# US 2020/0399599 A1 · Method for generating cells of the t cell lineage

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2020/0399599 A1
- Document ID: US20200399599A1
- Status: Published application
- Kind: A1
- Application number: 16/969,854
- Filed: 2019-02-14
- Published: 2020-12-24
- Earliest claimed: 2018-02-14
- Assignees: Sunnybrook Research Institute
- Applicants: Sunnybrook Research Institute
- Inventors: Juan Carlos Zuniga-Pflucker; Mahmood Mohtashami; Ashton Trotman-Grant
- Web page: https://patentgrep.com/patent/US20200399599A1

## CPC

- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C12N 5/0636: {T lymphocytes}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/40: characterised by antigens that are targeted or presented by cells of the immune system
- C12N 5/0075: {using microcarriers}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- C12N 2501/42: Notch; Delta; Jagged; Serrate
- C12N 2506/11: from blood or immune system cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- 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)
- 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)

## Abstract

A method of generating cells of the T cell lineage is provided comprising (a) culturing a sample comprising stem cells or progenitor cells with a Notch ligand conjugated to a suspension support and (b) isolating cells of the T cell lineage. In one embodiment, the cells of the T-cell lineage are progenitor T cells or mature T cells. Compositions, kits and uses thereof are also provided.

## Claims

### Claim 1

A method of generating a cell of the T cell lineage comprising (a) culturing a sample comprising stem cells or progenitor cells with a Notch ligand conjugated to a suspension support and (b) isolating cells of the T cell lineage.

### Claim 2 (depends on 1)

The method of claim 1, wherein the suspension support is a particle or a microbead.

### Claim 3

(canceled)

### Claim 4 (depends on 1)

The method of claim 1, wherein the stem cells or progenitor cells with the Notch ligand are cultured in suspension.

### Claim 5 (depends on 1)

The method of claim 1, wherein the stem cells are selected from hematopoietic stem/progenitor cells (HSPCs), embryonic stem cells and induced pluripotent stem cells (iPSCs).

### Claim 6 (depends on 1)

The method of claim 1, wherein the stem cells are CD34+ or CD34+ CD38−/lo HSPCs.

### Claim 7 (depends on 1)

The method of claim 1, wherein the stem cells are CD34+ hematopoietic precursor cells, optionally CD34+ hematopoietic precursor cells that have been differentiated from iPSCs.

### Claim 8 (depends on 1)

The method of claim 1, wherein the Notch ligand is DL4.

### Claim 9 (depends on 1)

The method of claim 1, wherein the cells of the T cell lineage are progenitor T (proT) cells.

### Claim 10 (depends on 9)

The method of claim 9, wherein the stem cells or progenitor cells are human cells and the proT cells have the phenotype CD34+ CD7+ or CD7+ CD5+ CD1a−.

### Claim 11 (depends on 10)

The method of claim 10, wherein the stem cells or progenitor cells are mouse cells, optionally lineage—CD117+ Sca-1+ mouse cells, and the proT cells have the phenotype CD25+ or CD25+CD90+.

### Claim 12 (depends on 1)

The method of claim 1, wherein the cells of the T-cell lineage are CD4+CD8+ double positive cells, CD4+CD8+CD3+ double positive cells, CD8+CD3+ single positive cells or CD4+CD3+ single positive cells.

### Claim 13 (depends on 1)

The method of claim 1, wherein the stem cells or progenitor cells are cultured in stromal cell-free media.

### Claim 14 (depends on 1)

The method of claim 1, wherein the stem cells or progenitor cells are cultured with at least one T cell co-stimulatory molecules attached to a suspension support, optionally wherein the at least one T cell co-stimulatory molecule is VCAM1.

### Claim 15 (depends on 1)

A cell of the T cell lineage, wherein the cell is generated by the method of claim 1, optionally wherein the cell is a progenitor T cell, CD4+CD8+ double positive cell, CD4+CD8+CD3+ double positive cell, CD8+CD3+ single positive cell or CD4+CD3+ single positive cell.

### Claim 16

(canceled)

### Claim 17

A suspension Notch ligand comprising (a) a Notch ligand and (b) a suspension support, optionally a microbead, wherein the Notch ligand is conjugated to the suspension support.

### Claim 18 (depends on 17)

The suspension Notch ligand of claim 17 wherein (i) the microbead has a diameter of 6.5 to 100 μm and/or (ii) the C-terminal of the Notch ligand is conjugated to the microbead.

### Claim 19

(canceled)

### Claim 20 (depends on 17)

A kit comprising (i) the suspension Notch ligand of claim 17 and (ii) instructions for use of the suspension Notch ligand for generating cells of the T cell lineage and/or a culture medium.

### Claim 21

(canceled)

### Claim 22 (depends on 20)

The kit of claim 20, wherein the suspension Notch ligand comprises DL4 conjugated to the microbead.

### Claim 23 (depends on 20)

The kit of claim 20, further comprising (iii) at least one T cell co-stimulatory molecule attached to a suspension support, optionally wherein the at least one T cell co-stimulatory molecule is VCAM1.

### Claim 24

A method of treating a subject having a condition requiring an increase in the number of T cells comprising:
(i) generating cells of the T cell lineage comprising (a) culturing a sample comprising stem cells or progenitor cells with a Notch ligand conjugated to a suspension support and (b) isolating cells of the T cell lineage, and
(ii) administering an effective amount of the cells of the T cell lineage to the subject.

### Claim 25 (depends on 24)

The method of claim 24, wherein the cells of the T cell lineage are progenitor T cells, CD4+CD8+ double positive cells, CD4+CD8+CD3+ double positive cells, CD8+CD3+ single positive cells, CD4+CD3+ single positive cells or mature T cells.

### Claim 26

(canceled)

### Claim 27

(canceled)