# US 12,215,134 B2 · Engineered immune cells with dominant signals

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- Number: US 12,215,134 B2
- Document ID: US12215134B2
- Status: Granted
- Kind: B2
- Application number: 17/480,473
- Filed: 2021-09-21
- Published: 2022-03-24
- Granted: 2025-02-04
- Earliest claimed: 2020-09-21
- Assignees: A2 Biotherapeutics, Inc.
- Applicants: A2 Biotherapeutics, Inc.
- Inventors: Alexander Kamb
- Web page: https://patentgrep.com/patent/US12215134B2

## CPC

- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- 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 40/32: T-cell receptors [TCR]
- A61K 40/4204: {Epidermal growth factor receptors [EGFR]}
- A61K 40/4268: {MAGE}
- A61K 40/4269: {NY-ESO}
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 14/70517: {CD8}
- C12N 5/0637: {Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg}
- C12N 15/85: for animal cells
- C07K 2317/53: Hinge
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/715: containing a domain for ligand dependent transcriptional activation, e.g. containing a steroid receptor domain
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure provides engineered immune cells and methods for their creation and use. The immune cells comprise activating and blocking receptors, in which the blocking receptor provides a signal that dominates a signal from the activating receptor.

## Claims

### Claim 1

A method for reducing tumor size or increasing survival time of a patient with a cancer, the method comprising:
providing an engineered immune cell to the patient, wherein the engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell, said blocking receptor comprising a hinge that comprises a peptide, wherein said peptide comprises a continuous stretch of at least 24 amino acids of SEQ ID NO: 84 derived from a leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), wherein:
when the engineered immune cell encounters a tumor cell, the activating receptor binds to a first ligand on the tumor cell and the activating receptor triggers an activating signal in the engineered immune cell that promotes a cytotoxic response by the engineered immune cell that results in a cytotoxic effect on the tumor cell; and
when the engineered immune cell encounters a normal cell, the activating receptor binds to the first ligand on the normal cell and the blocking receptor binds to a second ligand on the normal cell, wherein the activating receptor triggers the activating signal in the engineered immune cell and the blocking receptor triggers an inhibitory signal in the engineered immune cell that is stronger than the activating signal such that the inhibitory signal dominates and blocks the activating signal from the activating receptor, thereby preventing a localized cytotoxic response by the engineered immune cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein binding of the blocking receptor to the second ligand causes the engineered immune cell to exhibit reduced surface expression of the activating receptor.

### Claim 3 (depends on 2)

The method of claim 2, wherein the reduced surface expression is reversible.

### Claim 4 (depends on 1)

The method of claim 1, wherein the immune cell expresses different concentrations of the activating and blocking receptors based on a quantity of the first ligand and a quantity of the second ligand expressed in the normal cell of the patient.

### Claim 5 (depends on 4)

The method of claim 4, wherein a ratio of the concentration of the blocking receptors expressed to the activating receptors expressed is less than or equal to 1.

### Claim 6 (depends on 1)

The method of claim 1, wherein when the immune cell encounters the tumor cell, a first plurality of the activating receptors diffuses into a first region on the surface of the immune cell proximal to the tumor cell and forms a first micro-cluster that promotes the cytotoxic response by the immune cell that results in the cytotoxic effect on the tumor cell.

### Claim 7 (depends on 6)

The method of claim 6, wherein when the immune cell simultaneously encounters the normal cell, a plurality of the activating and blocking receptors diffuses into a second region on the surface of the immune cell proximal to the normal cell and forms a second micro-cluster causing the inhibitory signal from the blocking receptors to dominate the activating signal from the activating receptors in the second micro-cluster preventing the localized cytotoxic response by the engineered immune cell on the normal cell.

### Claim 8 (depends on 7)

The method of claim 7, wherein binding of the blocking receptors in the second micro-cluster to the second ligand prevents breakup of the second micro-cluster.

### Claim 9 (depends on 1)

The method of claim 1, wherein the blocking receptor does not bind to the second ligand until the activating receptor binds to the first ligand.

### Claim 10 (depends on 1)

The method of claim 1, wherein cross-talk between the activating receptor and the blocking receptor affects an activation threshold for the localized cytotoxic response.