# US 12,595,290 B2 · T cell receptors specific for mesothelin and their use in immunotherapy

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- Number: US 12,595,290 B2
- Document ID: US12595290B2
- Status: Granted
- Kind: B2
- Application number: 17/291,985
- Filed: 2019-11-08
- Published: 2022-01-13
- Granted: 2026-04-07
- Earliest claimed: 2018-11-09
- Assignees: Fred Hutchinson Cancer Center; Juno Therapeutics, Inc.
- Applicants: Fred Hutchinson Cancer Center; Juno Therapeutics, Inc.
- Inventors: Thomas M. Schmitt; Aude G. Chapuis; Philip D. Greenberg
- Web page: https://patentgrep.com/patent/US12595290B2

## CPC

- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/32: T-cell receptors [TCR]
- A61K 40/4255: {Mesothelin [MSLN]}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- C12N 5/0636: {T lymphocytes}
- C12N 15/86: Viral vectors
- A61K 2239/49: Breast
- C07K 2319/00: Fusion polypeptide
- 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

Binding proteins specific for Msln20-28 or Msln530-538 peptides are provided herein. Polynucleotides encoding the binding proteins, as well as compositions and recombinant host cells comprising the binding proteins or polynucleotides are also provided. The compositions and recombinant host cells may be used to treat a subject having mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, a cancer wherein an Msln20-28 peptide is expressed on a tumor cell of the cancer, or a cancer wherein an Msln530-538 peptide is expressed on a tumor cell of the cancer.

## Claims

### Claim 1

A binding protein comprising a T cell receptor (TCR) α-chain variable domain (Vα) and a TCR β-chain variable domain (Vβ), wherein:
(a) the Vα comprises the complementarity determining region (CDR)1α, CDR2α, and CDR3α amino acid sequences set forth in SEQ ID NOs: 93, 94, and 39, respectively and the Vβ comprises the CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 91, 92, and 40, respectively; or
(b) the Vα comprises the CDR1α, CDR2α, and CDR3α amino acid sequences set forth in SEQ ID NOs: 89, 90, and 37, respectively and the Vβ comprises the CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 87, 88, and 38, respectively
wherein the binding protein is capable of specifically binding to a SEQ ID NO:32:HLA-A: 0201 complex,
wherein the binding protein comprises:
(i) a TCR α-chain constant domain and a TCR β-chain constant domain, wherein the TCR α-chain constant domain, the TCR β-chain constant domain, or both comprise a non-native cysteine residue; and/or
(ii) a single chain TCR (scTCR) or a CAR.

### Claim 2 (depends on 1)

The binding protein of claim 1, comprising:
(a) the CDR1α amino acid sequence set forth in SEQ ID NO: 93;
(b) the CDR2α amino acid sequence set forth in SEQ ID NO: 94;
(c) the CDR3α amino acid sequence set forth in SEQ ID NO: 39;
(d) the CDR1β amino acid sequence set forth in SEQ ID NO: 91;
(e) the CDR2β amino acid sequence set forth in SEQ ID NO: 92; and
(f) the CDR3β amino acid sequence set forth in SEQ ID NO: 40.

### Claim 3 (depends on 2)

The binding protein of claim 2, wherein the Vα comprises an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 102, and wherein the Vβ comprises an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 101.

### Claim 4 (depends on 2)

The binding protein of claim 2, comprising a TCR α chain (TCRα) and a TCR β chain (TCRβ), wherein the TCRα comprises or consists of an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 110 or 29, and/or wherein the TCRβ comprises or consists of an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 109 or 28.

### Claim 5 (depends on 1)

The binding protein of claim 1, comprising:
(a) the CDR1α amino acid sequence set forth in SEQ ID NO: 89;
(b) the CDR2α amino acid sequence set forth in SEQ ID NO: 90;
(c) the CDR3α amino acid sequence set forth in SEQ ID NO: 37;
(d) the CDR1β amino acid sequence as set forth in SEQ ID NO: 87;
(e) the CDR2β amino acid sequence set forth in SEQ ID NO: 88; and
(f) the CDR3β amino acid sequence set forth in SEQ ID NO: 38.

### Claim 6 (depends on 5)

The binding protein of claim 5, wherein the Vα comprises an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO:100 and the Vβ comprises an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO:99.

### Claim 7 (depends on 5)

The binding protein of claim 5, comprising a TCR α chain (TCRα) and a TCR β chain (TCRβ), wherein the TCRα comprises or consists of an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 108 or 23, and/or wherein the TCRβ comprises or consists of an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 107 or 22.

### Claim 8 (depends on 1)

The binding protein of claim 1, wherein:
the Vα comprises or consists of the amino acid sequence set forth in SEQ ID NO: 102 and the Vβ comprises or consists of the amino acid sequence set forth in SEQ ID NO: 101; or
the Vα comprises or consists of the amino acid sequence set forth in SEQ ID NO: 100 and the Vβ comprises or consists of the amino acid sequence set forth in SEQ ID NO: 99.

