# US 10,865,408 B2 · Chimeric antigen receptors and methods of use

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,865,408 B2
- Document ID: US10865408B2
- Status: Granted
- Kind: B2
- Application number: 15/537,418
- Filed: 2015-12-18
- Published: 2019-11-14
- Granted: 2020-12-15
- Earliest claimed: 2014-12-19
- Assignees: ETH Zurich
- Applicants: ETH Zurich
- Inventors: Jan Kisielow; Franz-Josef Obermair; Manfred Kopf
- Web page: https://patentgrep.com/patent/US10865408B2

## CPC

- C12N 15/1055: {Protein x Protein interaction, e.g. two hybrid selection}
- 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 40/32: T-cell receptors [TCR]
- A61K 40/42: Cancer antigens
- A61K 40/46: Viral antigens
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- C12N 5/0636: {T lymphocytes}
- G01N 33/505: {involving T-cells}
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C07K 2319/61: containing an enzyme fusion for detection (lacZ, luciferase)
- C12N 2510/02: Cells for production
- G01N 2800/24: Immunology or allergic disorders
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention relates to a chimeric antigen-receptor polypeptide heterodimer comprising two polypeptides, wherein the first contains an extracellular part of the major histocompatibility complex I alpha chain and the second contains a 32-microglobulin domain, or the first contains an extracellular part of the major histocompatibility complex II alpha chain and the second contains a major histocompatibility complex II beta chain. One of the polypeptides further contains a transmembrane domain, a hinge region and an intracellular domain of the T cell receptor alpha chain and the other one contains a transmembrane domain, a hinge region and an intracellular domain of the T cell receptor beta chain, and additionally an antigen-peptide covalently linked to said extracellular MHC domain. The invention further relates to a method for the identification of a TCR recognizable peptide sequence making use of the heterodimer of the invention.

## Claims

### Claim 1

A chimeric antigen-receptor polypeptide heterodimer comprising a first polypeptide and a second polypeptide, wherein
a. said first polypeptide comprises an extracellular part of the major histocompatibility complex I (MHC class I) alpha chain, wherein said extracellular part of the MHC alpha chain retains the ability to interact with an oligopeptide and with CD8, and said second polypeptide comprises a β2-microglobulin domain, or
b. said first polypeptide comprises an extracellular part of the major histocompatibility complex II (MHC class II) alpha chain and said second polypeptide comprises an extracellular part of the major histocompatibility complex II (MHC class II) beta chain, wherein said extracellular parts of the MHC alpha and beta chain retain the ability to interact with an oligopeptide and CD4; and wherein
one of said first polypeptide of (a) or (b) and said second polypeptide (a) or (b) further comprises a hinge region, a transmembrane domain and an intracellular domain or intracellular tail of the T cell receptor alpha chain and the other one of said first polypeptide (a) or (b) and said second polypeptide (a) or (b) comprises a hinge region, a transmembrane domain and an intracellular domain of the T cell receptor beta chain.

### Claim 2 (depends on 1)

The chimeric antigen-receptor polypeptide heterodimer according to claim 1, wherein the extracellular part of the MHC molecule comprises each of a MHC class I alpha1, alpha2, and alpha3 domain forming part of said first polypeptide.

### Claim 3 (depends on 1)

The chimeric antigen-receptor polypeptide heterodimer according to claim 1, wherein an oligopeptide sequence is inserted after amino acid 1, 2, 3, 4 or 5 of the MHC alpha1 domain sequence.

### Claim 4 (depends on 1)

The chimeric antigen-receptor polypeptide heterodimer according to claim 1, comprising a first polypeptide with an amino acid sequence having at least ≥80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 008.

### Claim 5

A nucleic acid molecule encoding a chimeric antigen-receptor polypeptide heterodimer comprising a first polypeptide and a second polypeptide, wherein
a. said first polypeptide comprises an extracellular part of the major histocompatibility complex I (MHC class I) alpha chain, wherein said extracellular part of the MHC alpha chain retains the ability to interact with an oligopeptide and with CD8, and said second polypeptide comprises a β2-microglobulin domain, or
b. said first polypeptide comprises an extracellular part of the major histocompatibility complex II (MHC class II) alpha chain and said second polypeptide comprises an extracellular part of the major histocompatibility complex II (MHC class II) beta chain, wherein said extracellular parts of the MHC alpha and beta chain retain the ability to interact with an oligopeptide and CD4,
one of said first polypeptide (a) or (b) and said second polypeptide (a) or (b) further comprises a hinge region, a transmembrane domain and an intracellular domain or intracellular tail of the T cell receptor alpha chain and the other one of said first polypeptide (a) or (b) and said second polypeptide (a) or (b) comprises a hinge region, a transmembrane domain and an intracellular domain of the T cell receptor beta chain.

