# US 2021/0171908 A1 · Allogeneic t-cells and methods for production thereof

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- Number: US 2021/0171908 A1
- Document ID: US20210171908A1
- Status: Published application
- Kind: A1
- Application number: 17/088,759
- Filed: 2020-11-04
- Published: 2021-06-10
- Earliest claimed: 2019-11-05
- Assignees: Lonza Walkersville, Inc.; Lonza Israel Ltd.
- Applicants: Lonza Walkersville, Inc.; Lonza Israel Ltd.
- Inventors: Yonatan LEVINSON; Senthil RAMASWAMY; Alex SARGENT; Ariel GILERT; Alona FELDMAN; Frida GOTLIEB
- Web page: https://patentgrep.com/patent/US20210171908A1

## CPC

- C12N 5/0636: {T lymphocytes}
- C12N 15/635: {Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline}
- 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 40/4211: {CD19 or B4}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- A61K 40/50: characterised by the use of allogeneic cells
- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2500/10: Metals; Metal chelators
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2501/2307: Interleukin-7 (IL-7)
- C12N 2501/73: Hydrolases (EC 3.)
- C12N 2510/02: Cells for production
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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 present invention provides methods for producing allogeneic T-cells, including the use of an engineered nuclease under the control of a controllable promoter. By preparing T-cells with an inducible nuclease, large volumes of cells can be prepared, each of which contains the ability to individually produce the desired nuclease. These cells can then be modified as desired through the introduction of a gene of interest, or an undesired gene can be knocked-out. Also provided herein are allogeneic T-cells for use in various therapeutic applications.

## Claims

### Claim 1

A method of producing a T-cell line for use in an allogeneic application, comprising:
a. introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into a T-cell line;
b. integrating the nucleic acid molecule into the genome of the T-cell line; and
c. expanding the T-cell line.

### Claim 2 (depends on 1)

The method of claim 1, wherein the engineered nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector-based nuclease, and a CRISPR-associated nuclease.

### Claim 3 (depends on 2)

The method of claim 2, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

### Claim 4 (depends on 1)

The method of claim 1, wherein the controllable promoter is an inducible promoter, selected from the group consisting of a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element, and a glutamate inducible promoter.

### Claim 5

(canceled)

### Claim 6 (depends on 1)

The method of claim 1, wherein the controllable promoter comprises a Tet-on system and is a derepressible promoter, and wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO).

### Claim 7

(canceled)

### Claim 8 (depends on 6)

The method of claim 6, wherein the nucleic acid molecule further includes a tetracycline repressor protein.

### Claim 9

(canceled)

### Claim 10 (depends on 1)

The method of claim 1, wherein the T-cell line comprises at least about 109 T-cells and following the expanding, the method further comprises freezing the expanded T-cell line.

### Claim 11

(canceled)

### Claim 12

(canceled)

### Claim 13

A method of producing a genetically modified T-cell line, comprising:
a. introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T-cell line;
b. integrating the nucleic acid molecule into the genome of the T-cell line;
c. expanding the T-cell line;
d. inducing expression of the CRISPR-associated nuclease by activating the controllable promoter;
e. introducing a guide-RNA and a gene of interest into the expanded T-cell line;
f. knocking out expression of a T-cell receptor and introducing the gene of interest into the genome of the T-cell line; and
g. recovering the genetically modified T-cell line.

### Claim 14 (depends on 13)

The method of claim 13, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

### Claim 15 (depends on 13)

The method of claim 13, wherein the controllable promoter is an inducible promoter selected from the group consisting of a 4HT inducible promoter and a glutamate inducible promoter.

### Claim 16

(canceled)

### Claim 17 (depends on 13)

The method of claim 13, wherein the controllable promoter is a derepressible promoter, and wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO) and activating the derepressible promoter comprises adding doxycycline to the T-cell line.

### Claim 18

(canceled)

### Claim 19 (depends on 17)

The method of claim 17, wherein the nucleic acid molecule further includes a tetracycline repressor protein.

