# US 12,180,457 B2 · Closed-system manufacturing process for CAR-T cells

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- Number: US 12,180,457 B2
- Document ID: US12180457B2
- Status: Granted
- Kind: B2
- Application number: 16/965,964
- Filed: 2019-02-04
- Published: 2021-02-11
- Granted: 2024-12-31
- Earliest claimed: 2018-02-06
- Assignees: Seattle Children's Hospital
- Applicants: Seattle Children's Hospital
- Inventors: Michael C. Jensen; Joshua Gustafson
- Web page: https://patentgrep.com/patent/US12180457B2

## CPC

- 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 40/31: Chimeric antigen receptors [CAR]
- A61K 40/40: characterised by antigens that are targeted or presented by cells of the immune system
- C12N 2501/2307: Interleukin-7 (IL-7)
- C12N 2501/2315: Interleukin-15 (IL-15)
- C12N 2501/2321: Interleukin-21 (IL-21)
- C12N 2502/1114: T cells
- C12N 2510/00: Genetically modified cells
- G01N 33/54326: {Magnetic particles}
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Some embodiments provided herein relate to methods and compositions for making genetically modified T cells. In some such embodiments, CD4+ and CD8+ T cells are cultured in a single serum-free volume. In some embodiments, co-cultured CD4+ and CD8+ T cells can be transduced with a lentiviral vector, and a population of transduced T cells can be harvested within a shorter period of time than other conventional methods.

## Claims

### Claim 1

A method of manufacturing genetically modified T cells, wherein the genetically modified T cells comprise a chimeric antigen receptor (CAR), the method comprising:
(a) providing CD8+ T cells and CD4+ T cells in a culture media in a single culture vessel, wherein the culture media comprises IL-7, IL-15, and IL-21, and lacks serum, thereby obtaining co-cultured T cells;
(b) initiating the CD8+ T cells and CD4+ T cells for transduction, thereby obtaining initiated T cells, wherein the initiation is performed for a period within a range from about 12 hours to about 24 hours;
(c) transducing the initiated T cells with a vector encoding the CAR, thereby obtaining transduced T cells, wherein the transducing comprises:
(i) magnetically concentrating the initiated T cells, thereby obtaining concentrated T cells, and
(ii) incubating the concentrated T cells with a transduction media for 4 hours or less; and
(d) measuring an amount of the transduced T cells, wherein the amount is at least sufficient for a therapeutic dose of the transduced T cells in an adoptive cell transfer to a human subject, and harvesting the transduced T cells, thereby obtaining the genetically modified T cells, wherein step (d) is performed less than 7 days or less after step (b) is performed.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (b) comprises contacting the co-cultured T cells with magnetic beads coated with an anti-CD3 antibody and an anti-CD28 antibody antibodies or antigen binding fragments thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein step (i) further comprises removing the culture media from the vessel to obtain removed culture media.

### Claim 4 (depends on 3)

The method of claim 3, further comprising (iii) reintroducing the removed culture media into the vessel after the 4 hours or less.

### Claim 5 (depends on 1)

The method of claim 1, wherein the transduction media comprises a component selected from protamine sulfate, polybrene, an interleukin, or any combination thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the transduction media comprises protamine sulfate, Il-7, IL-15, IL-21, and the vector.

### Claim 7 (depends on 1)

The method of claim 1, wherein step (d) comprises cryofreezing the genetically modified T cells.

### Claim 8 (depends on 1)

The method of claim 1, wherein the method is performed outside of a biological safety cabinet (BSC).

### Claim 9 (depends on 1)

The method of claim 1, wherein the vector is a viral vector selected from a lentiviral vector, an adeno-associated vector, or an adenoviral vector.

### Claim 10 (depends on 1)

The method of claim 1, wherein the vector further comprises a nucleic acid encoding a genetic tag, or a protein for therapy.

### Claim 11 (depends on 1)

The method of claim 1, wherein the CD4+ T cells and CD8+ T cells are obtained from a single subject.

### Claim 12 (depends on 1)

The method of claim 1, wherein step (d) is performed 6 days after step (b) is performed.