### Claim 9 (depends on 1)

The binding protein of claim 1, wherein the binding protein comprises a TCR Cβ and a TCR Cα, wherein the TCR Cβ comprises a cysteine amino acid in place of a native serine at amino acid position 57 and the TCR Cα comprises a cysteine amino acid in place of a native threonine at amino acid position 48.

### Claim 10

A binding protein comprising a T cell receptor (TCR) α-chain variable domain (Vα) and a TCR β-chain variable domain (Vβ), wherein:
(a) the Vα comprises the CDR1α amino acid sequence set forth in SEQ ID NO: 80, the CDR2α amino acid sequence set forth in SEQ ID NO: 81 or 118, and CDR3α amino acid sequence set forth in SEQ ID NO: 33 and the Vβ comprises the CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 78, 79, and 34, respectively; or
(b) the Vα comprises the CDR1α amino acid sequence set forth in SEQ ID NO: 85, the CDR2α amino acid sequence set forth in SEQ ID NO: 86 or 119, and CDR3α amino acid sequence set forth in SEQ ID NO: 35 and the Vβ comprises the CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 82, 79, and 36, respectively,
wherein the binding protein is capable of specifically binding to a SEQ ID NO:31:HLA-A: 0201 complex,
wherein the binding protein comprises:
(i) a TCR α-chain constant domain and a TCR β-chain constant domain, wherein the TCR α-chain constant domain, the TCR β-chain constant domain, or both comprise a non-native cysteine residue; and/or
(ii) a scTCR or a CAR.

### Claim 11 (depends on 10)

The binding protein of claim 10, comprising:
(a) the CDR1α amino acid sequence set forth in SEQ ID NO: 80;
(b) the CDR2α amino acid sequence set forth in SEQ ID NO: 81 or 118;
(c) the CDR3α amino acid sequence set forth in SEQ ID NO: 33;
(d) the CDR1β amino acid sequence as set forth in SEQ ID NO: 78;
(e) the CDR2β amino acid sequence set forth in SEQ ID NO: 79; and
(f) the CDR3β amino acid sequence set forth in SEQ ID NO: 34.

### Claim 12 (depends on 11)

The binding protein of claim 11, wherein the Vα comprises an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 96, and wherein the Vβ comprises an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 95.

### Claim 13 (depends on 11)

The binding protein of claim 11, comprising a TCR α chain (TCRα) and a TCR β chain (TCRβ), wherein the TCRα comprises or consists of an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 104 or 7, and/or wherein the TCRβ comprises or consists of an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 103 or 6; or (ii) wherein the TCRα comprises or consists of an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 106 or 15, and/or wherein the TCR β comprises or consists of an amino acid sequence having at least about 85% identity to the amino acid sequence set forth in SEQ ID NO: 105 or 14.

### Claim 14 (depends on 10)

The binding protein of claim 10, comprising:
(a) the CDR1α amino acid sequence set forth in SEQ ID NO: 85;
(b) the CDR2α amino acid sequence set forth in SEQ ID NO: 86 or 119;
(c) the CDR3α amino acid sequence set forth in SEQ ID NO: 35;
(d) the CDR1β amino acid sequence set forth in SEQ ID NO: 82;
(e) the CDR2β amino acid sequence set forth in SEQ ID NO: 79; and
(f) the CDR3β amino acid sequence set forth in SEQ ID NO: 36.

### Claim 15 (depends on 10)

The binding protein of claim 10, wherein:
the Vα comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96 and the Vβ comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95; or
the Vα comprises or consists of the amino acid sequence set forth in SEQ ID NO: 98 and the Vβ comprises or consists of the amino acid sequence set forth in SEQ ID NO: 97.

### Claim 16 (depends on 1)

A polynucleotide encoding the binding protein of claim 1.

### Claim 17 (depends on 10)

A polynucleotide encoding the binding protein of claim 10.

### Claim 18 (depends on 16)

A recombinant expression vector comprising the polynucleotide of claim 16 operably linked to an expression control sequence.

### Claim 19 (depends on 17)

A recombinant expression vector comprising the polynucleotide of claim 17 operably linked to an expression control sequence.

### Claim 20 (depends on 16)

A recombinant host cell comprising a heterologous polynucleotide of claim 16, wherein the recombinant host cell is capable of expressing on its cell surface the encoded binding protein.

### Claim 21 (depends on 17)

A recombinant host cell comprising a heterologous polynucleotide of claim 17, wherein the recombinant host cell is capable of expressing on its cell surface the encoded binding protein.

### Claim 22 (depends on 20)

A method of treating a disease or disorder associated with mesothelin expression and/or activity in a subject, the method comprising:
administering to the subject an effective amount of a composition comprising (a) the recombinant host cell of claim 20 and (b) a pharmaceutically acceptable carrier, excipient, or diluent.

### Claim 23 (depends on 22)

The method of claim 22, wherein the disease or disorder associated with mesothelin expression and/or activity is a cancer.

### Claim 24 (depends on 23)

The method of claim 23, wherein the cancer is a solid cancer or a hematological malignancy.

