# US 11,993,660 B2 · Chimeric polypeptides and methods of altering the membrane localization of the same

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- Number: US 11,993,660 B2
- Document ID: US11993660B2
- Status: Granted
- Kind: B2
- Application number: 17/402,009
- Filed: 2021-08-13
- Published: 2021-12-23
- Granted: 2024-05-28
- Earliest claimed: 2017-03-28
- Assignees: Cell Design Labs, Inc.
- Applicants: Cell Design Labs, Inc.
- Inventors: Stephen Santoro; Scott Coyle; Levi Rupp; Peter Emtage
- Web page: https://patentgrep.com/patent/US11993660B2

## CPC

- C07K 16/30: from tumour cells
- A61K 38/177: {Receptors; Cell surface antigens; Cell surface determinants}
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 39/001112: {CD19 or B4}
- 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 40/4211: {CD19 or B4}
- A61K 40/4215: {Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- 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 14/7153: {for colony-stimulating factors [CSF]}
- C07K 14/721: {Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 5/0637: {Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- A61K 2039/5156: {expressing foreign proteins}
- A61K 2039/5158: {Antigen-pulsed cells, e.g. T-cells}
- A61K 2239/11: Antigen recognition domain
- A61K 2239/22: Intracellular domain
- A61K 2239/23: On/off switch
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/035: containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
- C12N 5/0636: {T lymphocytes}
- 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

Methods and compositions are provided for reversibly localizing proteins to the exterior part of the cell surface. Compositions provided herein can include nucleic acids that encode polypeptides of interest and the ligand binding domain (LBD) of a nuclear hormone receptor. Medical applications are provided, including controlling the toxicity of CAR T cells.

## Claims

### Claim 1

A method of inducing membrane localization of at least a portion of a single-chain chimeric polypeptide to an extracellular side of the membrane in a mammalian cell, the method comprising:
contacting a mammalian cell expressing a single-chain chimeric polypeptide with an amount of amoxifen sufficient to induce localization of at least a portion of the single-chain chimeric polypeptide to an extracellular side of the membrane of the cell;
wherein the single-chain chimeric polypeptide comprises the amino sequences of SEQ ID NO: 31, SEQ ID NO: 33 or SEQ ID NO: 35.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
introducing a nucleic acid encoding the single-chain chimeric polypeptide into a mammalian cell to generate the mammalian cell expressing the single-chain chimeric antigen receptor.

### Claim 3 (depends on 2)

The method of claim 2, wherein the mammalian cell is a T cell.

### Claim 4 (depends on 3)

The method of claim 3, wherein the T cell is selected from the group consisting of: a CD8+ T cell, a CD4+ T cell, a memory T cell, a Treg cell, and a natural killer T cell.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mammalian cell is a mammalian cell previously obtained from a subject.

### Claim 6 (depends on 5)

The method of claim 5, wherein the subject has been identified or diagnosed as having a blood cancer.

### Claim 7 (depends on 1)

The method of claim 1, wherein the contacting step is performed in vitro.

### Claim 8 (depends on 7)

The method of claim 7, wherein the mammal has a blood cancer.

### Claim 9 (depends on 1)

The method of claim 1, wherein the contacting step is performed in a mammal.

### Claim 10

The method of 9, wherein the contacting step results in the treatment of the blood cancer in the mammal.

### Claim 11

A method of reversibly altering membrane localization of a single-chain chimeric polypeptide in a mammalian cell, the method comprising:
(a) contacting a mammalian cell expressing a single-chain chimeric polypeptide with an amount of amoxifen sufficient to induce localization of at least a portion of the single-chain chimeric polypeptide to an extracellular side of the plasma membrane of the cell; and
(b) contacting the mammalian cell with a reduced amount of amoxifen that results in a decreased level of the single-chain chimeric polypeptide on an extracellular side of the membrane of the mammalian cell;
wherein the single-chain chimeric polypeptide comprises the amino sequences of SEQ ID NO: 31, SEQ ID NO: 33 or SEQ ID NO: 35.

### Claim 12 (depends on 11)

The method of claim 11, further comprising:
(c) contacting the mammalian cell with an amount of tamoxifen that results in an increased level of at least a portion of the single-chain chimeric polypeptide to the extracellular side of the plasma membrane of the cell as compared to the level in (b).

### Claim 13

A method of inducing plasma membrane localization of at least a portion of a single-chain chimeric polypeptide to an extracellular side of the plasma membrane in a mammalian cell, the method comprising:
contacting a mammalian cell expressing a single-chain chimeric polypeptide with an amount of tamoxifen sufficient to induce localization of at least a portion of the single-chain chimeric polypeptide to an extracellular side of the plasma membrane of the cell;
wherein the single-chain chimeric polypeptide comprises the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 33 or SEQ ID NO: 35.

### Claim 14 (depends on 13)

The method of claim 13, further comprising, before step (a):
introducing a nucleic acid encoding the single-chain chimeric polypeptide into a mammalian cell to generate the mammalian cell expressing the single-chain chimeric polypeptide.

### Claim 15 (depends on 13)

The method of claim 13, wherein the mammalian cell is a T cell.

### Claim 16 (depends on 13)

The method of claim 13, wherein the mammalian cell is from a subject identified or diagnosed as having blood cancer.

### Claim 17 (depends on 13)

The method of claim 13, further comprising obtaining the mammalian cell from the subject.

### Claim 18 (depends on 13)

The method of claim 13, wherein the contact step is performed in vitro.