# US 11,898,207 B2 · Methods of isolating neoantigen-specific T cell receptor sequences

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- Number: US 11,898,207 B2
- Document ID: US11898207B2
- Status: Granted
- Kind: B2
- Application number: 16/495,508
- Filed: 2018-03-28
- Published: 2020-02-20
- Granted: 2024-02-13
- Earliest claimed: 2017-03-31
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Inventors: Yong-Chen Lu; Peter Fitzgerald; Zhili Zheng; Steven A. Rosenberg
- Web page: https://patentgrep.com/patent/US11898207B2

## CPC

- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- 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 40/42: Cancer antigens
- A61K 40/4201: {Neoantigens}
- A61K 40/4253: {GTPases, e.g. Ras or Rho}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C12N 5/0636: {T lymphocytes}
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/686: Polymerase chain reaction [PCR]
- G01N 33/56972: {White blood cells}
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 30/10: Sequence alignment; Homology search
- G16B 30/20: Sequence assembly
- C12N 2502/30: tumour cells
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)
- 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)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

Disclosed are methods of isolating paired T cell receptor (TCR) alpha and beta chain sequences, or an antigen-binding portion thereof. Also disclosed are methods of automatically identifying the TCR alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. Methods of preparing a population of cells that express paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof, are also disclosed. Isolated pairs of TCR alpha and beta chain sequences and isolated populations of cells prepared by the methods are also disclosed.

## Claims

### Claim 1

A method of isolating nucleic acid(s) comprising one or more nucleotide sequences encoding a T cell receptor (TCR) comprising a TCR alpha chain polypeptide and a TCR beta chain polypeptide, or an antigen-binding portion thereof, the method comprising:
(a) isolating, from a biological sample, T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation;
(b) co-culturing the isolated T cells with antigen presenting cells (APCs) that present the mutated amino acid sequence so that the T cells express one or more T cell activation markers;
(c) sorting the co-cultured T cells into separate single T cell samples;
(d) isolating mRNA from each separate single T cell sample;
(e) sequencing the mRNA from each separate single T cell sample, wherein the sequencing comprises:
(i) producing cDNA from the mRNA and amplifying the cDNA;
(ii) producing multiple fragments of the amplified cDNA and tagging the multiple fragments;
(iii) amplifying the tagged, multiple fragments of the cDNA; and
(iv) sequencing the amplified, tagged multiple fragments of the cDNA;
wherein the sequencing identifies the sequences of each of the multiple fragments of cDNA;
(f) aligning the sequences of each of the multiple fragments of cDNA to a known sequence of the one or more T cell activation markers to identify which single T cell sample contained a single T cell which expressed the one or more T cell activation markers;
(g) aligning the sequences of each of the multiple fragments of cDNA to a reference TCR sequence database to identify TCR alpha chain variable (V) segment sequences and TCR beta chain V segment sequences of the multiple fragments of cDNA of each separate single T cell sample which was identified in (f) to express one or more T cell activation markers;
(h) identifying TCR complementarity determining region 3 (CDR3) sequences in the multiple fragments of cDNA containing the TCR alpha chain V segment sequences identified in (g) and in the multiple fragments of cDNA containing the TCR beta chain V segment sequences identified in (g);
(i) counting the number of multiple fragments of cDNA which share the same alpha chain CDR3 amino acid sequence and the number of multiple fragments of cDNA which share the same beta chain CDR3 amino acid sequence;
(j) collecting the highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, the highest number of multiple fragments of cDNA which encode the same beta chain CDR3 sequence and, optionally, the second highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, wherein the alpha chain CDR3 sequence encoded by the second highest number of multiple fragments of cDNA is different from the alpha chain CDR3 sequence encoded by the highest number of multiple fragments of cDNA
to identify the TCR alpha and beta chain CDR3 sequences;
(k) identifying the TCR alpha chain V segment sequence of the highest number of multiple fragments of cDNA collected in (j), the TCR beta chain V segment sequence of the highest number of multiple fragments of cDNA collected in (j) and, optionally, the TCR alpha chain V segment sequence of the second highest number of multiple fragments of cDNA collected in (j)
to identify the TCR alpha and beta chain V segment sequences; and
(l) assembling one or more nucleic acid(s) comprising nucleotide sequences encoding:
a TCR alpha chain polypeptide comprising the TCR alpha chain V segment sequence identified in (k) and the TCR alpha chain CDR3 sequence collected in (j) and
a TCR beta chain polypeptide comprising the TCR beta chain V segment sequence identified in (k) and the TCR beta chain CDR3 sequence collected in (j),
optionally assembling a second one or more nucleic acid(s) comprising nucleotide sequences encoding:
a second TCR alpha chain polypeptide comprising the TCR alpha chain V segment sequence of the second highest number of multiple fragments of cDNA identified in (k) and the TCR alpha chain CDR3 sequence of the second highest number of multiple fragments of cDNA collected in (j) and
the TCR beta chain polypeptide comprising the TCR beta chain V segment sequence identified in (k) and the TCR beta chain CDR3 sequence collected in (j)
to produce isolated nucleic acid(s) comprising one or more nucleotide sequences encoding the TCR comprising the TCR alpha chain polypeptide and the TCR beta chain polypeptide, or an antigen-binding portion thereof, wherein the TCR has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the one or more T cell activation markers comprise one or more of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor alpha (TNF-α), programmed cell death 1 (PD-1), lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1BB, OX40, CD107a, granzyme B, granulocyte/monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

