# US 12,180,296 B2 · Cross species single domain antibodies targeting mesothelin for treating solid tumors

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- Number: US 12,180,296 B2
- Document ID: US12180296B2
- Status: Granted
- Kind: B2
- Application number: 17/421,334
- Filed: 2020-01-02
- Published: 2022-03-03
- Granted: 2024-12-31
- Earliest claimed: 2019-01-08
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The U.S.A., as represented by the Secretary, Department of Health and Human Services
- Inventors: Mitchell Ho; Ira H. Pastan; Jessica D. Hong; Nan Li
- Web page: https://patentgrep.com/patent/US12180296B2

## CPC

- C07K 16/30: from tumour cells
- A61K 39/39558: {against tumor tissues, cells, antigens}
- 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 40/4255: {Mesothelin [MSLN]}
- A61K 47/6851: {the antibody targeting a determinant of a tumour cell}
- A61K 47/6929: {the form being a nanoparticle, e.g. an immuno-nanoparticle}
- A61P 35/00: Antineoplastic agents
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 14/71: for growth factors; for growth regulators
- C07K 14/7153: {for colony-stimulating factors [CSF]}
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 47/68: the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K 2039/505: {comprising antibodies}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/55: Lung
- C07K 2317/22: from camelids, e.g. camel, llama or dromedary
- C07K 2317/31: multispecific
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- 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

Camel single-domain monoclonal antibodies that specifically bind human and mouse mesothelin are described. Chimeric antigen receptor (CAR) T cells and antibody conjugates based on the mesothelin-specific antibodies are also described. The disclosed CAR T cells, mesothelin-specific antibodies and conjugates thereof can be used, for example, in the diagnosis or treatment of mesothelin-positive cancers.

## Claims

### Claim 1

A single-domain monoclonal antibody that specifically binds mesothelin, wherein the monoclonal antibody comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 2 or SEQ ID NO: 4.

### Claim 2 (depends on 1)

The monoclonal antibody of claim 1, wherein the CDR sequences are defined using the Kabat, IMGT or Paratome numbering schemes, or a combination of the Kabat, IMGT and Paratome numbering schemes.

### Claim 3 (depends on 1)

The monoclonal antibody of claim 1, wherein:
the CDR1, CDR2 and CDR3 sequences of the monoclonal antibody respectively comprise residues 31-35, 50-66 and 99-108 of SEQ ID NO: 2;
the CDR1, CDR2 and CDR3 sequences of the monoclonal antibody respectively comprise residues 26-33, 51-58 and 97-108 of SEQ ID NO: 2; or
the CDR1, CDR2 and CDR3 sequences of the monoclonal antibody respectively comprise residues 27-35, 47-61 and 97-108 of SEQ ID NO: 2.

### Claim 4 (depends on 1)

The monoclonal antibody of claim 1, wherein:
the CDR1, CDR2 and CDR3 sequences of the monoclonal antibody respectively comprise residues 31-35, 50-66 and 99-106 of SEQ ID NO: 4;
the CDR1, CDR2 and CDR3 sequences of the monoclonal antibody respectively comprise residues 26-33, 51-58 and 97-106 of SEQ ID NO: 4; or
the CDR1, CDR2 and CDR3 sequences of the monoclonal antibody respectively comprise residues 27-35, 47-60 and 98-106 of SEQ ID NO: 4.

### Claim 5 (depends on 1)

The monoclonal antibody of claim 1, wherein:
the amino acid sequence of the monoclonal antibody is at least 95% identical to SEQ ID NO: 2 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2; or
the amino acid sequence of the monoclonal antibody is at least 95% identical to SEQ ID NO: 4 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 4.

### Claim 6 (depends on 1)

The monoclonal antibody of claim 1, wherein the amino acid sequence of the monoclonal antibody comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 4.

### Claim 7 (depends on 1)

The monoclonal antibody of claim 1, wherein the monoclonal antibody is a camel antibody, a humanized antibody, or a chimeric antibody.

### Claim 8 (depends on 1)

A chimeric antigen receptor (CAR) comprising the monoclonal antibody of claim 1.

### Claim 9 (depends on 8)

The CAR of claim 8, further comprising a hinge region, a transmembrane domain, a costimulatory signaling moiety, a signaling domain, or any combination thereof.

### Claim 10 (depends on 9)

The CAR of claim 9, wherein
the hinge region comprises a CD8α hinge region;
the transmembrane domain comprises a CD8α transmembrane domain;
the costimulatory signaling moiety comprises a 4-1BB signaling moiety; and/or
the signaling domain comprises a CD3ξ signaling domain.

### Claim 11 (depends on 8)

An isolated cell expressing the CAR of claim 8.

### Claim 12 (depends on 11)

The isolated cell of claim 11, which is a cytotoxic T lymphocyte (CTL) or a natural killer (NK) cell.