### Claim 20

(canceled)

### Claim 21 (depends on 13)

The method of claim 13, wherein the controllable promoter comprises a Tet-on system, and wherein the activating the Tet-on system comprises adding doxycycline to the T-cell line.

### Claim 22

(canceled)

### Claim 23 (depends on 13)

The method of claim 13, wherein the gene of interest encodes a chimeric antigen receptor (CAR).

### Claim 24 (depends on 13)

The method of claim 13, wherein the genetically modified T-cell line comprises at least about 109 T-cells and following expanding of the T-cells, and wherein the method further comprises freezing the T-cell line and thawing prior to the inducing.

### Claim 25

(canceled)

### Claim 26

A method of producing a chimeric antigen receptor (CAR) T-cell line, comprising:
a. introducing a nucleic acid molecule encoding a Cas9 nuclease or a Cas12 nuclease under the control of a controllable promoter into a T-cell line;
b. integrating the nucleic acid molecule into the genome of the T-cell line;
c. expanding the T-cell line;
d. inducing expression of the Cas9 nuclease or Cas12 nuclease by activating the controllable promoter;
e. introducing a guide-RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the expanded T-cell line;
f. knocking out expression of a T-cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T-cell line; and
g. recovering the CAR T-cell line.

### Claim 27 (depends on 26)

The method of claim 26, wherein the controllable promoter is an inducible promoter selected from the group consisting of a 4HT inducible promoter and a glutamate inducible promoter.

### Claim 28

(canceled)

### Claim 29 (depends on 26)

The method of claim 26, wherein the controllable promoter is a derepressible promoter, and wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO) and activating the derepressible promoter comprises adding doxycycline to the T-cell line.

### Claim 30

(canceled)

### Claim 31 (depends on 29)

The method of claim 29, wherein the nucleic acid molecule further includes a tetracycline repressor protein.

### Claim 32

(canceled)

### Claim 33 (depends on 26)

The method of claim 26, wherein the controllable promoter comprises a Tet-on system, and wherein the activating the Tet-on system comprises adding doxycycline to the T-cell line.

### Claim 34

(canceled)

### Claim 35 (depends on 26)

The method of claim 26, further comprising freezing the T-cell line following the expanding in c, and thawing prior to the inducing in d.

### Claim 36 (depends on 26)

The method of claim 26, wherein the CAR T-cell line comprises at least about 109 T-cells.

### Claim 37

An allogeneic T-cell line, comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T-cell line.

### Claim 38 (depends on 37)

The allogeneic T-cell line of claim 37, wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

### Claim 39 (depends on 37)

The allogeneic T-cell line of claim 37, wherein the controllable promoter is an inducible promoter selected from the group consisting of a 4HT inducible promoter and a glutamate inducible promoter.

### Claim 40

(canceled)

### Claim 41 (depends on 37)

The allogeneic T-cell line of claim 37, wherein the controllable promoter is a derepressible promoter, and wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO).

### Claim 42

(canceled)

### Claim 43 (depends on 41)

The allogeneic T-cell line of claim 41, wherein T-cell further includes a nucleic acid encoding a tetracycline repressor protein.

### Claim 44 (depends on 37)

The allogeneic T-cell line of claim 37, comprising at least about 109 T-cells and, wherein the controllable promoter comprises a Tet-on system.

### Claim 45

(canceled)

### Claim 46 (depends on 44)

The allogeneic T-cell line of claim 44, comprising at least about 1010 T-cells.

### Claim 47 (depends on 44)

The allogeneic T-cell line of claim 44, further comprising a transposon.

### Claim 48 (depends on 1)

The method of claim 1, wherein the integrating the nucleic acid molecule into the genome of the T-cell line includes the use of a transposon.

### Claim 49 (depends on 13)

The method of claim 13, wherein the integrating the nucleic acid molecule into the genome of the T-cell line includes the use of a transposon.

### Claim 50 (depends on 26)

The method of claim 26, wherein the integrating the nucleic acid molecule into the genome of the T-cell line includes the use of a transposon.