# US 2018/0322244 A1 · Method for identifying antigen-specific t cell receptors in primate

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- Number: US 2018/0322244 A1
- Document ID: US20180322244A1
- Status: Published application
- Kind: A1
- Application number: 16/021,024
- Filed: 2018-06-28
- Published: 2018-11-08
- Earliest claimed: 2016-07-13
- Assignees: Qyuns Therapeutics Co., Ltd.
- Applicants: Qyuns Therapeutics Co.,Ltd.
- Inventors: Guo-liang Yu
- Web page: https://patentgrep.com/patent/US20180322244A1

## CPC

- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/2818: {against CD28 or CD152}
- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/21: from primates, e.g. man
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- G16B 30/10: Sequence alignment; Homology search
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- 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)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

Provided are methods for identifying T-cell receptors that bind to an antigen in primates. In certain embodiments, the methods comprise: sequencing the cDNA prepared from a combined population of T-cells to obtain a plurality of T-cell receptor alpha chain sequences and a plurality of T-cell receptor beta chain sequences; grouping the obtained alpha chain sequences into alpha chain groups based on their CDR3 sequences; grouping the obtained beta chain sequences into beta chain groups based on their CDR3 sequences; pairing the alpha chain groups with the beta chain groups based on the number of alpha chain or beta chain sequences in each group; pairing one alpha chain sequence and one beta chain sequence in each paired alpha chain group and beta chain group; and testing candidate T-cell receptor comprising the paired alpha chain and beta chain sequences for binding to the antigen.

## Claims

### Claim 1

A method comprising steps of:
a) obtaining a population of T cells from a non-human primate that has been immunized by an antigen;
b) preparing cDNA from the population of T cells without separating the population of T cells into single cells;
c) sequencing the cDNA to obtain:
(c1) a plurality of T-cell receptor (TCR) α-chain sequences and
(c2) a plurality of TCR β-chain sequences,
wherein each of the TM α-chain sequences comprises an α-chain framework, CDR-α1, CDR-α2 and CDR-α3, and wherein each of the TCR β-chain sequences comprises a β-chain framework, CDR-β1, CDR-β2 and CDR-β3;
d) grouping said obtained plurality of TCR α-chain sequences into a plurality of α-chain groups, wherein the CDR-α3 of said α-chain sequences in each α-chain group contain 0, 1 or 2 amino acid differences relative to one another;
e) grouping said obtained plurality of TCRβ-chain sequences into a plurality of β-chain groups, wherein the CDR-β3 of said β-chain sequences in each β-chain group contain 0, 1 or 2 amino acid differences relative to one another;
f) ranking the plurality of α-chain groups according to the number of TCR β-chain sequences in each α-chain group, wherein
a first α-chain group has the most members of TCR α-chain sequences and a
second α-chain group has the second most members of TCR α-chain sequences,
and so on-for the plurality of α-chain groups;
g) ranking the plurality of β-chain groups according to the number of TCR β-chain sequences in each β-chain group, wherein
a first β-chain group has the most members of TCR β-chain sequences and a
second β-chain group has the second most members of TCR β-chain sequences,
and so on for the plurality of β-chain groups;
h) pairing the first α-chain group with the first β-chain group;
i) pairing a first TCR α-chain sequence from the first α-chain group and a first TCR β-chain sequence from the first β-chain group,
wherein
the first TCR α-chain sequence comprises a first α-chain framework, first CDR-α1, first CDR-α2 and first CDR-α3, and
the first TCR β-chain sequence comprises a first β-chain framework, first CDR-β1, first CDR-β2 and first CDR-β3, and
j) testing a first candidate TCR comprising the first TCR α-chain sequence from the first α-chain group and the first TCR β-chain sequence from the first β-chain group for binding to the antigen.

### Claim 2 (depends on 1)

The method of claim 1, wherein the antigen is a human antigen.

### Claim 3 (depends on 1)

The method of claim 1, wherein the non-human primate is an old-world monkey, an orangutan, a gorilla, or a chimpanzee.

### Claim 4 (depends on 1)

The method of claim 1, wherein the non-human primate is a crab-eating macaque, a rhesus macaque or a pig-tailed macaque.

### Claim 5 (depends on 1)

The method of claim 1, wherein the α-chain groups and the β-chain groups contain at least 2 members.

### Claim 6 (depends on 1)

The method of claim 1, wherein the step j) comprises testing the candidate TCR in a blocking assay, a neutralization assay, an agonist assay or an antagonist assay.

### Claim 7 (depends on 1)

The method of claim 1, wherein the step j) comprises testing the candidate TCR in an ELISA.

### Claim 8 (depends on 1)

The method of claim 1, wherein the plurality of TCR α-chain sequences comprises at least 100 different TCR α-chain sequences and the plurality of TCR β-chain sequences comprises at least 100 different TCR β-chain sequences.

### Claim 9 (depends on 1)

The method of claim 1, wherein the cells are obtained from bone marrow, thymus, lymph node or peripheral blood.

### Claim 10 (depends on 1)

The method of claim 1, further comprising:
k) aligning the first TCR α-chain sequence with a human TCR α-chain sequence comprising a human α-chain framework, wherein the human α-chain framework is at least 90% identical to the first α-chain framework of the first TCR α-chain sequence;
l) aligning the first TCR β-chain sequence with a human TCR β-chain sequence comprising a human β-chain framework, wherein the human β-chain framework is at least 90% identical to the first β-chain framework of the first TCR β-chain sequence;
m) substituting at least one amino acid residue in the first TCR α-chain sequence with the corresponding amino acid of the human TCR α-chain sequence or at least one amino acid residue in the first TCR β-chain sequence with the corresponding amino acid of the human TCR β-chain sequence, thereby producing a humanized TCR.

### Claim 11 (depends on 5)

The method of claim 5, further comprising:
n) pairing a second TCR α-chain sequence from the first α-chain group and a second TCR β-chain sequence from the first β-chain group,
wherein
the second TCR α-chain sequence comprises a second α-chain framework, a second CDR-α1, a second CDR-α2 and a second CDR-α3 containing 0, 1 or 2 amino acid differences relative to the first CDR-α3, and
the second TCR β-chain sequence comprises a second β-chain framework, a second CDR-β1, a second CDR-β2 and a second CDR-β3 containing 0, 1 or 2 amino acid differences relative to the first CDR-β3, and
o) testing a second TCR comprising the second TCR α-chain sequence from the first α-chain group and the second TCR β-chain sequence from the first β-chain group for binding to the antigen.

### Claim 12 (depends on 1)

The method of claim 1, wherein in step c) the cDNA is sequenced using high throughput sequencing technology.

### Claim 13 (depends on 1)

The method of claim 1, further comprising
pairing the second α-chain group with the second β-chain group;
pairing an TCR α-chain sequence from the second α-chain group and an TCR β-chain sequence from the second β-chain group, wherein
the TCR α-chain sequence from the second α-chain group comprises a second α-chain framework, a second CDR-α1, a second CDR-α2 and a second CDR-α3, and the TCR β-chain sequence from the second β-chain group comprises a second β-chain framework, a second CDR-β1, a second CDR-β2 and a second CDR-β3; and
testing a second candidate TCR comprising the TCR α-chain sequence from the second α-chain group and the TCR β-chain sequence from the second β-chain group for binding to the antigen.