### Claim 3 (depends on 1)

The method according to claim 1, further comprising labeling the mRNA from each separate single T cell sample with a different tag for each separate single T cell sample.

### Claim 4 (depends on 1)

The method according to claim 1, wherein (h) comprises identifying TCR CDR3 sequences by identifying cDNA sequences which encode conserved amino acid residues positioned near the C-terminus of the amino acid sequence which is encoded by the V segment of the alpha and beta chains.

### Claim 5 (depends on 1)

The method according to claim 1, wherein (k) further comprises identifying the TCR alpha chain constant (C) region sequence of the highest number of multiple fragments of cDNA collected in (j) and the TCR beta chain C region sequence of the highest number of multiple fragments of cDNA collected in (j).

### Claim 6 (depends on 5)

The method according to claim 5, wherein (l) comprises assembling nucleic acid(s) comprising one or more nucleotide sequences encoding a TCR alpha chain polypeptide comprising the TCR alpha chain V segment sequence identified in (k), the TCR alpha chain C region sequence identified in (k), and the TCR alpha chain CDR3 sequence collected in (j) and a TCR beta chain polypeptide comprising the TCR beta chain V segment sequence identified in (k), the TCR beta chain C region sequence identified in (k), and the TCR beta chain CDR3 sequence collected in (j).

### Claim 7 (depends on 1)

The method according to claim 1, wherein (l) comprises assembling nucleic acid(s) comprising one or more nucleotide sequences encoding a TCR alpha chain polypeptide comprising the TCR alpha chain V segment sequence identified in (k), an exogenous TCR alpha chain C region sequence, and the TCR alpha chain CDR3 sequence collected in (j) and a TCR beta chain polypeptide comprising the TCR beta chain V segment sequence identified in (k), an exogenous TCR beta chain C region sequence, and the TCR beta chain CDR3 sequence collected in (j).

### Claim 8 (depends on 1)

The method according to claim 1, further comprising receiving, at a user computing device, the sequences of the multiple fragments of cDNA of the single T cell identified in (f);
wherein (g) comprises performing computerized alignment of the sequences of each of the multiple fragments of cDNA to a reference TCR sequence database to identify TCR alpha chain variable (V) segment sequences and TCR beta chain V segment sequences of the multiple fragments of cDNA of the single T cell identified in (f);
wherein (h) comprises performing computerized identification of TCR CDR3 sequences in the multiple fragments of cDNA containing the TCR alpha chain V segment sequences identified in (g) and in the multiple fragments of cDNA containing the TCR beta chain V segment sequences identified in (g);
wherein (i) comprises performing computerized counting of the number of multiple fragments of cDNA which share the same alpha chain CDR3 amino acid sequence and the number of multiple fragments of cDNA which share the same beta chain CDR3 amino acid sequence;
wherein (j) comprises performing computerized collecting of the highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, the highest number of multiple fragments of cDNA which encode the same beta chain CDR3 sequence and, optionally, the second highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, wherein the alpha chain CDR3 sequence encoded by the second highest number of multiple fragments of cDNA is different from the alpha chain CDR3 sequence encoded by the highest number of multiple fragments of cDNA
to identify the TCR alpha and beta chain CDR3 sequences; and
wherein (k) comprises performing computerized identification of the TCR alpha chain V segment sequence of the highest number of multiple fragments of cDNA collected in (j), the TCR beta chain V segment sequence of the highest number of multiple fragments of cDNA collected in (j) and, optionally, the TCR alpha chain V segment sequence of the second highest number of multiple fragments of cDNA collected in (j)
to identify the TCR alpha and beta chain V segment sequences.

### Claim 9 (depends on 1)

A method of preparing a population of cells that express a TCR, or an antigen-binding portion thereof, the method comprising:
isolating nucleic acid(s) comprising one or more nucleotide sequences encoding a TCR comprising a TCR alpha chain polypeptide and a TCR beta chain polypeptide, or an antigen-binding portion thereof, according to the method of claim 1, and
introducing the nucleic acid(s) into host cells to obtain cells that express the TCR, or the antigen-binding portion thereof.

### Claim 10 (depends on 9)

The method of claim 9, further comprising expanding the numbers of host cells that express the TCR, or the antigen-binding portion thereof.