# US 10,765,699 B2 · Methods for enhancing efficacy of therapeutic immune cells

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- Number: US 10,765,699 B2
- Document ID: US10765699B2
- Status: Granted
- Kind: B2
- Application number: 15/548,577
- Filed: 2016-02-05
- Published: 2018-01-11
- Granted: 2020-09-08
- Earliest claimed: 2015-02-06
- Assignees: National University of Singapore
- Applicants: National University of Singapore
- Inventors: Dario Campana; Takahiro Kamiya
- Web page: https://patentgrep.com/patent/US10765699B2

## CPC

- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- 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}
- A61K 40/4224: {Molecules with a "CD" designation not provided for elsewhere}
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/2833: {against MHC-molecules, e.g. HLA-molecules}
- C07K 16/289: {against CD45}
- C12N 5/0636: {T lymphocytes}
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2319/03: containing a transmembrane segment
- 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

The present invention relates to a method of using a receptor (e.g., chimeric antigen receptor—CAR) that activates an immune response upon binding a cancer cell ligand in conjunction with a target-binding molecule that targets a protein or molecule CI for removal or neutralization to generate enhanced anti-cancer immune cells. The present invention also relates to engineered immune cells having enhanced therapeutic efficacy and uses thereof.

## Claims

### Claim 1

An engineered NK cell or T cell comprising: a first nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a second nucleic acid comprising a nucleotide sequence encoding a single-chain variable fragment (scFv) linked to a localizing domain,
wherein the scFv binds a target expressed by the cell and selected from the group consisting of killer cell immunoglobulin-like receptors 2DL1 (KIR2DL1) and 2DL2/DL3 (KIR2DL2/DL3), and NKG2A,
wherein the scFv that binds KIR2DL1 and KIR2DL2/DL3 comprises a variable heavy chain of SEQ ID NO:36 and a variable light chain of SEQ ID NO:37, and the scFv that binds NKG2A comprises a variable heavy chain of SEQ ID NO:32 and a variable light chain of SEQ ID NO:33,
wherein the localization domain comprise an endoplasmic reticulum (ER) or Golgi retention sequence comprising an amino acid sequence selected from the group consisting of KKMP, EEKKMP, and AEKDEL, and
wherein the scFv linked to the localizing domain is expressed by the cell, retained within the cell, and downregulates or suppresses surface expression of the target in the engineered cell rendering the target inactive.

### Claim 2 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the CAR is an anti-CD19-4-1BB-CD3ζ CAR.

### Claim 3

An engineered NK cell or T cell comprising: a chimeric antigen receptor (CAR) comprising an anti-CD19-4-1BB-CD3ζ CAR, and an anti-NKG2A scFv linked to a localizing domain comprising an ER or Golgi retention sequence comprising EEKKMP,
wherein the scFv comprises a variable heavy chain comprising SEQ ID NO:32 and a variable light chain comprising SEQ ID NO:33, and the scFv linked to the localizing domain is expressed by the cell, retained within the cell, and downregulates or suppresses surface expression of NKG2A in the engineered cell rendering NKG2A inactive.

### Claim 4

An engineered NK cell or T cell comprising: a chimeric antigen receptor (CAR) comprising an anti-CD19-4-1BB-CD3ζ CAR, and an anti-KIR2DL1 and KIR2DL2/DL3 scFv linked to a localizing domain comprising an ER or Golgi retention sequence comprising EEKKMP or AEKDEL,
wherein the scFv comprises a variable heavy chain comprising SEQ ID NO:36 and a variable light chain comprising SEQ ID NO:37, and the scFv linked to the localizing domain is expressed by the cell, retained within the cell, and downregulates or suppresses surface expression of KIR2DL1 and KIR2DL2/DL3 in the engineered cell rendering KIR2DL1 and KIR2DL2/DL3 inactive.

### Claim 5 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the scFv binds NKG2A.

### Claim 6 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the scFv binds KIR2DL1 and KIR2DL2/DL3.

### Claim 7 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the ER or Golgi retention sequence comprises EEKKMP.

### Claim 8 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the ER or Golgi retention sequence comprises AEKDEL.

### Claim 9 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the scFv binds NKG2A and the ER or Golgi retention sequence comprises EEKKMP.

### Claim 10 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the scFv binds NKG2A and the ER or Golgi retention sequence comprises AEKDEL.

### Claim 11 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the scFv binds KIR2DL1 and KIR2DL2/DL3 and the ER or Golgi retention sequence comprises EEKKMP.

### Claim 12 (depends on 1)

The engineered NK cell or T cell of claim 1, wherein the scFv binds KIR2DL1 and KIR2DL2/DL3 and the ER or Golgi retention sequence comprises AEKDEL.