# US 12,005,082 B2 · Composition and method of use recombinant fusion protein to generate CAR-immune cells

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- Number: US 12,005,082 B2
- Document ID: US12005082B2
- Status: Granted
- Kind: B2
- Application number: 18/185,805
- Filed: 2023-03-17
- Published: 2023-09-21
- Granted: 2024-06-11
- Earliest claimed: 2022-03-17
- Assignees: Singular Immune, Inc.
- Applicants: Singular Immune, Inc.
- Inventors: Youngwook Won; Seungmin Han; David Bull
- Web page: https://patentgrep.com/patent/US12005082B2

## CPC

- 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 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4224: {Molecules with a "CD" designation not provided for elsewhere}
- A61P 35/00: Antineoplastic agents
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- A61K 2239/10: characterized by the structure of the chimeric antigen receptor [CAR]
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/49: Breast
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/50: containing protease site
- 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)
- 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 a recombinant chimeric antigen receptor (CAR) fusion protein, a method of modifying an immune cell into a CAR immune cell by treating the immune cell with the recombinant CAR fusion protein, and a method of treating cancer by administering the CAR immune cell to a subject in need thereof.

## Claims

### Claim 1

A recombinant chimeric antigen receptor (CAR) fusion protein comprising, in order:
an immune cell targeting domain, the immune cell targeting domain being a NK cell targeting domain, or a T cell targeting domain,
a cleavable peptide,
a membrane targeting domain, the membrane targeting domain being a peptide from interleukin-1 receptor type 1, 4F2 cell-surface antigen heavy chain, a linker for activation of T-cells family member 1, junctophilin-1, antilisterial bacteriocin subtilosin biosynthesis protein AlbG, calcitonin receptor, gamma-secretase subunit APH-1A, or adipnectin receptor protein 2,
an extracellular cancer targeting domain,
a transmembrane domain, and
an intracellular signaling domain.

### Claim 2 (depends on 1)

The recombinant CAR fusion protein of claim 1, wherein the NK cell targeting domain is a NK cell-targeting-scFv antibody, or a peptide comprising an anti-NKp46 VH, IgG hinge, IgG CH2 and IgG CH3.

### Claim 3 (depends on 1)

The recombinant CAR fusion protein of claim 1, wherein the cleavable peptide is a peptide which is configured to be enzymatically cleaved.

### Claim 4 (depends on 1)

The recombinant CAR fusion protein of claim 1, wherein the extracellular cancer targeting domain is an anti-PD-L1 Vh.

### Claim 5 (depends on 1)

The recombinant CAR fusion protein of claim 1 further comprising a hinge region between the extracellular cancer targeting domain and the transmembrane domain.

### Claim 6 (depends on 1)

The recombinant CAR fusion protein of claim 1, wherein the transmembrane domain is NKG2D.

### Claim 7 (depends on 1)

The recombinant CAR fusion protein of claim 1, wherein the intracellular signaling domain comprises a CD3 zeta (CD3ξ) signaling domain and a co-stimulatory signaling region selected from the group consisting of 2B4, DAP12, GITR, CD137, OX40.

### Claim 8 (depends on 1)

The recombinant CAR fusion protein of claim 1 further comprising a tag sequence.

### Claim 9 (depends on 8)

The recombinant CAR fusion protein of claim 8, wherein the tag sequence is selected from the group consisting of a histidine tag, glutathione-S-transferase tag, or hemagglutinin tag.

### Claim 10 (depends on 1)

A method of modifying an immune cell into a chimeric antigen receptor (CAR) immune cell, comprising
treating the immune cell with the recombinant CAR fusion protein of claim 1.

### Claim 11 (depends on 10)

The method of claim 10, wherein the recombinant CAR fusion protein is in a concentration of about 1,000 nM to about 2,000 nM.

### Claim 12 (depends on 10)

A method of treating cancer comprising administering the CAR immune cell of claim 10 to a subject in need thereof.

### Claim 13 (depends on 1)

A method of treating cancer comprising administering the recombinant CAR fusion protein of claim 1 to a subject in need thereof.

### Claim 14 (depends on 13)

The method of claim 13, wherein the recombinant CAR fusion protein is in a concentration of about 1,000 nM to about 2,000 nM.

### Claim 15 (depends on 1)

A method of treating cancer comprising administering to a subject in need thereof:
(i) the recombinant chimeric antigen receptor (CAR) fusion protein of claim 1; and
(ii) a CAR immune cell.

### Claim 16 (depends on 15)

The method of claim 15, wherein the CAR immune cell is prepared by treating an immune cell with the recombinant CAR fusion protein.