# US 12,600,793 B2 · Chimeric antigen receptors with MAGE-A4 specificity and uses thereof

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- Number: US 12,600,793 B2
- Document ID: US12600793B2
- Status: Granted
- Kind: B2
- Application number: 17/629,562
- Filed: 2020-07-24
- Published: 2022-09-08
- Granted: 2026-04-14
- Earliest claimed: 2019-07-24
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Kevin Bray; Frank Delfino; David DiLillo; Thomas Craig Meagher
- Web page: https://patentgrep.com/patent/US12600793B2

## CPC

- C07K 16/30: from tumour cells
- 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 40/4268: {MAGE}
- A61P 35/00: Antineoplastic agents
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/2833: {against MHC-molecules, e.g. HLA-molecules}
- C12N 5/0636: {T lymphocytes}
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/876: {Skin, melanoma}
- A61K 2239/13: Antibody-based
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/46: characterised by the cancer treated
- A61K 2239/57: Skin; melanoma
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2317/55: Fab or Fab'
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2319/02: containing a signal sequence
- 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 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

MAGE-A4, or Melanoma-Associated Antigen A4, is a cancer-testis antigen (CTA) on the X chromosome. The present disclosure provides MAGE-A4-specific chimeric antigen receptors and cells expressing such chimeric antigen receptors. In certain embodiments, engineered cells expressing the chimeric antigen receptors of the present disclosure are capable of inhibiting the growth of tumors expressing MAGE-A4. The engineered cells of the present disclosure are useful for the treatment of diseases and disorders in which an upregulated or induced MAGE-A4-targeted immune response is desired and/or therapeutically beneficial. For example, engineered cells expressing the MAGE-A4-specific chimeric antigen receptors of the present disclosure are useful for the treatment of various cancers.

## Claims

### Claim 1

A Melanoma-Associated Antigen A4 (MAGE-A4)-specific chimeric antigen receptor comprising, from N-terminus to C-terminus:
(a) an extracellular ligand-binding domain comprising an anti-MAGE-A4 antigen-binding domain;
(b) a hinge;
(c) a transmembrane domain; and
(d) a cytoplasmic domain comprising a costimulatory domain, and a signaling domain;
wherein the extracellular ligand-binding domain is an anti-MAGE-A4 single chain variable fragment (scFv) domain comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) joined by a linker,
wherein:
(A) the LCVR comprises LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 12, 14, and 16, respectively, and the HCVR comprises HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively, or
(B) the LCVR comprises LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 61, 63, and 65, respectively, and the HCVR comprises HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 53, 55, and 57, respectively.

### Claim 2 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 12, 14, and 16, respectively, and the HCVR comprises HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 4, 6, and 8, respectively.

### Claim 3 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 61, 63, and 65, respectively, and the HCVR comprises HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 53, 55, and 57, respectively.

### Claim 4 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the hinge and the transmembrane domain are from a CD28 polypeptide, the costimulatory domain comprises a CD28 costimulatory domain, and the signaling domain comprises a CD3zeta signaling domain.

### Claim 5 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the hinge and the transmembrane domain are from a CD8α polypeptide, the costimulatory domain comprises a 4-1BB costimulatory domain, and the signaling domain comprises a CD3zeta signaling domain.

### Claim 6 (depends on 2)

The chimeric antigen receptor of claim 2, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 37 and the HCVR comprises the amino acid sequence of SEQ ID NO: 2.

### Claim 7 (depends on 3)

The chimeric antigen receptor of claim 3, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 59 and the HCVR comprises the amino acid sequence of SEQ ID NO: 51.

### Claim 8 (depends on 1)

The chimeric antigen receptor of claim 1, further comprising a peptide linker between the extracellular ligand-binding domain and the hinge.

### Claim 9 (depends on 1)

The chimeric antigen receptor of claim 1, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26.

### Claim 10 (depends on 1)

The chimeric antigen receptor of claim 1, wherein:
(a) the hinge comprises the amino acid sequence of SEQ ID NO: 41;
(b) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 43;
(c) the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 45; and/or
(d) the signaling domain comprises the amino acid sequence of SEQ ID NO: 30.

