# US 2020/0399390 A1 · Methods for producing lymphocyte progenitors

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- Number: US 2020/0399390 A1
- Document ID: US20200399390A1
- Status: Published application
- Kind: A1
- Application number: 16/492,036
- Filed: 2018-03-08
- Published: 2020-12-24
- Earliest claimed: 2017-03-08
- Assignees: Murdoch Childrens Research Institute
- Applicants: Murdoch Childrens Research Institute
- Inventors: Edouard Guy STANLEY; Andrew ELEFANTY; Ali MOTAZEDIAN
- Web page: https://patentgrep.com/patent/US20200399390A1

## CPC

- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/22: Immunosuppressive or immunotolerising
- A61K 40/418: {Antigens related to induction of tolerance to non-self}
- C07K 14/5418: {IL-7}
- C07K 16/22: against growth factors {; against growth regulators}
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C12N 5/0635: {B lymphocytes}
- C12N 5/0636: {T lymphocytes}
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/42: Notch; Delta; Jagged; Serrate
- C12N 2506/02: from embryonic 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

The invention relates to a method for producing a lymphocyte progenitor, the method comprising culturing a pluripotent stem cell (PSC)-derived CD34+ cell at an air-liquid interface (ALI). The invention also relates to a method for producing a B-cell progenitor, the method comprising co-culturing a PSC-derived CD34+ cell and a stromal cell in a medium comprising a CD117 activator and a NOTCH1 inhibitor. The invention further relates to a T-cell progenitor and a B-cell progenitor when produced by the methods of the invention and to use of the T-cell progenitor in the manufacture of T cell with defined antigen specificity, optionally a chimeric antigen receptor (CAR) T cell, and use of the B-cell progenitor in the manufacture of an antibody.

## Claims

### Claim 1

A method for producing a lymphocyte progenitor, the method comprising culturing a pluripotent stem cell (PSC)-derived CD34+ cell at an air-liquid interface (ALI).

### Claim 2 (depends on 1)

The method of claim 1, wherein culturing the CD34+ cell excludes co-culturing the CD34+ cells with an exogenous stromal cell.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method excludes purifying the CD34+ cell before culturing.

### Claim 4 (depends on 1)

The method of claim 1, comprising generating an embryoid body.

### Claim 5 (depends on 1)

The method of claim 1, comprising culturing at the ALI for about 2 weeks to about 5 weeks.

### Claim 6 (depends on 1)

The method of claim 1, wherein the PSC is human, optionally an embryonic stem cell (ESC) or induced PSC (iPSC).

### Claim 7 (depends on 1)

The method of claim 1, wherein the lymphocyte progenitor is a T-cell progenitor, and wherein the method comprises culturing the CD34+ cell at the ALI in a medium comprising vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF, FGF2), stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (FLT3L), and interleukin-7 (IL-7), and optionally IL-3.

### Claim 8 (depends on 7)

The method of claim 7, wherein the medium excludes IL-3 or IL-6.

### Claim 9 (depends on 7)

The method of claim 7, wherein an embryoid body is generated by culturing the PSC cell in a medium comprising a WNT agonist, optionally CHIR99021, and BMP4 and Activin A.

### Claim 10 (depends on 9)

The method of claim 9, wherein the embryoid body is generated by culturing the PSC in a medium comprising a WNT agonist, BMP4 and Activin A from about day zero to about day 2, or from about day 2 to about day 4, or from about day zero to about day 4.

### Claim 11 (depends on 1)

The method of claim 1, wherein the lymphocyte progenitor is a B-cell progenitor, and comprising culturing the CD34+ cell in a medium comprising a NOTCH1 inhibitor during or after an embryoid body comprising the CD34+ cell is cultured at the ALI.

### Claim 12 (depends on 11)

The method of claim 11, comprising culturing the embryoid body comprising the CD34+ cell in a medium comprising the NOTCH1 inhibitor from about day 15, optionally to about day 32, during culturing at the ALI.

### Claim 13 (depends on 11)

The method of claim 11, comprising culturing the embryoid body comprising the CD34+ cell at the ALI in a medium comprising a CD117 activator.

### Claim 14 (depends on 13)

The method of claim 13, comprising culturing the embryoid body comprising the CD34+ cell in the medium comprising a CD117 activator.

### Claim 15 (depends on 13)

The method of claim 13, further comprising culturing in a medium comprising IL-7 from about day 8.

### Claim 16 (depends on 15)

The method of claim 15, comprising culturing in a medium comprising IL-7 for up to 35 days, optionally 30 days.

### Claim 17 (depends on 11)

The method of claim 11, wherein the NOTCH1 inhibitor is N—[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

### Claim 18 (depends on 13)

The method of claim 13, wherein the CD117 activator is stem cell factor (SCF).

### Claim 19 (depends on 1)

A lymphocyte progenitor when produced by the method of claim 1.

### Claim 20 (depends on 1)

Use of a lymphocyte progenitor when produced by the method of claim 1 in the manufacture of (i) a T cell with defined antigen specificity, optionally a chimeric antigen receptor (CAR) T cell, or (ii) an antibody.

### Claim at position 21

-39. (canceled)