# US 11,306,134 B2 · Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof

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- Number: US 11,306,134 B2
- Document ID: US11306134B2
- Status: Granted
- Kind: B2
- Application number: 17/477,485
- Filed: 2021-09-16
- Published: 2022-02-03
- Granted: 2022-04-19
- Earliest claimed: 2015-07-28
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: The Trustees of the University of Pennsylvania
- Inventors: Saar Gill; Michael Klichinsky; Carl H. June
- Web page: https://patentgrep.com/patent/US11306134B2

## CPC

- C07K 14/70517: {CD8}
- A61K 40/17: Monocytes; Macrophages
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4205: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4}
- A61K 40/4211: {CD19 or B4}
- A61K 40/4255: {Mesothelin [MSLN]}
- A61P 37/02: Immunomodulators
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70535: {Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)}
- C07K 14/7056: {Lectin superfamily, e.g. CD23, CD72}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/30: from tumour cells
- C07K 16/32: against translation products of oncogenes
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 5/0645: {Macrophages, e.g. Kuepfer cells in the liver; Monocytes}
- A61K 35/14: Blood; Artificial blood (perfluorocarbons A61K31/02; umbilical cord blood A61K35/51; haemoglobin A61K38/42)
- 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 2039/505: {comprising antibodies}
- A61K 2039/892: {Reproductive system [uterus, ovaries, cervix, testes]}
- A61P 35/00: Antineoplastic agents
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- 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

The present invention includes methods and compositions for treating cancer, whether a solid tumor or a hematologic malignancy. By expressing a chimeric antigen receptor in a monocyte, macrophage or dendritic cell, the modified cell is recruited to the tumor microenvironment where it acts as a potent immune effector by infiltrating the tumor and killing the target cells. One aspect includes a modified cell and pharmaceutical compositions comprising the modified cell for adoptive cell therapy and treating a disease or condition associated with immunosuppression.

## Claims

### Claim 1

A modified cell comprising a chimeric antigen receptor (CAR),
wherein the CAR comprises an antigen binding domain, a transmembrane domain and an intracellular domain of a stimulatory and/or co-stimulatory molecule, and
wherein the modified cell expresses the CAR and possesses targeted effector activity,
wherein the modified cell comprises a macrophage or a monocyte, and
wherein the modified cell comprises a lentiviral component.

### Claim 2 (depends on 1)

The modified cell of claim 1, wherein the modified cell exhibits reduced SIRPα activity relative to an unmodified monocyte or macrophage.

### Claim 3 (depends on 1)

The modified cell of claim 1, wherein the antigen binding domain of the CAR comprises an antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, human antibody, humanized antibody, single domain antibody, single chain variable fragment, and antigen-binding fragments thereof.

### Claim 4 (depends on 1)

The modified cell of claim 1, wherein the antigen binding domain of the CAR is selected from the group consisting of an anti-CD19 antibody, an anti-HER2 antibody, an anti-mesothelin antibody, an anti-PSMA antibody, and a fragment thereof.

### Claim 5 (depends on 1)

The modified cell of claim 1, wherein the transmembrane domain of the CAR comprises a CD8 or CD28 transmembrane domain.

### Claim 6 (depends on 1)

The modified cell of claim 1, wherein the intracellular domain of the CAR comprises dual signaling domains.

### Claim 7 (depends on 1)

The modified cell of claim 1, wherein the intracellular domain of the CAR comprises a CD3 zeta intracellular domain.

### Claim 8 (depends on 1)

The modified cell of claim 1, wherein the targeted effector activity is directed against a target cell comprising an antigen that specifically binds the antigen binding domain of the CAR.

### Claim 9 (depends on 1)

The modified cell of claim 1, wherein the targeted effector activity is selected from the group consisting of phagocytosis, targeted cellular cytotoxicity, antigen presentation, and cytokine secretion.

### Claim 10 (depends on 1)

The modified cell of claim 1, further comprising an agent selected from the group consisting of a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or antibody fragments thereof, a growth factor, a cytokine, an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule, an organic molecule, a carbohydrate or the like, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combination thereof.

### Claim 11 (depends on 1)

The modified cell of claim 1, wherein the modified cell has at least one upregulated M1 marker and at least one downregulated M2 marker.

### Claim 12 (depends on 1)

The modified cell of claim 1, wherein the modified cell is genetically modified to express the CAR.

### Claim 13 (depends on 1)

The modified cell of claim 1, wherein the targeted effector activity is enhanced by inhibition of CD47 or SIRPα activity.

### Claim 14 (depends on 1)

A pharmaceutical composition comprising the cell of claim 1 and a pharmaceutically acceptable carrier.

### Claim 15 (depends on 14)

The pharmaceutical composition of claim 14, wherein at least 35% of the cells in the composition express a CAR.

### Claim 16 (depends on 1)

A method for stimulating an immune response to a target tumor cell or tumor tissue in a subject comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the modified cell of claim 1.

### Claim 17

A method of modifying a cell, comprising:
introducing a chimeric antigen receptor (CAR) into the cell, wherein the CAR comprises an antigen binding domain, a transmembrane domain and an intracellular domain of a stimulatory and/or co-stimulatory molecule, and
wherein the modified cell expresses the CAR and possesses targeted effector activity,
wherein the modified cell comprises a macrophage or a monocyte, and
wherein the modified cell comprises a lentiviral component.

### Claim 18 (depends on 17)

The method of claim 17, wherein introducing the CAR into the cell comprises introducing a nucleic acid sequence encoding the CAR.

### Claim 19 (depends on 17)

The method of claim 17, wherein introducing the nucleic acid sequence comprises electroporating an mRNA encoding the CAR.

### Claim 20 (depends on 17)

The method of claim 17, wherein introducing the nucleic acid sequence comprises transducing the cell with a viral vector comprising a nucleic acid sequence encoding the CAR.

### Claim 21 (depends on 17)

The method of claim 17, wherein the targeted effector activity is selected from the group consisting of phagocytosis, targeted cellular cytotoxicity, antigen presentation, and cytokine secretion.

### Claim 22 (depends on 17)

The method of claim 17, further comprising inhibiting CD47 or SIRPα activity to enhance the targeted effector activity.

### Claim 23 (depends on 22)

The method of claim 22, wherein inhibiting CD47 or SIRPα activity comprises contacting the cell with a blocking anti-CD47 antibody or a blocking anti-SIRPα antibody.

### Claim 24 (depends on 17)

The method of claim 17, wherein the intracellular domain of the CAR comprises dual signaling domains.

### Claim 25 (depends on 17)

The method of claim 17, wherein the intracellular domain of the CAR comprises a CD3 zeta intracellular domain.

### Claim 26 (depends on 17)

The method of claim 17, wherein the antigen binding domain of the CAR comprises an antibody selected from the group consisting of a synthetic antibody, human antibody, humanized antibody, single domain antibody, single chain variable fragment, and antigen-binding fragments thereof.

### Claim 27 (depends on 17)

The method of claim 17, wherein the antigen binding domain of the CAR is selected from the group consisting of an anti-CD19 antibody, an anti-HER2 antibody, an anti-mesothelin antibody, an anti-PSMA antibody, and a fragment thereof.