### Claim 11 (depends on 2)

The chimeric antigen receptor of claim 2, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 47.

### Claim 12 (depends on 3)

The chimeric antigen receptor of claim 3, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 73.

### Claim 13 (depends on 1)

An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

### Claim 14 (depends on 11)

An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 11, wherein the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 48.

### Claim 15 (depends on 12)

An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 12, wherein the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 72.

### Claim 16 (depends on 13)

A vector comprising the nucleic acid molecule of claim 13.

### Claim 17 (depends on 16)

The vector of claim 16, wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenovirus vector, or a retroviral vector.

### Claim 18 (depends on 16)

A cell comprising the vector of claim 16.

### Claim 19 (depends on 1)

An engineered cell comprising a chimeric antigen receptor of claim 1.

### Claim 20 (depends on 19)

The engineered cell of claim 19, wherein the engineered cell is a T lymphocyte.

### Claim 21 (depends on 20)

The engineered cell of claim 20, wherein the T lymphocyte is an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte.

### Claim 22 (depends on 21)

The engineered cell of claim 21 that is a CD8+ cytotoxic T lymphocyte.

### Claim 23 (depends on 1)

A population of engineered cells, which is obtained by:
(a) providing a population of immune cells, which have been obtained from a subject;
(b) introducing into the immune cells a nucleic acid molecule encoding a chimeric antigen receptor of claim 1;
(c) culturing the immune cells under conditions to express the nucleic acid molecules; and
(d) isolating the immune cells expressing the chimeric antigen receptor at the cells' surface.

### Claim 24 (depends on 1)

A pharmaceutical composition comprising a genetically-modified human T cell and a pharmaceutically acceptable carrier, wherein the genetically-modified human T cell comprises a chimeric antigen receptor of claim 1.

### Claim 25 (depends on 24)

A method for treating a MAGE-A4-expressing cancer, the method comprising administering the pharmaceutical composition of claim 24 to a subject.

### Claim 26 (depends on 25)

The method of claim 25, wherein the MAGE-A4-expressing cancer is selected from the group consisting of multiple myeloma, synovial sarcoma, esophageal cancer, head and neck cancer, lung cancer, bladder cancer, ovarian cancer, uterine cancer, stomach cancer, cervical cancer, breast cancer, and melanoma.

### Claim 27 (depends on 26)

The method of claim 26, wherein the MAGE-A4-expressing cancer is multiple myeloma.

### Claim 28 (depends on 1)

A method of engineering a population of cells to express a chimeric antigen receptor, comprising:
(a) providing a population of immune cells;
(b) introducing into the immune cells a nucleic acid molecule encoding a chimeric antigen receptor of claim 1;
(c) culturing the immune cells under conditions to express the nucleic acid molecule; and
(d) isolating the immune cells expressing the chimeric antigen receptor at the cells' surface.

### Claim 29 (depends on 28)

The method of claim 28, wherein the population of immune cells in (a) is obtained from a subject.

### Claim 30

An antibody or antigen-binding fragment thereof that specifically binds Melanoma-Associated Antigen A4 (MAGE-A4), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions, HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NOs: 4, 6 and 8, respectively, and a light chain variable region (LCVR) comprising three light chain complementarity determining regions, LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NOs: 12, 14 and 16.

### Claim 31 (depends on 30)

The antibody or antigen-binding fragment thereof of claim 30, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 2, and the LCVR comprises the amino acid sequence of SEQ ID NO: 37.

### Claim 32

An antibody or antigen-binding fragment thereof that specifically binds Melanoma-Associated Antigen A4 (MAGE-A4), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions, HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NOs: 53, 55 and 57, respectively, and a light chain variable region (LCVR) comprising three light chain complementarity determining regions, LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NOs: 61, 63 and 65.

### Claim 33 (depends on 32)

The antibody or antigen-binding fragment thereof of claim 32, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 51, and the LCVR comprises the amino acid sequence of SEQ ID NO: 59.

### Claim 34 (depends on 11)

An engineered cell comprising a chimeric antigen receptor of claim 11.

### Claim 35 (depends on 12)

An engineered cell comprising a chimeric antigen receptor of claim 12.