# US 11,078,481 B1 · Methods for screening for cancer targets

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- Number: US 11,078,481 B1
- Document ID: US11078481B1
- Status: Granted
- Kind: B1
- Application number: 15/668,652
- Filed: 2017-08-03
- Granted: 2021-08-03
- Earliest claimed: 2016-08-03
- Assignees: KSQ Therapeutics, Inc.
- Applicants: KSQ Therapeutics, Inc.
- Inventors: Micah Benson
- Web page: https://patentgrep.com/patent/US11078481B1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/1086: {Preparation or screening of expression libraries, e.g. reporter assays}
- 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 40/4205: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4}
- A61K 40/4211: {CD19 or B4}
- A61P 35/00: Antineoplastic agents
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/70503: {Immunoglobulin superfamily}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 14/7153: {for colony-stimulating factors [CSF]}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C07K 16/32: against translation products of oncogenes
- C12N 5/0636: {T lymphocytes}
- C12N 5/0693: {Tumour cells; Cancer cells}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- A61K 2039/505: {comprising antibodies}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/03: containing a transmembrane segment
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/11: for the determination of target sites, i.e. of active nucleic acids
- C12N 2330/31: Libraries, arrays
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2501/53: CD2
- C12N 2501/599: with CD designations not provided for elsewhere
- C12N 2502/1114: T cells
- C12N 2502/30: tumour cells
- C12N 2510/00: Genetically modified cells
- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- 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)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

This invention describes a novel CRISPR/Cas9 target identification platform permitting the discovery of novel genes and pathways involved in the ability of T cells and NK cells to react against and generate an anti-tumor response.

## Claims

### Claim 1

A method for obtaining T cells that are resistant to tumor suppression or deactivation, said method comprising:
(a) obtaining a population of tumor cells that are engineered to express an extracellular protein target;
(b) obtaining a primary cell population of T cells that are engineered to express:
(i) a chimeric antigen receptor (CAR), said CAR comprising an intracellular signaling domain and an extracellular target-binding domain that binds to the extracellular protein target expressed by the population of tumor cells of (a), to form a population of CAR-T cells that express the extracellular target-binding domain on the cell surface, and
(ii) a Cas9 protein and a gRNA library that selectively edit the CAR-T cells at one or more locations of the CAR-T cell genome to form a subpopulation of edited CAR-T cells;
(c) co-culturing the subpopulation of edited CAR-T cells of (b) with the population of tumor cells of (a); and
(d) isolating the edited CAR-T cells that accumulate over the co-culture period, wherein the isolated CAR-T cells have been edited in a manner as to make the CAR-T cells resistant to tumor suppression or deactivation, thereby obtaining T cells that are resistant to tumor suppression or deactivation.

### Claim 2 (depends on 1)

The method of claim 1 wherein the subpopulation of edited CAR-T cells and the population of tumor cells are co-cultured for at least 7 days.

### Claim 3 (depends on 1)

The method of claim 1 wherein said CAR-T cells have been engineered with a nucleic acid encoding the Cas9 protein.

### Claim 4 (depends on 3)

The method of claim 3, wherein the nucleic acid encoding the Cas9 protein is mRNA and is introduced via electroporation.

### Claim 5 (depends on 3)

The method of claim 3, wherein the nucleic acid encoding the Cas9 protein is encoded by a viral vector.

### Claim 6 (depends on 5)

The method of claim 5, wherein the viral vector is a lentiviral vector and is introduced at a titer of at least about 1×106 infectious particles/mL.

### Claim 7 (depends on 1)

The method of claim 1, wherein the Cas9 protein is a dCas9.

### Claim 8 (depends on 7)

The method of claim 7, wherein the dCas9 is fused with a repressor domain.

### Claim 9 (depends on 8)

The method of claim 8, wherein the repressor domain is selected from the group consisting of MAX-interacting protein 1 (MXI1), Krüppel-associated box (KRAB) domain and four concatenated mSin3 domains (SID4X).

### Claim 10 (depends on 7)

The method of claim 7, wherein the dCas9 is fused with a transcriptional activator.

### Claim 11 (depends on 10)

The method of claim 10, wherein the transcriptional activator is selected from the group consisting of (i) one or multiple repeats of the herpes simplex VP16 activation domain selected from the group consisting of VP64 and VP160, and (ii) the nuclear factor-κB (NF-κB) transactivating subunit activation domain p65AD.

### Claim 12 (depends on 11)

The method of claim 11, wherein the Cas9 protein comprises a dCas9-VP64 fusion protein.

### Claim 13 (depends on 3)

The method of claim 3, wherein the nucleic acid encoding the Cas9 protein further encodes a selectable marker.

### Claim 14 (depends on 13)

The method of claim 13, wherein the selectable marker is a fluorophore or an antibiotic resistance gene.

### Claim 15

A method for obtaining NK cells or NKT cells that are resistant to tumor suppression or deactivation, said method comprising:
(a) obtaining a population of tumor cells that are engineered to express an extracellular protein target;
(b) obtaining a primary cell population of NK cells or NKT cells that are engineered to express:
(i) a chimeric antigen receptor (CAR), said CAR comprising an intracellular signaling domain and an extracellular target-binding domain that binds to the extracellular protein target expressed by the population of tumor cells of (a), to form a population of CAR-NK cells or CAR-NKT cells that express the extracellular target-binding domain on the cell surface, and
(ii) a Cas9 protein and a gRNA library that selectively edit the CAR-NK cells or CAR-NKT cells in one or more locations of the CAR-NK cell genome or CAR-NKT cell genome to form a subpopulation of edited CAR-NK cells or edited CAR-NKT cells;
(c) co-culturing the subpopulation of edited CAR-NK cells or edited CAR-NKT cells of (b) with the population of tumor cells of (a);
(d) isolating the edited CAR-NK cells or edited CAR-NKT cells that accumulate over the co-culture period;
wherein the isolated CAR-NK cells or CAR-NKT cells have been edited in a manner as to make said cells resistant to suppression or deactivation by said tumor, thereby obtaining NK cells or NKT cells that are resistant to tumor suppression or deactivation.

### Claim 16 (depends on 1)

The method of claim 1, wherein the extracellular protein target is selected from the group consisting of HER2, EGFR, ERBB2, ERBB3, ERBB4, CD19, and CD20.