### Claim 13 (depends on 1)

An immunoconjugate comprising the monoclonal antibody of claim 1 and an effector molecule.

### Claim 14 (depends on 13)

The immunoconjugate of claim 13, wherein the effector molecule is a toxin, a photon absorber, or a detectable label.

### Claim 15 (depends on 1)

An antibody-drug conjugate (ADC) comprising a drug conjugated to the monoclonal antibody of claim 1.

### Claim 16 (depends on 15)

The ADC of claim 15, wherein the drug is an anti-microtubule agent, an anti-mitotic agent and/or a cytotoxic agent.

### Claim 17 (depends on 1)

A multi-specific antibody comprising the monoclonal antibody of claim 1 and at least one additional monoclonal antibody or antigen-binding fragment thereof.

### Claim 18 (depends on 17)

The multi-specific antibody of claim 17, wherein the at least one additional monoclonal antibody or antigen binding fragment thereof specifically binds a component of the T cell receptor or a natural killer (NK) cell activating receptor.

### Claim 19 (depends on 1)

An antibody-nanoparticle conjugate, comprising a nanoparticle conjugated to the monoclonal antibody of claim 1.

### Claim 20 (depends on 1)

A fusion protein comprising the monoclonal antibody of claim 1 and a heterologous protein or peptide.

### Claim 21 (depends on 20)

The fusion protein of claim 20, wherein the heterologous protein is an Fc protein.

### Claim 22 (depends on 1)

An isolated nucleic acid molecule encoding the monoclonal antibody of claim 1.

### Claim 23 (depends on 22)

The isolated nucleic acid molecule of claim 22, comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a degenerate variant thereof.

### Claim 24 (depends on 22)

The isolated nucleic acid molecule of claim 22, operably linked to a promoter.

### Claim 25 (depends on 22)

A vector comprising the nucleic acid molecule of claim 22.

### Claim 26 (depends on 1)

A nucleic acid molecule encoding a chimeric antigen receptor (CAR), comprising in the 5′ to 3′ direction:
a nucleic acid encoding a first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss);
a nucleic acid encoding the monoclonal antibody of claim 1;
a nucleic acid encoding an extracellular hinge region;
a nucleic acid encoding a transmembrane domain;
a nucleic acid encoding an intracellular co-stimulatory domain;
a nucleic acid encoding a intracellular signaling domain;
a nucleic acid encoding a self-cleaving 2A peptide;
a nucleic acid encoding a second GMCSFRss; and
a nucleic acid encoding a truncated human epidermal growth factor receptor (huEGFRt).

### Claim 27 (depends on 26)

The nucleic acid molecule of claim 26, further comprising a human elongation factor 1α (EF1α) promoter sequence 5′ of the nucleic acid encoding the first GMCSFRss.

### Claim 28 (depends on 26)

A vector comprising the nucleic acid molecule of claim 26.

### Claim 29 (depends on 25)

An isolated cell comprising the claim 25.

### Claim 30 (depends on 1)

A composition comprising a pharmaceutically acceptable carrier and the monoclonal antibody of claim 1.

### Claim 31 (depends on 1)

A method of treating a mesothelin-positive cancer in a subject, comprising administering to the subject the monoclonal antibody of claim 1.

### Claim 32 (depends on 1)

A method of inhibiting tumor growth or metastasis of a mesothelin-positive cancer in a subject, comprising administering to the subject the monoclonal antibody of claim 1.

### Claim 33 (depends on 31)

The method of claim 31, wherein the mesothelin-positive cancer is a solid tumor.

### Claim 34 (depends on 31)

The method of claim 31, wherein the mesothelin-positive cancer is a mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer or ovarian cancer.

### Claim 35 (depends on 1)

A method of detecting expression of mesothelin in a sample, comprising:
contacting the sample with the monoclonal antibody of claim 1; and
detecting binding of the antibody to the sample, thereby detecting expression of mesothelin in the sample.

### Claim 36 (depends on 35)

The method of claim 35, wherein the monoclonal antibody is directly labeled.

### Claim 37 (depends on 35)

The method of claim 35, further comprising:
contacting the monoclonal antibody with a detection antibody, and
detecting the binding of the detection antibody to the monoclonal antibody, thereby detecting expression of mesothelin in the sample.

### Claim 38 (depends on 35)

The method of claim 35, wherein the sample is obtained from a subject suspected of having a mesothelin-positive cancer.

### Claim 39 (depends on 35)

The method of claim 35, wherein the sample is a tumor biopsy.

### Claim 40 (depends on 1)

A method of diagnosing a subject as having a mesothelin-positive cancer, comprising:
contacting a sample obtained from the subject with the monoclonal antibody of claim 1; and
detecting binding of the antibody to the sample, thereby diagnosing the subject as having a mesothelin-positive cancer.