# US 10,870,864 B2 · Methods for engineering T cells for immunotherapy by using RNA-guided Cas nuclease system

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- Number: US 10,870,864 B2
- Document ID: US10870864B2
- Status: Granted
- Kind: B2
- Application number: 16/793,896
- Filed: 2020-02-18
- Published: 2020-09-03
- Granted: 2020-12-22
- Earliest claimed: 2013-05-29
- Assignees: Cellectis
- Applicants: Cellectis
- Inventors: Philippe Duchateau; André Choulika; Laurent Poirot
- Web page: https://patentgrep.com/patent/US10870864B2

## CPC

- C12N 15/85: for animal cells
- 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 40/31: Chimeric antigen receptors [CAR]
- A61K 40/32: T-cell receptors [TCR]
- A61K 40/418: {Antigens related to induction of tolerance to non-self}
- A61K 40/4211: {CD19 or B4}
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- C12N 5/0636: {T lymphocytes}
- C12N 5/0637: {Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg}
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- A61K 40/50: characterised by the use of allogeneic cells
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2510/00: Genetically modified cells
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES

## Abstract

The present invention relates to methods of developing genetically engineered, preferably non-alloreactive T-cells for immunotherapy. This method involves the use of RNA-guided endonucleases, in particular Cas9/CRISPR system, to specifically target a selection of key genes in T-cells. The engineered T-cells are also intended to express chimeric antigen receptors (CAR) to redirect their immune activity towards malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer and viral infections.

## Claims

### Claim 1

A method of preparing and administering T-cells for cancer immunotherapy comprising the steps of:
(a) providing primary human T-cells from a donor,
(b) genetically modifying the primary human T-cells to eliminate expression of a gene encoding an immune checkpoint protein, comprising expressing in the cells
(i) a Cas9 endonuclease fused to a nuclear localization signal (NLS), and
(ii) a guide RNA that directs said endonuclease to at least one targeted locus in the gene encoding the immune checkpoint protein in the T-cell genome,
(c) expanding the genetically modified T-cells, and
(d) administering at least 104 of the expanded genetically modified T-cells to a cancer patient.

### Claim 2 (depends on 1)

The method of claim 1, comprising administering at least 105 of the expanded genetically modified T-cells to the cancer patient.

### Claim 3 (depends on 1)

The method of claim 1, comprising administering at least 106 of the expanded genetically modified T-cells to the cancer patient.

### Claim 4 (depends on 1)

The method of claim 1, comprising administering at least 107 of the expanded genetically modified T-cells to the cancer patient.

### Claim 5 (depends on 1)

The method of claim 1, comprising administering at least 108 of the expanded genetically modified T-cells to the cancer patient.

### Claim 6 (depends on 1)

The method of claim 1, comprising administering at least 109 of the expanded genetically modified T-cells to the cancer patient.

### Claim 7

A method of preparing and administering T-cells for cancer immunotherapy comprising the steps of:
(a) providing primary human T-cells from a donor,
(b) genetically modifying the primary human T-cells to eliminate expression of the CD52 gene, comprising expressing in the cells
(i) a Cas9 endonuclease fused to a nuclear localization signal (NLS), and
(ii) a guide RNA that directs said endonuclease to at least one targeted locus in the CD52 gene in the T-cell genome,
(c) expanding the genetically modified T-cells, and
(d) administering at least 104 of the expanded genetically modified T-cells to a cancer patient.

### Claim 8 (depends on 1)

The method of claim 1, wherein the gene is the PD1 gene.

### Claim 9 (depends on 1)

The method of claim 1, wherein the gene is the CTLA4 gene.

### Claim 10 (depends on 1)

The method of claim 1, wherein the gene is the LAG3 gene.

### Claim 11 (depends on 1)

The method of claim 1, further comprising introducing into the T-cells a chimeric antigen receptor (CAR).

### Claim 12 (depends on 11)

The method of claim 11, wherein the CAR is an anti-CD19 CAR.

### Claim 13 (depends on 7)

The method of claim 7, further comprising introducing into the T-cells a chimeric antigen receptor (CAR).

### Claim 14 (depends on 13)

The method of claim 13, wherein the CAR is an anti-CD19 CAR.

### Claim 15 (depends on 8)

The method of claim 8, further comprising introducing into the T-cells a chimeric antigen receptor (CAR).

### Claim 16 (depends on 15)

The method of claim 15, wherein the CAR is an anti-CD19 CAR.

### Claim 17 (depends on 9)

The method of claim 9, further comprising introducing into the T-cells a chimeric antigen receptor (CAR).

### Claim 18 (depends on 17)

The method of claim 17, wherein the CAR is an anti-CD19 CAR.