# US 2021/0230548 A1 · Natural killer cells engineered to express chimeric antigen receptors with immune checkpoint blockade

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- Number: US 2021/0230548 A1
- Document ID: US20210230548A1
- Status: Published application
- Kind: A1
- Application number: 17/050,775
- Filed: 2019-05-03
- Published: 2021-07-29
- Earliest claimed: 2018-05-03
- Assignees: Board of Regents, The University of Texas System
- Applicants: Board of Regents, The University of Texas System
- Inventors: May Daher; Rafet Basar; Elizabeth Shpall; Katy Rezvani
- Web page: https://patentgrep.com/patent/US20210230548A1

## CPC

- C07K 14/4703: {Inhibitors; Suppressors}
- A61K 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/35: Cytokines
- A61K 40/36: Immune checkpoint inhibitors
- A61K 40/4211: {CD19 or B4}
- A61P 35/00: Antineoplastic agents
- C07K 14/54: Interleukins [IL]
- C07K 14/5434: {IL-12}
- C07K 14/5443: {IL-15}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2319/00: Fusion polypeptide
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2510/00: Genetically modified cells
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are methods for producing NK cells expressing chimeric antigen receptors and having no expression of CISH. Further provided are methods for treating diseases by administering the CAR NK cells.

## Claims

### Claim 1

An isolated natural killer (NK) cell engineered to express (1) a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR) and (2) human IL-15 (hIL-15) and to have essentially no expression of CISH.

### Claim 2 (depends on 1)

The NK cell of claim 1, wherein the NK cell is engineered to express a CAR.

### Claim 3 (depends on 1)

The NK cell of claim 1, wherein the NK cell is engineered to express a TCR.

### Claim 4 (depends on 1)

The NK cell of claim 1, wherein the NK cell is engineered to express a CAR and TCR.

### Claim 5 (depends on 1)

The NK cell of claim 1, wherein the NK cell is derived from cord blood, peripheral blood, bone marrow, CD34+ cells, or iPSCs.

### Claim 6 (depends on 1)

The NK cell of claim 1, wherein the NK cell is derived from cord blood.

### Claim 7 (depends on 6)

The NK cell of claim 6, wherein the cord blood has previously been frozen.

### Claim 8 (depends on 1)

The NK cell of claim 1, wherein the CAR and/or TCR has antigenic specificity for CD19, CD319/CS1, ROR1, CD20, CD5, CD7, CD22, CD70, CD30, BCMA, CD25, NKG2D ligands, MICA/MICB, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, EGFRvIII, TRAIL/DR4, and/or VEGFR2.

### Claim 9 (depends on 1)

The NK cell of claim 1, wherein the CAR has antigenic specificity for CD19.

### Claim 10 (depends on 1)

The method of claim 1, wherein the NK cell further expresses a second cytokine.

### Claim 11 (depends on 10)

The method of claim 10, wherein the cytokine is IL-21 or IL-12.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method of any of claims 1-11, wherein the NK cell has activated mammalian target of rapamycin (mTOR) signaling.

### Claim 13 (depends on 12)

The method of claim 12, wherein the NK cell has increased JAK/STAT signaling.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

A method for producing NK cells of any one of claims 1-14 comprising:
(a) obtaining a starting population of NK cells;
(b) culturing the starting population of NK cells in the presence of artificial presenting cells (APCs);
(c) introducing a CAR and/or TCR expression vector into the NK cells;
(d) expanding the NK cells in the presence of APCs, thereby obtaining expanded NK cells; and
(e) disrupting the expression of CISH in the expanded NK cells.

### Claim 15 (depends on 14)

The method of claim 14, wherein disrupting expression comprises using CRISPR-mediated gene silencing.

### Claim 16 (depends on 15)

The method of claim 15, wherein CRISPR-mediated gene silencing comprises contacting the CAR NK cells with sgRNA and Cas9.

### Claim 17 (depends on 11)

The method of claim 11, wherein the sgRNA targets exon 4 of CISH.

### Claim 18 (depends on 17)

The method of claim 17, wherein the sgRNA comprises SEQ ID NOs:1-2.

### Claim 19 (depends on 14)

The method of claim 14, wherein the starting population of NK cells is obtained by isolating mononuclear cells using a ficoll-paque density gradient.

### Claim 20 (depends on 14)

The method of claim 14, wherein the APCs are gamma-irradiated APCs.

### Claim 21 (depends on 14)

The method of claim 14, wherein the APCs are universal APCs (uAPCs).

### Claim 22 (depends on 14)

The method of claim 14, wherein the APCs are engineered to express 41BB and IL-21.

