# US 2021/0171602 A1 · Chimeric Antigen Receptor Polypeptides and Methods of Using Same

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- Number: US 2021/0171602 A1
- Document ID: US20210171602A1
- Status: Published application
- Kind: A1
- Application number: 17/254,618
- Filed: 2019-07-03
- Published: 2021-06-10
- Earliest claimed: 2018-07-06
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Crystal Mackall; Michael Lin; Robbie Majzner; Hokyung Chung; Louai Labanieh
- Web page: https://patentgrep.com/patent/US20210171602A1

## CPC

- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/3084: {against tumour-associated gangliosides}
- A61K 31/426: 1,3-Thiazoles
- A61K 31/498: Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
- A61K 39/12: Viral 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 40/421: {Immunoglobulin superfamily}
- A61P 35/00: Antineoplastic agents
- C07K 14/005: from viruses
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- C12N 9/506: {derived from RNA viruses}
- C12Y 304/21098: Hepacivirin (3.4.21.98)
- A61K 2039/585: {wherein the target is cancer}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/47: Brain; Nervous system
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- 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
- C07K 2319/50: containing protease site
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C12N 2770/24222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2770/24234: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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)
- C12Y: ENZYMES

## Abstract

Provided are polypeptides that include, from N-terminus to C-terminus, a chimeric antigen receptor (CAR), a protease, and a degron, where the polypeptide further includes a cleavage site for the protease disposed between the CAR and the degron. Also provided are cells that include such polypeptides (e.g., where the cells express the CAR on their surface) and pharmaceutical compositions including such cells. Nucleic acids that encode the polypeptides, cells including such nucleic acids, and pharmaceutical compositions including such cells, are also provided. Also provided are methods for controlling the expression of a CAR on the surface of a cell, and methods of using the cells of the present disclosure, including methods of using such cells to administer a regulatable CAR cell-based therapy (e.g., a regulatable CAR T cell therapy) to an individual.

## Claims

### Claim 1

A polypeptide comprising from N-terminus to C-terminus:
a chimeric antigen receptor (CAR);
a protease; and
a degron,
wherein the polypeptide further comprises a cleavage site for the protease disposed between the CAR and the degron.

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein the cleavage site is between the CAR and the protease.

### Claim 3 (depends on 1)

The polypeptide of claim 1, wherein the cleavage site is between the protease and the degron.

### Claim 4 (depends on 2)

The polypeptide of claim 2, further comprising a spacer domain between the CAR and the cleavage site.

### Claim 5 (depends on 3)

The polypeptide of claim 3, further comprising a spacer domain between the CAR and the cleavage site.

### Claim 6 (depends on 4, 5)

The polypeptide of claim 4 or claim 5, wherein the spacer domain comprises a linker.

### Claim 7 (depends on 4, 5, 6)

The polypeptide of any one of claims 4 to 6, wherein the spacer domain comprises a reporter.

### Claim 8 (depends on 7)

The polypeptide of claim 7, wherein the reporter is a bioluminescence reporter.

### Claim 9 (depends on 8)

The polypeptide of claim 8, wherein the bioluminescence reporter is a luciferase.

### Claim 10 (depends on 4, 5, 6, 7, 8, 9)

The polypeptide of any one of claims 4 to 9, wherein the spacer domain decreases the response time of the polypeptide to an inhibitor of the protease.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The polypeptide of any one of claims 1 to 10, wherein the antigen-binding portion of the CAR is an antibody.

### Claim 12 (depends on 11)

The polypeptide of claim 11, wherein the antibody is a single chain variable fragment (scFv).

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The polypeptide of any one of claims 1 to 12, wherein the antigen-binding portion of the CAR specifically binds an antigen on the surface of a cancer cell.

### Claim 14 (depends on 13)

The polypeptide of claim 13, wherein the antigen on the surface of the cancer cell is a tumor-associated antigen or a tumor-specific antigen.

### Claim 15 (depends on 13)

The polypeptide of claim 13, wherein the antigen on the surface of the cancer cell is selected from the group consisting of: B7-H3 (CD276), CD19, GD2, CD22, and HER2.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The polypeptide of any one of claims 1 to 15, wherein the protease is a viral protease.

### Claim 17 (depends on 16)

The polypeptide of claim 16, wherein the protease is derived from hepatitis C virus (HCV) nonstructural protein 3 (NS3).

### Claim 18 (depends on 17)

The polypeptide of claim 17, wherein the protease further comprises a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A).