### Claim 6 (depends on 1)

A mammalian cell comprising
i. the chimeric antigen-receptor polypeptide heterodimer according to claim 1, and
ii. an effector function functionally linked to said chimeric antigen-receptor polypeptide heterodimer.

### Claim 7 (depends on 4)

The chimeric antigen-receptor polypeptide heterodimer of claim 4, wherein at least one of said first and said second polypeptide chain additionally comprises an oligopeptide that can be recognized by a T cell receptor, said oligopeptide being covalently linked to said extracellular WIC domain.

### Claim 8

A chimeric antigen-receptor polypeptide heterodimer comprising a first polypeptide and a second polypeptide, wherein said first polypeptide comprises an extracellular part of the major histocompatibility complex II (MHC class II) alpha chain and said second polypeptide comprises an extracellular part of the major histocompatibility complex II (MHC class II) beta chain;
wherein the extracellular part of the MHC molecule comprises an MHC class II alpha1 and an MHC class II alpha2 domain forming part of said first polypeptide, and an MHC class II beta1 and an MHC class II beta2 domain forming part of said second polypeptide; and
wherein said first polypeptide and said second polypeptide comprises a hinge region, a transmembrane domain and an intracellular domain of the T cell receptor beta chain.

### Claim 9 (depends on 1)

The chimeric antigen-receptor polypeptide heterodimer of claim 1, wherein the extracellular part of the MHC molecule is selected from the members of the human major histocompatibility complex gene family HLA.

### Claim 10 (depends on 1)

The chimeric antigen-receptor polypeptide heterodimer of claim 1, wherein:
(a) the extracellular part of the MEW molecule comprises each of a MEW class I alpha1, alpha2, and alpha3 domain forming part of said first polypeptide; and
(b) the extracellular part of the MEW molecule is selected from the members of the human major histocompatibility complex gene family HLA.

### Claim 11 (depends on 1)

The chimeric antigen-receptor polypeptide heterodimer of claim 1, wherein:
(a) wherein the extracellular part of the MHC molecule comprises an MHC class II alpha1 and an MHC class II alpha2 domain forming part of said first polypeptide, and an MHC class II beta1 and an MEW class II beta2 domain forming part of said second polypeptide; and
(b) the extracellular part of the MHC molecule is selected from the members of the human major histocompatibility complex gene family HLA.

### Claim 12 (depends on 3)

The chimeric antigen-receptor polypeptide heterodimer according to claim 3, wherein said oligopeptide sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 amino acids in length.

### Claim 13 (depends on 3)

The chimeric antigen-receptor polypeptide heterodimer according to claim 3, wherein said oligopeptide sequence is inserted after amino acid 1, 2, 3, 4 or 5 of the MHC alpha1 domain sequence and is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 amino acids in length.

### Claim 14 (depends on 6)

The mammalian cell according to claim 6, wherein the effector function is:
i. a reporter protein, and/or
ii. a protein, or a plurality of proteins, capable of inducing cell death in cells bound to the chimeric antigen-receptor polypeptide.

### Claim 15 (depends on 14)

The mammalian cell according to claim 14, wherein the reporter protein is selected from:
iii. a fluorescent protein, or
iv. a luciferase protein, or
v. a protein encoded by an antibiotic resistance gene, or
vi. a Cre recombinase, or
vii. a CAS-9 nuclease, or
viii. a CAS-9 chimeric transcriptional suppressor or activator.

### Claim 16 (depends on 1)

The chimeric antigen-receptor polypeptide heterodimer of claim 1, wherein the extracellular part of the MHC molecule comprises an MHC class II alpha1 and an MHC class II alpha2 domain forming part of said first polypeptide, and an MHC class II beta1 and an MHC class II beta2 domain forming part of said second polypeptide.

### Claim 17

A chimeric antigen-receptor polypeptide heterodimer comprising a first polypeptide and a second polypeptide, wherein said first polypeptide comprises an extracellular part of the major histocompatibility complex I (MHC class I) alpha chain and said second polypeptide comprises an extracellular part of the major histocompatibility complex I (WIC class I) β2-microglobulin chain;
wherein the extracellular part of the MHC molecule comprises an MHC class I alpha 1 domain, an MHC class I alpha 2 domain and an MHC class I alpha 3 domain forming part of said first polypeptide; and
wherein said first polypeptide further comprises a hinge region, a transmembrane domain and an intracellular domain or intracellular tail of the T cell receptor alpha chain and said second polypeptide comprises a hinge region, a transmembrane domain and an intracellular domain of the T cell receptor beta chain.

### Claim 18 (depends on 14)

The mammalian cell according to claim 14, wherein the effector function is:
i. a reporter protein, and a protein or a plurality of proteins, capable of inducing apoptotic cell death, in cells bound to the chimeric antigen-receptor polypeptide, or
ii. a protein or a plurality of proteins, capable of inducing apoptotic cell death, in cells bound to the chimeric antigen-receptor polypeptide.