7, and
i. an inhibitor of the mTOR Complex 1, preferably rapamycin or a rapamycin analogue, or
ii. an inhibitor of interleukin 2 (IL-2)—interleukin-2-receptor (IL-2R) interaction.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the concentration of
inhibitor of the mTOR complex 1 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 nmol/l, or
inhibitor of IL-2-IL-2R interaction is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 μg/ml.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the inhibitor of IL-2-IL-2R interaction is a monoclonal antibody targeting CD25, particularly Daclizumab or Basiliximab.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the lymphoid cells are isolated from whole blood peripheral mononuclear cells.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the culturing step lasts between 10 and 25 days, between 15 and 21 days, or about 18 days.

### Claim 6 (depends on 1)

A T cell preparation obtained by a method according to claim 1, wherein the percentage of CD45RA− CCR7+ cells (total central memory) is greater than (>) 10%, >15%, or >20% in reference to the sum of all CD4+ cells and CD8+ cells within the preparation.

### Claim 7 (depends on 1)

A T cell preparation obtained by a method according to claim 1, wherein the ratio of cells expressing CD8 to cells expressing CD4 is smaller than (<) 6, <5, <4,5 or around 4.

### Claim 8 (depends on 6)

The T cell preparation according to claim 6, wherein the percentage of CD4+, CCR7+ CD45RA− (CM) cells is >15%, calculated in reference to the sum of all CD4+ cells and CD8+ cells within the preparation.

### Claim 9 (depends on 6)

The T cell preparation according to claim 6, wherein the percentage of CD8+, CCR7+ and CD45RA− (CM) cells is >7%, calculated in reference to the sum of all CD4+ cells and CD8+ cells within the preparation.

### Claim 10 (depends on 6)

The T cell preparation according to claim 6, wherein the percentage of cells expressing all of IFNγ, TNFα and IL2 is greater than (>) 10%.

### Claim 11 (depends on 6)

The T cell preparation according to claim 6, wherein more than 50% of all cells positive for CD4+ or CD8+ express CCR7 and CD62L, and more than 50% of cells expressed CD127 at day 2, 3, 4, 5, or 6 of the culturing step.

### Claim 12

(canceled)

### Claim 13

(canceled)

### Claim 14

(canceled)

### Claim 15 (depends on 1)

A pharmaceutical composition comprising the T cell preparation prepared by the method of claim 1; and a pharmaceutically acceptable carrier, excipient, medium or diluent.