# US 2020/0400680 A1 · Ligand discovery for t cell receptors

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- Number: US 2020/0400680 A1
- Document ID: US20200400680A1
- Status: Published application
- Kind: A1
- Application number: 17/011,911
- Filed: 2020-09-03
- Published: 2020-12-24
- Earliest claimed: 2014-04-04
- Assignees: The Board of Trustees of the Leland Stanford Junior University; California Institute of Technology
- Applicants: The Board of Trustees of the Leland Stanford Junior University; California Institute of Technology
- Inventors: Michael Edward Birnbaum; Juan Luis Mendoza; Michael Thomas Bethune; David Baltimore; Kenan Christopher Garcia
- Web page: https://patentgrep.com/patent/US20200400680A1

## CPC

- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- G01N 2333/7051: T-cell receptor (TcR)-CD3 complex
- G01N 2333/70539: MHC-molecules, e.g. HLA-molecules
- 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)
- 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)

## Abstract

Compositions and methods are provided for the identification of peptide sequences that are ligands for a T cell receptor (TCR) of interest, in a given MHC context.

## Claims

### Claim at position 1

-27. (canceled)

### Claim 28

A method of creating a cell library of diverse candidate peptide ligands of a T-cell receptor (TCR), the method comprising:
providing a population of host cells;
introducing into the host cells nucleic acids encoding polypeptides comprising the diverse candidate peptide ligands;
wherein the polypeptides are configured to be displayed on a surface of the host cells; and
allowing the host cells to express and display the diverse candidate peptide ligands on the surface of the host cells.

### Claim 29 (depends on 28)

The method of claim 28, wherein the polypeptides are single chain polypeptides each comprising (a) a peptide ligand and (b) a binding domain of an MHC protein.

### Claim 30 (depends on 29)

The method of claim 29, wherein the single chain polypeptides each have a structure of P-L1-β-L2-α-L3-T, wherein
P is the peptide ligand;
each of L1, L2 and L3 are flexible linkers of from about 4 to about 12 amino acids in length;
α is a soluble form of an α domain of a human class I MHC protein or of a human class II MHC protein;
β is a soluble form of human class I MHC β2 microglobulin or a soluble form of a β domain of a human class II MHC β protein;
when a is the soluble form of an α domain of a human class I MHC protein, then β is the soluble form of human class I MHC β2 microglobulin;
when α is the soluble form of an α domain of a human class II MHC protein, then β is the soluble form of a β domain of a human class II MHC β protein; and
T is a domain that tethers the single chain polypeptide to the surface of a host cell in the population of host cells.

### Claim 31 (depends on 29)

The method of claim 29, wherein the MHC binding domain comprises α1 and α2 domains of a class I MHC α protein, and β2 microglobulin.

### Claim 32 (depends on 29)

The method of claim 29, wherein the MHC binding domain comprises α1 and β1 domains of a class II MHC protein.

### Claim 33 (depends on 32)

The method of claim 32, wherein the binding domain is encoded by an allele of HLA-DRA and an allele of HLA-DRB4.

### Claim 34 (depends on 32)

The method of claim 32, wherein the binding domain is encoded by an allele of HLA-DRA and an allele of HLA-DRB15.

### Claim 35 (depends on 28)

The method of claim 28, further comprising mutagenizing the nucleic acids using error-prone PCR.

### Claim 36 (depends on 29)

The method of claim 29, wherein the peptide ligand is from about 8 to about 20 amino acids in length.

### Claim 37 (depends on 28)

The method of claim 28, further comprising mutagenizing the nucleic acids along the candidate peptide ligands using mutagenic primers.

### Claim 38 (depends on 29)

The method of claim 29, wherein the peptide ligand is randomized at multiple positions, and wherein the peptide ligand has limited diversity at MHC anchor positions.

### Claim 39 (depends on 28)

The method of claim 28, wherein the cell library is created in the cells using homologous recombination.

### Claim 40 (depends on 29)

The method of claim 29, wherein the cell library comprises at least 106 diverse single chain polypeptides.

### Claim 41 (depends on 28)

The method of claim 28, wherein the host cells are yeast cells.

### Claim 42 (depends on 29)

The method of claim 29, wherein the host cells are yeast cells.

### Claim 43 (depends on 42)

The method of claim 42, wherein the T is Aga2.

### Claim 44 (depends on 29)

The method of claim 29, wherein the flexible linkers are Gly-Ser linkers.

### Claim 45 (depends on 28)

The method of claim 28, wherein
the polypeptides are single chain polypeptides each comprising (a) a peptide ligand and (b) a binding domain of an MHC protein; and
the peptide ligand is from about 8 to about 20 amino acids in length and is randomized at multiple positions and has limited diversity at MHC anchor positions.

### Claim 46 (depends on 29)

The method of claim 29, wherein
the peptide ligand is from about 8 to about 20 amino acids in length and is randomized at multiple positions and has limited diversity at MHC anchor positions;
the α is the soluble form of an α domain of a human class I MHC protein comprising α1 and α2 domains of the human class I MHC protein;
the β is the soluble form of the human class I MHC β2 microglobulin;
the T is Aga2; and
the host cells are yeast cells.

### Claim 47 (depends on 29)

The method of claim 29, wherein
the peptide ligand is from about 8 to about 20 amino acids in length and is randomized at multiple positions and has limited diversity at MHC anchor positions;
the α is the soluble form of an α domain of a human class II MHC protein;
the β is a soluble form of the human class II MHC β protein;
the T is Aga2; and
the host cells are yeast cells.