### Claim 19 (depends on 17, 18)

The polypeptide of claim 17 or claim 18, wherein the protease is engineered to include one or more amino acid substitutions relative to a wild-type HCV NS3 protease.

### Claim 20 (depends on 19)

The polypeptide of claim 19, wherein the protease comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical, to the amino acid sequence set forth in SEQ ID NO:2, which protease comprises an alanine at position 54, and wherein numbering is as in SEQ ID NO:2.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The polypeptide of any one of claims 1 to 20, wherein the cleavage site for the protease is selected from the group consisting of: an NS4A/4B junction cleavage site, an NS3/NS4A junction cleavage site, an NS4A/NS4B junction cleavage site, an NS4B/NS5A junction cleavage site, and an NS5A/NS5B junction cleavage site.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)

The polypeptide of any one of claims 1 to 21, wherein the degron comprises portions of the HCV nonstructural proteins NS3 and NS4A.

### Claim 23 (depends on 22)

The polypeptide of claim 22, wherein the degron comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical, to the amino acid sequence set forth in SEQ ID NO:1.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

A cell comprising the polypeptide of any one of claims 1 to 23.

### Claim 25 (depends on 24)

The cell of claim 24, wherein the cell is a mammalian cell.

### Claim 26 (depends on 25)

The cell of claim 25, wherein the cell is a human cell.

### Claim 27 (depends on 24, 25, 26)

The cell of any one of claims 24 to 26, wherein the cell is an immune cell.

### Claim 28 (depends on 27)

The cell of claim 27, wherein the cell is a T cell.

### Claim 29 (depends on 24, 25, 26, 27, 28)

The cell of any one of claims 24 to 28, wherein the CAR is expressed on the surface of the cell.

### Claim 30 (depends on 24, 25, 26, 27, 28, 29)

A pharmaceutical composition, comprising:
the cell of any one of claims 24 to 29; and
a pharmaceutically-acceptable carrier.

### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

A nucleic acid encoding the polypeptide of any one of claims 1 to 23.

### Claim 32 (depends on 31)

An expression vector comprising the nucleic acid of claim 31.

### Claim 33 (depends on 31, 32)

A cell comprising the nucleic acid of claim 31 or the expression vector of claim 32.

### Claim 34 (depends on 33)

The cell of claim 33, wherein the cell is a mammalian cell.

### Claim 35 (depends on 34)

The cell of claim 34, wherein the cell is a human cell.

### Claim 36 (depends on 33, 34, 35)

The cell of any one of claims 33 to 35, wherein the cell is an immune cell.

### Claim 37 (depends on 36)

The cell of claim 36, wherein the cell is a T cell.

### Claim 38 (depends on 31, 32, 33, 34, 35, 36, 37)

A method of making the cell of any one of claims 33 to 37, comprising introducing the nucleic acid of claim 31 or the expression vector of claim 32 into the cell.

### Claim 39 (depends on 33, 34, 35, 36, 37)

A method for controlling the expression of a CAR on the surface of a cell, comprising:
contacting the cell of any one of claims 33 to 37 with an inhibitor of the protease when expression of the CAR on the surface of the cell is not desired.

### Claim 40 (depends on 39)

The method according to claim 39, wherein the protease is derived from HCV NS3, and wherein the inhibitor of the protease is selected from the group consisting of: asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), grazoprevir (GPV), and any combination thereof.

### Claim 41 (depends on 39, 40)

The method according to claim 39 or claim 40, wherein prior to the contacting, the cell is allowed to express the CAR on the surface of the cell.

### Claim 42 (depends on 39, 40, 41)

The method according to any one of claims 39 to 41, further comprising ceasing the contacting to express the CAR on the surface of the cell.

### Claim 43 (depends on 39, 40, 41, 42)

The method according to any one of claims 39 to 42, wherein the method is performed in vitro.

### Claim 44 (depends on 39, 40, 41, 42)

The method according to any one of claims 39 to 42, wherein the method is performed ex vivo.

### Claim 45 (depends on 39, 40, 41, 42)

The method according to any one of claims 39 to 42, wherein the method is performed in vivo.

### Claim 46 (depends on 33, 34, 35, 36, 37)

A pharmaceutical composition, comprising:
the cell of any one of claims 33 to 37; and
a pharmaceutically-acceptable carrier.