### Claim 23 (depends on 21)

The method of claim 21, wherein the uAPCs are engineered to express (1) CD48 and/or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL).

### Claim 24 (depends on 14)

The method of claim 14, wherein the NK cells and APCs are present at a 1:2 ratio.

### Claim 25 (depends on 14)

The method of claim 14, wherein the NK cells of step (b) and (d) are further expanded in the presence of IL-2.

### Claim 26 (depends on 25)

The method of claim 25, wherein the IL-2 is present at a concentration of 100-300 U/mL.

### Claim 27 (depends on 25)

The method of claim 25, wherein IL-2 is present at a concentration of 200 U/mL.

### Claim 28 (depends on 14)

The method of claim 14, wherein introducing comprises transduction.

### Claim 29 (depends on 14)

The method of claim 14, wherein the CAR and/or TCR expression construct is a lentiviral vector or retroviral vector.

### Claim 30 (depends on 14)

The method of claim 14, wherein the NK cells are GMP-compliant.

### Claim 31 (depends on 14)

The method of claim 14, wherein the NK cells are allogeneic.

### Claim 32 (depends on 14)

The method of claim 14, wherein the NK cells are autologous.

### Claim 33 (depends on 14)

The method of claim 14, wherein the CAR and/or TCR has antigenic specificity for CD19, CD319/CS1, ROR1, CD20, CD5, CD7, CD22, CD70, CD30, BCMA, CD25, NKG2D ligands, MICA/MICB, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, EGFRvIII, TRAIL/DR4, and/or VEGFR2.

### Claim 34 (depends on 14)

The method of claim 14, wherein the CAR has antigenic specificity for CD19.

### Claim 35 (depends on 14)

The method of claim 14, wherein the CAR and/or TCR expression construct further expresses a cytokine.

### Claim 36 (depends on 35)

The method of claim 35, wherein the cytokine is IL-15, IL-21, or IL-12.

### Claim 37 (depends on 14)

The method of claim 14, further comprising cryopreserving the population of expanded NK cells.

### Claim 38 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

A pharmaceutical composition comprising a population of NK cells of any one of claims 1-11 or NK cells produced by the method of any one of claims 14-37 and a pharmaceutically acceptable carrier.

### Claim 39 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

A composition comprising an effective amount of NK cells of any one of claims 1-11 or NK cells produced by the method of any one of claims 14-37 for use in the treatment of a disease or disorder in a subject.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The use of a composition comprising an effective amount of NK cells of any one of claims 1-11 or NK cells produced by the method of any one of claims 14-37 for the treatment of an immune-related disorder in a subject.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

A method of treating an immune-related disorder in a subject comprising administering an effective amount of NK cells of any one of claims 1-11 or NK cells produced by the method of any one of claims 14-37 to the subject.

### Claim 42 (depends on 41)

The method of claim 41, wherein the immune-related disorder is a cancer, autoimmune disorder, graft versus host disease, allograft rejection, or inflammatory condition.

### Claim 43 (depends on 41)

The method of claim 41, wherein the immune-related disorder is an inflammatory condition and the immune cells have essentially no expression of glucocorticoid receptor.

### Claim 44 (depends on 43)

The method of claim 43, wherein the subject has been or is being administered a steroid therapy.

### Claim 45 (depends on 41)

The method of claim 41, wherein the NK cells are autologous.

### Claim 46 (depends on 41)

The method of claim 41, wherein the NK cells are allogeneic.

### Claim 47 (depends on 41)

The method of claim 41, wherein the immune-related disorder is a cancer.

### Claim 48 (depends on 47)

The method of claim 47, wherein the cancer is a solid cancer or a hematologic malignancy.

### Claim 49 (depends on 41)

The method of claim 41, further comprising administering at least a second therapeutic agent.

### Claim 50 (depends on 49)

The method of claim 49, wherein the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, or biotherapy.

### Claim 51 (depends on 49)

The method of claim 49, wherein the NK cells and/or the at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.

### Claim 52 (depends on 49)

The method of claim 49, wherein the NK cells with essentially no expression of CISH have enhanced function as compared to NK cells with expression of CISH.

### Claim 53 (depends on 52)

The method of claim 52, wherein the enhanced function is measured by intracellular staining for IFN-γ and TNF-α, CD107a degranulation, and tumor killing by 51Cr release assay.

### Claim 54 (depends on 52)

The method of claim 52, wherein the enhanced function is measured by increased expression of granzyme-b, perforin, TRAIL, CD3z, Eomes, T-bet, DAP12, DNAM, CD25 and/or Ki67.