# US 10,765,701 B2 · Elimination of CD19-positive lymphoid malignancies by CD19-CAR expressing NK cells

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- Number: US 10,765,701 B2
- Document ID: US10765701B2
- Status: Granted
- Kind: B2
- Application number: 16/529,251
- Filed: 2019-08-01
- Published: 2020-04-30
- Granted: 2020-09-08
- Earliest claimed: 2018-10-31
- Assignees: NantKwest, Inc.
- Applicants: Nantkwest, Inc.
- Inventors: Hans G. Klingemann; Laurent H. Boissel; Patrick Soon-Shiong
- Web page: https://patentgrep.com/patent/US10765701B2

## CPC

- C07K 14/70535: {Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4211: {CD19 or B4}
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- C07K 14/5443: {IL-15}
- C07K 14/55: IL-2
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- 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 2039/54: {characterised by the route of administration}
- A61K 2039/804: {Blood cells [leukemia, lymphoma]}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2510/00: Genetically modified cells
- 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 compositions of NK-92® cells that express a CD19 CAR, CD16 and IL2, and the method of using these cells to and treat cancer in a patient.

## Claims

### Claim 1

A recombinant isolated natural killer (NK)-92 cell expressing an anti-cluster of differentiation 19 (CD19) chimeric antigen receptor (CAR) and a fragment crystallizable (Fc) receptor, wherein the anti-CD19 CAR has the amino acid sequence of SEQ ID NO:12, wherein the NK-92 cell has an accession number selected from the group consisting of CRL-2407, PTA 6670, CRL-2408, CRL-2409, PTA-6967, PTA-8837 and PTA-8836.

### Claim 2 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein the NK-92 cell comprises a multi-cistronic construct and wherein the multi-cistronic construct encodes the anti-CD19 CAR and the Fc receptor, and wherein the anti-CD19 CAR has an single-chain variable fragment (scFv) ectodomain that is encoded by SEQ ID NO:9.

### Claim 3 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein the Fc receptor is a cluster of differentiation 16 (CD16).

### Claim 4 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein the Fc receptor comprises SEQ ID NO: 2.

### Claim 5 (depends on 2)

The recombinant isolated NK-92 cell of claim 2, wherein the multi-cistronic transgene further comprises a sequence that encodes an interleukin-2 (IL-2) or a variant thereof.

### Claim 6 (depends on 5)

The recombinant isolated NK-92 cell of claim 5, wherein the IL-2 variant is endoplasmic reticulum-directed (er)IL-2.

### Claim 7 (depends on 6)

The recombinant isolated NK-92 cell of claim 6, wherein the coding sequences for one or more of the anti-CD19 CAR, the Fc receptor, or erIL-2 or erIL-15 are codon-optimized for expression in a human system.

### Claim 8 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein NK-92 cell is capable of killing a CD19-expressing cell.

### Claim 9 (depends on 8)

The recombinant isolated NK-92 cell of claim 8, wherein the CD19-expressing cell is a tumor cell.

### Claim 10 (depends on 9)

The recombinant isolated NK-92 cell of claim 9, wherein the tumor cell is a SUP-B15 cell.

### Claim 11 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein the NK-92 cell is transfected with a nucleic acid having a sequence of SEQ ID NO:13 that encodes the anti-CD19 CAR and the Fc receptor.

### Claim 12 (depends on 2)

The recombinant isolated NK-92 cell of claim 2, wherein the multi-cistronic transgene further comprises a sequence that encodes an interleukin-15 (IL-15) or a variant thereof.

### Claim 13 (depends on 12)

The recombinant isolated NK-92 cell of claim 12, wherein the variant thereof is erIL-15.

### Claim 14 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein the NK-92 cell comprises a sequence encoding a self-cleaving peptide, wherein the sequence is located between the anti-CD19 CAR and the Fc receptor, and wherein the sequence allows equimolar expression of the anti-CD19 CAR and the Fc receptor.

### Claim 15 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein the NK-92 cell comprises an internal ribsosomal entry sequence (IRES) between a sequence encoding the Fc receptor and the sequence encoding IL-2 or a variant thereof.

### Claim 16 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein a direct cytotoxicity of the NK-92 cell on CD19-expressing cells is 70-100% when the effector to target ratio is 10.

### Claim 17 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein an antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the NK-92 cell is at 30%-90% when the effector to target ratio is 10.

### Claim 18 (depends on 1)

The recombinant isolated NK-92 cell of claim 1, wherein the anti-CD19 CAR comprises a cytoplasmic signaling domain.

### Claim 19 (depends on 18)

The recombinant isolated NK-92 cell of claim 18, wherein the cytoplasmic signaling domain comprises at last one of Fc epsilon receptor gamma (FcεRIγ) and a cluster of differentiation 3 (CD3) zeta signaling domain.

### Claim 20 (depends on 1)

A method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition to the subject, the composition comprising a plurality of recombinant isolated NK-92 cells according to claim 1, and where the cancer has cancer cells expressing CD19.

### Claim 21 (depends on 20)

The method of claim 20, wherein about 1×108 to about 1×1011 modified cells per m2 of body surface area of the subject are administered to the subject.

### Claim 22 (depends on 20)

The method of claim 20, wherein the cancer is a leukemia or a lymphoma.

### Claim 23 (depends on 20)

The method of claim 20, wherein the cancer is one or more of B-cell malignancy, B-cell malignancy post-HSCT, CLL, B-ALL, acute lymphoblastic leukemia (ALL), B-lineage lymphoid malignancy post-UCBT, chronic lymphocytic leukemia (CLL), B-Non-Hodgkin's Lymphoma (B-NHL), ALL post-HSCT; lymphoma, refractory follicular lymphoma, or Lymphoblastic leukemia.

### Claim 24 (depends on 23)

The method of claim 23, wherein the B-cell malignancy is a Mantle cell lymphoma.

### Claim 25 (depends on 20)

The method of claim 20, wherein the plurality of the NK-92 cells are administered intravenously and/or intratumorally.