Methods of isolating neoantigen-specific T cell receptor sequences

Patent metadata

Field	Value
Publication number	US-11898207-B2
Application number	US-201816495508-A
Country	US
Kind code	B2
Filing date	Mar 28, 2018
Priority date	Mar 31, 2017
Publication date	Feb 13, 2024
Grant date	Feb 13, 2024

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.

First claim

The invention claimed is:

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.

2. 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.

3. 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.

4. 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.

5. 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).

6. 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 CDR 3 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 CDR 3 sequence collected in (j).

7. 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 CDR 3 sequence collected in (j).

8. 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 CDR 3 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 perf

Assignees

• Us Health

Inventors

• Lu Yong-Chen
• Fitzgerald Peter
• Zheng Zhili
• Rosenberg Steven A

Classifications

• A61K40/4253

  GTPases, e.g. Ras or Rho · CPC title

• A61K40/4201

  Neoantigens · CPC title

• A61K40/42

  Cancer antigens · CPC title

• A61K40/32

  T-cell receptors [TCR] · CPC title

• A61K40/11

  T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells · CPC title