### Claim 47 (depends on 32, 46)

A method of making the pharmaceutical composition of claim 46, comprising introducing the expression vector of claim 32 into cells obtained from an individual.

### Claim 48 (depends on 46)

A method of administering a regulatable CAR cell-based therapy to an individual in need thereof, comprising administering to the individual the pharmaceutical composition of claim 46.

### Claim 49 (depends on 48)

The method according to claim 48, further comprising contacting the administered cells or progeny thereof with an inhibitor of the protease when expression of the CAR on the surface of the administered cells or progeny thereof is not desired, wherein the contacting comprises administering the inhibitor of the protease to the individual.

### Claim 50 (depends on 49)

The method according to claim 49, wherein the inhibitor of the protease is administered to the individual prior to administration of the pharmaceutical composition.

### Claim 51 (depends on 49, 50)

The method according to claim 49 or claim 50, wherein the pharmaceutical composition and the inhibitor of the protease are co-administered to the individual.

### Claim 52 (depends on 49, 50, 51)

The method according to any one of claims 49 to 51, wherein the inhibitor of the protease is administered to the individual to prevent or delay the onset of cell exhaustion resulting from CAR activity.

### Claim 53 (depends on 49)

The method according to claim 49, comprising:
administering the pharmaceutical composition to the individual prior to the contacting to allow expression of the CAR on the surface of the cells or progeny thereof in the individual; and
administering the inhibitor of the protease to the individual when expression of the CAR on the surface of the cells or progeny thereof is no longer desired.

### Claim 54 (depends on 53)

The method according to claim 53, wherein the inhibitor of the protease is administered to the individual to delay or prevent cell exhaustion resulting from CAR activity.

### Claim 55 (depends on 53, 54)

The method according to claim 53 or claim 54, wherein the inhibitor of the protease is administered to the individual to reduce adverse side effects caused by the cells or progeny thereof.

### Claim 56 (depends on 49, 50, 51, 52, 53, 54, 55)

The method according to any one of claims 49 to 55, further comprising ceasing the contacting to express the CAR on the surface of the cells or progeny thereof.

### Claim 57 (depends on 49, 50, 51, 52, 53, 54, 55, 56)

The method according to any one of claims 49 to 56, wherein the protease is derived from HCV NS3, and wherein the inhibitor of the protease is selected from the group consisting of: asunaprevir (ASV), danoprevir (DPV), simeprevir (SPV), and grazoprevir (GPV).

### Claim 58 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57)

The method according to any one of claims 48 to 57, further comprising administering to the individual a pharmacokinetic enhancer of the inhibitor of the protease.

### Claim 59 (depends on 58)

The method according to claim 58, wherein the pharmacokinetic enhancer is an inhibitor of an enzyme that metabolizes the inhibitor of the protease.

### Claim 60 (depends on 59)

The method according to claim 59, wherein the pharmacokinetic enhancer is an inhibitor of a cytochrome P450 (CYP) enzyme that metabolizes the inhibitor of the protease.

### Claim 61 (depends on 60)

The method according to claim 60, wherein the pharmacokinetic enhancer is ritonavir.

### Claim 62 (depends on 61)

The method according to claim 61, wherein the inhibitor of the protease is grazoprevir.

### Claim 63 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)

The method according to any one of claims 48 to 62, further comprising producing the pharmaceutical composition.

### Claim 64 (depends on 32, 63)

The method according to claim 63, wherein producing the pharmaceutical composition comprises introducing the expression vector of claim 32 into cells or progeny thereof obtained from the individual.

### Claim 65 (depends on 64)

The method according to claim 64, comprising contacting the cells obtained from the individual, or progeny thereof, with an inhibitor of the protease prior to administering the pharmaceutical composition to the individual.

### Claim 66 (depends on 31, 32)

A kit, comprising:
the nucleic acid of claim 31 or the expression vector of claim 32; and
instructions for introducing the nucleic acid or expression vector into a cell.

### Claim 67 (depends on 66)

The kit of claim 66, wherein the instructions further comprise instructions for regulatable expression of the CAR on the surface of the cell or progeny thereof.

### Claim 68 (depends on 67)

The kit of claim 67, wherein the instructions comprise instructions for contacting the cell or progeny thereof with an inhibitor of the protease when expression of the CAR on the surface of the cell or progeny thereof is not desired.

### Claim 69 (depends on 66, 67, 68)

The kit of any one of claims 66 to 68, further comprising an inhibitor of the protease.