# US 2018/0021379 A1 · Methods for engineering allogeneic and immunosuppressive resistant t cell for immunotherapy

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- Number: US 2018/0021379 A1
- Document ID: US20180021379A1
- Status: Published application
- Kind: A1
- Application number: 15/711,289
- Filed: 2017-09-21
- Published: 2018-01-25
- Earliest claimed: 2012-05-25
- Assignees: Cellectis
- Applicants: Cellectis
- Inventors: Roman GALETTO; Agnes GOUBLE; Stephanie GROSSE; Cecile MANNIOUI; Laurent POIROT; Andrew SCHARENBERG; Julianne SMITH
- Web page: https://patentgrep.com/patent/US20180021379A1

## CPC

- C12N 15/85: for animal cells
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- 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}
- A61P 5/38: of the suprarenal hormones
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/02: Immunomodulators
- A61P 37/04: Immunostimulants
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/2803: {against the immunoglobulin superfamily}
- C12N 5/0636: {T lymphocytes}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C12N 2501/39: Steroid hormones
- C12N 2501/51: B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
- C12N 2501/515: CD3, T-cell receptor complex
- C12N 2501/599: with CD designations not provided for elsewhere
- C12N 2502/99: genetically modified cells
- C12N 2510/00: Genetically modified cells
- 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
- 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)

## Abstract

Methods for developing engineered T-cells for immunotherapy that are both non-alloreactive and resistant to immunosuppressive drugs. The present invention relates to methods for modifying T-cells by inactivating both genes encoding target for an immunosuppressive agent and T-cell receptor, in particular genes encoding CD52 and TCR. This method involves the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonuclease) and polynucleotides encoding such polypeptides, to precisely target a selection of key genes in T-cells, which are available from donors or from culture of primary cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.

## Claims

### Claim at position 1

1-25. (canceled)

### Claim 26

A method for treating a cancer patient using immunotherapy comprising:
providing primary human T cells from a single donor;
co-electroporating into said primary human T-cells:
(a) RNAs encoding two half TALE-nucleases that cleave a first gene, and
(b) RNAs encoding two half TALE-nucleases that cleave a second gene,
to generate a population of transfected human T cells comprising at least 15.5% doubly-inactivated human T-cells having both first and second genes inactivated;
modifying the population of doubly inactivated human T-cells to express a chimeric antigen receptor; and
infusing the population of doubly inactivated human T-cells into said cancer patient for immunotherapy treatment.

### Claim 27 (depends on 26)

The method of claim 26, wherein the first gene is CD52 or glucocorticoid receptor (GR).

### Claim 28 (depends on 26)

The method of claim 26, wherein the second gene encodes T-cell receptor alpha.

### Claim 29 (depends on 27)

The method of claim 27, wherein the second gene encodes T-cell receptor alpha.

### Claim 30

A method for treating a cancer patient using immunotherapy comprising:
providing primary human T cells from a single donor;
modifying the primary human T cells to express a chimeric antigen receptor;
co-electroporating into said modified primary human T-cells:
(a) RNAs encoding two half TALE-nucleases that cleave a first gene, and
(b) RNAs encoding two half TALE-nucleases that cleave a second gene,
to generate a population of transfected human T cells comprising at least 15.5% doubly-inactivated human T-cells having both first and second genes inactivated; and
infusing the population of doubly inactivated human T-cells into said cancer patient for immunotherapy treatment.

### Claim 31 (depends on 30)

The method of claim 30, wherein the first gene is CD52 or glucocorticoid receptor (GR).

### Claim 32 (depends on 30)

The method of claim 30, wherein the second gene encodes T-cell receptor alpha.

### Claim 33 (depends on 31)

The method of claim 31, wherein the second gene encodes T-cell receptor alpha.

### Claim 34

A method for treating a cancer patient using immunotherapy comprising:
providing a population of transfected human T cells from a single donor comprising at least 15.5% doubly-inactivated human T-cells having both first and second genes inactivated;
wherein said cells are further modified to express a chimeric antigen receptor; and
infusing the population of doubly inactivated human T-cells into said cancer patient for immunotherapy treatment.

### Claim 35 (depends on 34)

The method of claim 34, wherein the first gene is CD52 or glucocorticoid receptor (GR).

### Claim 36 (depends on 34)

The method of claim 34, wherein the second gene encodes T-cell receptor alpha.

### Claim 37 (depends on 35)

The method of claim 35, wherein the second gene encodes T-cell receptor alpha.