### Claim 25 (depends on 23)

The method of claim 23, wherein the cancer comprises biliary cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal cancer, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, hepatic cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ-cell tumor, urothelial cancer, uterine sarcoma, or uterine cancer.

### Claim 26 (depends on 21)

A method of treating a disease or disorder associated with mesothelin expression and/or activity in a subject, the method comprising:
administering to the subject an effective amount of a composition comprising (a) the recombinant host cell of claim 21 and (b) a pharmaceutically acceptable carrier, excipient, or diluent.

### Claim 27 (depends on 26)

The method of claim 26, wherein the disease or disorder associated with mesothelin expression and/or activity is a cancer.

### Claim 28 (depends on 27)

The method of claim 27, wherein the cancer is a solid cancer or a hematological malignancy.

### Claim 29 (depends on 27)

The method of claim 27, wherein the cancer comprises biliary cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal cancer, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, hepatic cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, oVαrian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ-cell tumor, urothelial cancer, uterine sarcoma, or uterine cancer.

### Claim 30

An isolated polynucleotide that encodes a binding protein that is capable of specifically binding to a SEQ ID NO:32:HLA-A: 02*01 complex, wherein the polynucleotide comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 120.

### Claim 31 (depends on 30)

An expression vector comprising the polynucleotide of claim 30 operably linked to an expression control sequence.

### Claim 32 (depends on 30)

A recombinant host cell comprising the polynucleotide of claim 30, wherein the recombinant host cell is capable of expressing on its cell surface the encoded binding protein.

### Claim 33 (depends on 20)

A method of making the recombinant host cell of claim 20, the method comprising transfecting or transducing the host cell in vitro or ex vivo with the polynucleotide encoding the binding protein.

### Claim 34 (depends on 21)

A method of making the recombinant host cell of claim 21, the method comprising transfecting or transducing the host cell in vitro or ex vivo with the polynucleotide encoding the binding protein.

### Claim 35

A T cell receptor (TCR) comprising an alpha-chain (TCRα) and a beta-chain (TCRβ), wherein the TCRα and the TCRβ comprise the amino acid sequences set forth in SEQ ID NOs:
(i) 110 and 109, respectively;
(ii) 108 and 107, respectively;
(iii) 104 and 103, respectively; or
(v) 106 and 105, respectively.

### Claim 36

A polynucleotide encoding a T cell receptor (TCR),
wherein the encoded TCR comprises an alpha-chain (TCRα) and a beta-chain (TCRβ), wherein the TCRα and the TCRβ comprise the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in:
(i) 93, 94, 39, 91, 92, and 40, respectively, wherein the binding protein is capable of specifically binding to a SEQ ID NO:32:HLA-A: 0201 complex;
(ii) 89, 90, 37, 87, 88, and 38, respectively, wherein the binding protein is capable of specifically binding to a SEQ ID NO:32:HLA-A: 0201 complex;
(iii) 80, 81 or 118, 33, 78, 79, and 34, respectively, wherein the binding protein is capable of specifically binding to a SEQ ID NO:31:HLA-A: 0201 complex; or
(iv) 85, 86 or 119, 35, 82, 79, and 36, respectively, wherein the binding protein is capable of specifically binding to a SEQ ID NO:31:HLA-A: 0201 complex;
and wherein:
(1) the polynucleotide comprises a sequence encoding a viral 2A peptide, an internal ribosome entry site (IRES), a furin cleavage site, or any combination thereof, disposed between a sequence encoding the TCRβ and a sequence encoding the TCRα; and/or
(2) the polynucleotide further encodes a CD8 co-receptor α-chain and a CD8 co-receptor β-chain; and/or
(3) the polynucleotide is comprised in a viral vector.

### Claim 37 (depends on 36)

The polynucleotide of claim 36, wherein:
(a) the TCRα and the TCRβ comprise the Vα and Vβ amino acid sequences set forth in SEQ ID NOs: 102 and 101, respectively; 100 and 99, respectively; 96 and 95, respectively; or 98 and 97, respectively; and/or
(b) the polynucleotide comprises a sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 113-117, disposed between a sequence encoding the TCRβ and a sequence encoding the TCRα; and/or
(c) the polynucleotide encodes the amino acid sequence set forth in any one of SEQ ID NOs: 8, 16, 24, and 30.

### Claim 38

A method of treating a cancer associated with mesothelin expression and/or activity in a subject, the method comprising administering to the subject an effective amount of T cells comprising CD8+ T cells, wherein
(1) cells of the cancer express a SEQ ID NO:32:HLA-A: 0201 complex, and the T cells express an αβ T cell receptor (TCR) comprising the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 93, 94, 39, 91, 92, and 40, respectively, or set forth in SEQ ID NOs: 89, 90, 37, 87, 88, and 38, respectively; or
(2) cells of the cancer express a SEQ ID NO:31:HLA-A: 0201 complex, and the T cells express an αβ T cell receptor (TCR) comprising the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in 80, 81 or 118, 33, 78, 79, and 34, respectively, or set forth in SEQ ID NOs: 85, 86 or 119, 35, 82, 79, and 36, respectively.