# US 9,751,928 B2 · Enhanced affinity T cell receptors and methods for making the same

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- Number: US 9,751,928 B2
- Document ID: US9751928B2
- Status: Granted
- Kind: B2
- Application number: 14/398,206
- Filed: 2013-05-02
- Published: 2015-04-30
- Granted: 2017-09-05
- Earliest claimed: 2012-05-03
- Assignees: Fred Hutchinson Cancer Research Center
- Applicants: Fred Hutchinson Cancer Research Center
- Inventors: Thomas M. Schmitt; Philip D. Greenberg
- Web page: https://patentgrep.com/patent/US9751928B2

## CPC

- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- A61K 38/1774: {Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/32: T-cell receptors [TCR]
- A61K 40/4243: {Wilms tumor 1 [WT1]}
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 5/0636: {T lymphocytes}
- C12N 5/065: {Thymocytes}
- G01N 33/56966: {Animal cells}
- C12N 2501/42: Notch; Delta; Jagged; Serrate
- C12N 2502/1394: Bone marrow stromal cells; whole marrow
- C12N 2502/99: genetically modified cells
- G01N 2333/7051: T-cell receptor (TcR)-CD3 complex
- 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)
- 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 present disclosure provides methods for generating enhanced affinity T cell receptors by agonist selection of hematopoietic progenitor cells expressing an antigen specific TCRα cultured with stromal cells expressing Delta-like-1 or Delta-like-4, compositions prepared from such methods, and uses of thereof.

## Claims

### Claim 1

A method for generating an enhanced affinity T cell receptor (TCR), comprising:
a. contacting one or more hematopoietic progenitor cells with one or more stromal cells and a peptide antigen, under conditions and for a time sufficient to induce differentiation of at least one of the hematopoietic progenitor cells into a double negative (DN) TCRαβ30 thymocyte,
wherein the one or more hematopoietic progenitor cells comprise a non-endogenous polynucleotide encoding a TCRα chain from a parent TCR specific for the peptide antigen, and
wherein the one or more stromal cells comprise a non-endogenous polynucleotide encoding Delta-like-1 or Delta-like-4 and a polynucleotide encoding an MHC molecule;
b. introducing at least one polynucleotide encoding a TCRβ chain isolated from the DN TCRαβ+thymocyte generated in step (a) into one or more cells that are capable of expressing a TCR on the cell surface and comprise the non-endogenous polynucleotide encoding the TCRα chain from step (a), thereby generating one or more cells expressing a candidate TCR; and
c. identifying an enhanced affinity TCR expressed by at least one of the one or more cells generated in step (b),
wherein identifying the enhanced affinity TCR comprises comparing the binding affinity of the candidate TCR to the binding affinity of the parent TCR.

### Claim 2 (depends on 1)

The method of claim 1, wherein the hematopoietic progenitor cells comprise thymocyte progenitor cells, or the hematopoietic progenitor cells are derived from hematopoietic stem cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the hematopoietic progenitor cells comprise hematopoietic stem cells derived from bone marrow or cord blood.

### Claim 4 (depends on 1)

The method of claim 1, wherein a viral vector is used to introduce the non-endogenous polynucleotide encoding the TCRα chain specific for the peptide antigen into the hematopoeitic progenitor cells.

### Claim 5 (depends on 4)

The method of claim 4, wherein the viral vector is a retroviral vector or a lentiviral vector.

### Claim 6 (depends on 1)

The method of claim 1, wherein the stromal cell expresses Delta-like-1.

### Claim 7 (depends on 1)

The method of claim 1, wherein the stromal cell is derived from OP9.

### Claim 8 (depends on 1)

The method of claim 1, wherein the method further comprises selecting the cells that are capable of expressing a TCR on the cell surface resulting from step (b) with MHC-peptide tetramer staining one or more times.

### Claim 9 (depends on 1)

The method of claim 1, wherein a viral vector is used to introduce the polynucleotides encoding the TCRβ chains from step (b) into the cells that are capable of expressing TCR on the cell surface.

### Claim 10 (depends on 9)

The method of claim 9, wherein the viral vector is a retroviral vector or a lentiviral vector.

### Claim 11 (depends on 1)

The method of claim 1, wherein the cells that are capable of expressing a TCR on the cell surface are derived from TCRα−/β−58 T cell hybridoma.

### Claim 12 (depends on 1)

The method of claim 1, wherein the enhanced affinity TCR is a human TCR.

### Claim 13 (depends on 1)

The method of claim 1, wherein the MHC molecule comprises HLA-A2 and human beta-2-microglobulin (β2M).

### Claim 14 (depends on 1)

The method of claim 1, wherein the peptide antigen is selected from the group consisting of: a viral antigen, a bacterial antigen, a cancer antigen, and an autoimmune antigen.

### Claim 15 (depends on 14)

The method of claim 14, wherein the peptide antigen is WT1 peptide antigen or mesothelin peptide antigen.

### Claim 16 (depends on 15)

The method of claim 15, wherein the WT1 peptide antigen comprises an amino acid sequence RMFPNAPYL (SEQ ID NO:2).

### Claim 17 (depends on 15)

The method of claim 15, wherein the mesothelin peptide antigen comprises an amino acid sequence GQKMNAQAI (SEQ ID NO:31).

### Claim 18 (depends on 1)

The method of claim 1, wherein the peptide antigen is added to the hematopoietic progenitor cell and stromal cell in culture.

### Claim 19 (depends on 1)

The method of claim 1, wherein the stromal cell comprises a polynucleotide encoding a peptide antigen.

### Claim 20 (depends on 1)

The method of claim 1, further comprising prior to the introducing of step (b), isolating the one or more polynucleotides encoding one or more TCRβ chains from the DN TCRαβ+ thymocytes, wherein the isolating comprises selecting one or more TCRβ chains comprising the same Vβ gene as the β chain of the parent TCR and wherein the introducing comprises introducing at least one of the one or more polynucleotides encoding a selected TCRβ chain into the cell.