# US 11,028,142 B2 · Compositions and methods for TCR reprogramming using fusion proteins

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- Number: US 11,028,142 B2
- Document ID: US11028142B2
- Status: Granted
- Kind: B2
- Application number: 16/989,602
- Filed: 2020-08-10
- Published: 2020-11-19
- Granted: 2021-06-08
- Earliest claimed: 2015-05-18
- Assignees: Tcr2 Therapeutics Inc.
- Applicants: TCR2 Therapeutics Inc.
- Inventors: Patrick Baeuerle; Gregory Sieczkiewicz; Robert Hofmeister
- Web page: https://patentgrep.com/patent/US11028142B2

## CPC

- A61P 35/00: Antineoplastic agents
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- A61K 39/0011: {Cancer 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 40/32: T-cell receptors [TCR]
- A61K 40/4211: {CD19 or B4}
- A61K 40/4215: {Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30}
- A61P 35/02: specific for leukemia
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 16/30: from tumour cells
- C07K 16/40: against enzymes
- C12N 5/0636: {T lymphocytes}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/5158: {Antigen-pulsed cells, e.g. T-cells}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- 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 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C12N 2510/00: Genetically modified cells
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are T-cell receptor (TCR) fusion proteins (TFPs), T-cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases, including cancer.

## Claims

### Claim 1

A T cell from a human subject, wherein the T cell comprises a recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising:
(a) a TCR subunit comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and
(b) a murine, human or humanized scFv or single domain antibody comprising an antigen binding domain; and a pharmaceutically acceptable carrier;
wherein the TCR subunit and the antigen binding domain are operatively linked;
wherein the extracellular domain, the transmembrane domain and the intracellular signaling domain are derived from a single subunit, wherein the single subunit is CD3 epsilon or wherein the single subunit is CD3 gamma;
wherein the extracellular domain comprises an extracellular domain of the single subunit;
wherein the TFP functionally interacts with an endogenous TCR when expressed in a T cell; and
wherein the T cell exhibits increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the antigen binding domain compared to a T cell not containing the TFP.

### Claim 2 (depends on 1)

The T cell of claim 1, wherein the single subunit comprises at least a portion of CD3 epsilon.

### Claim 3 (depends on 1)

The T cell of claim 1, wherein the single subunit comprises the extracellular domain of CD3 epsilon.

### Claim 4 (depends on 1)

The T cell of claim 1, wherein the single subunit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 57.

### Claim 5 (depends on 2)

The T cell of claim 2, wherein the single subunit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 56.

### Claim 6 (depends on 2)

The T cell of claim 2, wherein the single subunit comprises an amino acid sequence that is at least 89% identical to SEQ ID NO: 56.

### Claim 7 (depends on 3)

The T cell of claim 3, wherein the extracellular domain of CD3 epsilon has an amino acid sequence identical to amino acids 23-126 of SEQ ID NO: 56.

### Claim 8 (depends on 2)

The T cell of claim 2, wherein the single subunit comprises a transmembrane domain that has an amino acid sequence identical to amino acids 127-152 of SEQ ID NO: 56.

### Claim 9 (depends on 2)

The T cell of claim 2, wherein the single subunit comprises an intracellular domain that has an amino acid sequence identical to amino acids 153-207 of SEQ ID NO: 56.

### Claim 10 (depends on 1)

The T cell of claim 1, wherein the single subunit comprises an amino acid sequence identical to amino acids 23-207 of SEQ ID NO: 56.

### Claim 11 (depends on 1)

The T cell of claim 1, wherein the single subunit consists of an amino acid sequence identical to amino acids 23-207 of SEQ ID NO: 56.

### Claim 12 (depends on 1)

The T cell of claim 1, wherein the antigen binding domain comprises an anti-CD19 binding domain.

### Claim 13 (depends on 12)

The T cell of claim 12, wherein the anti-CD19 binding domain comprises (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively; (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 35, respectively; or (iii) a combination thereof.

### Claim 14 (depends on 1)

The T cell of claim 1, wherein the TFP comprises the murine, human or humanized scFv.

### Claim 15 (depends on 1)

The T cell of claim 1, wherein the single subunit comprises an amino acid sequence that is at least 89% identical to SEQ ID NO: 57.

### Claim 16 (depends on 1)

The T cell of claim 1, wherein the extracellular domain of CD3 gamma has an amino acid sequence identical to amino acids 23-116 of SEQ ID NO: 57.

### Claim 17 (depends on 1)

The T cell of claim 1, wherein the TFP comprises the murine, human or humanized single domain antibody.

### Claim 18 (depends on 17)

The T cell of claim 17, wherein the murine, human or humanized single domain antibody is a VHH domain.

### Claim 19 (depends on 1)

The T cell of claim 1, wherein the antigen binding domain is connected to the TCR extracellular domain by a linker.

### Claim 20 (depends on 19)

The T cell of claim 19, wherein the linker comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 4.

### Claim 21 (depends on 1)

The T cell of claim 1, wherein the TFP lacks a costimulatory domain.

### Claim 22 (depends on 1)

The T cell of claim 1, wherein the TFP lacks a heterologous stimulatory domain.

### Claim 23 (depends on 1)

The T cell of claim 1, wherein the single subunit comprises an amino acid sequence identical to amino acids 23-182 of SEQ ID NO: 57.

### Claim 24 (depends on 1)

The T cell of claim 1, wherein the T cell is formulated as a pharmaceutical composition.

### Claim 25 (depends on 1)

The T cell of claim 1, wherein the T cell is a CD8+ human T cell, a CD4+ human T cell, or a combination thereof.

### Claim 26 (depends on 1)

The T cell of claim 1, wherein the single subunit comprises a transmembrane domain that has an amino acid sequence identical to amino acids 117-137 of SEQ ID NO: 57.

### Claim 27 (depends on 1)

The T cell of claim 1, wherein the single subunit comprises an intracellular domain that has an amino acid sequence identical to amino acids 138-182 of SEQ ID NO: 57.

### Claim 28 (depends on 1)

The T cell of claim 1, wherein production of IFNγ by the T cell is increased compared to a T cell not containing the TFP in the presence of a human cell expressing an antigen that specifically interacts with the antigen binding domain.

### Claim 29

A pharmaceutical composition comprising a T cell from a human subject, wherein the T cell comprises a recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising:
(a) a TCR subunit comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and
(b) a murine, human or humanized single domain antibody comprising an antigen binding domain; and a pharmaceutically acceptable carrier;
wherein the TCR subunit and the antigen binding domain are operatively linked;
wherein the extracellular domain, the transmembrane domain and the intracellular signaling domain are derived from a single subunit, wherein the single subunit is CD3 epsilon;
wherein the single subunit consists of an amino acid sequence with amino acids 23-207 of SEQ ID NO: 56;
wherein the TFP functionally interacts with an endogenous TCR when expressed in a T cell;
wherein the T cell exhibits increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the antigen binding domain compared to a T cell not containing the TFP;
wherein the TFP lacks a costimulatory domain and lacks a heterologous stimulatory domain;
wherein the antigen binding domain is connected to the TCR extracellular domain by a linker; and
wherein the linker comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 4.

### Claim 30

A pharmaceutical composition comprising a T cell from a human subject, wherein the T cell comprises a recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising:
(a) a TCR subunit comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and
(b) a murine, human or humanized scFv comprising an antigen binding domain; and a pharmaceutically acceptable carrier;
wherein the TCR subunit and the antigen binding domain are operatively linked;
wherein the extracellular domain, the transmembrane domain and the intracellular signaling domain are derived from a single subunit, wherein the single subunit is CD3 epsilon;
wherein the single subunit consists of an amino acid sequence with amino acids 23-207 of SEQ ID NO: 56;
wherein the TFP functionally interacts with an endogenous TCR when expressed in a T cell;
wherein the T cell exhibits increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the antigen binding domain compared to a T cell not containing the TFP;
wherein the TFP lacks a costimulatory domain and lacks a heterologous stimulatory domain;
wherein the antigen binding domain is connected to the TCR extracellular domain by a linker;
wherein the linker comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 4;
wherein the antigen binding domain comprises an anti-CD19 binding domain; and
wherein the anti-CD19 binding domain comprises (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively; (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 35, respectively.