System for identification of antigens recognized by T cell receptors expressed on tumor infiltrating lymphocytes

Patent metadata

Field	Value
Publication number	US-11603565-B2
Application number	US-201917252222-A
Country	US
Kind code	B2
Filing date	Jun 14, 2019
Priority date	Jun 15, 2018
Publication date	Mar 14, 2023
Grant date	Mar 14, 2023

Abstract

The invention is a method of identifying a cognate antigen for a T-cell receptor using neoantigens from a patient's tumor cells combined with the patient's T-cells and using cell sorting, genome sequencing, expressing TCR genes, presenting tumor neoantigens on MHC complex and uniquely barcoding the T-cells where TCR recognition occurs to tag all components of the TCR recognition complex.

First claim

The invention claimed is:

1. A method of identifying a cognate antigen for a T-cell receptor (TCR) composed of an alpha chain (TCRα) and a beta chain (TCRβ), the method comprising:
(a) obtaining a population of T-cells from a patient wherein each T-cell comprises a TCR comprising TCRα and TCRβ chains;
(b) barcoding nucleic acids from each T-cell with a cell-specific barcode;
(c) sequencing the barcoded nucleic acids to obtain TCRA and TCRB gene sequences coding for the TCRα and TCRβ chains;
(d) identifying TCRA and TCRB genes having the same barcodes as gene pairs encoding a functional TCR;
(e) introducing the TCRA and TCRB gene pairs identified in step (d) into a T-cell, by means of expression vectors, transcriptionally active DNA fragments, or mRNA;
(f) obtaining a population of tumor cells from the patient each possibly comprising one or more tumor neoantigen;
(g) identifying tumor neoantigen genes;
(h) synthesizing neoantigen peptides from the tumor neoantigen genes identified in step (g), by transcribing and translating the tumor neoantigen genes;
(i) combining the neoantigen peptides from step (h) with an MHC-1 antigen presenting complex and a barcode to form a barcoded MHC-neoantigen peptide complex;
(j) contacting the T-cells from step (e) with the barcoded MHC-neoantigen peptide complexes from step (i) to form cell-bound MHC-neoantigen-TCR complexes;
(k) further barcoding the TCR genes and the barcoded MHC-neoantigen peptide complex from each of the cell-bound MHC-neoantigen-TCR complexes formed from step (j) with a cell-specific barcode;
(l) sequencing the TCR genes associated with the cell-specific barcodes and sequencing the MHC-neoantigen complex barcodes associated with the cell-specific barcodes in the T-cells from step (k); and
(m) identifying the neoantigen as the cognate antigen for the TCR if at least one of the TCR genes and MHC-neoantigen complex barcode are associated with the same cell-specific barcode.

2. The method of claim 1 , wherein the population of T-cells is obtained by dissociation of tumor tissue.

3. The method of claim 1 , wherein the TCRA and TCRB gene sequences coding for the TCRα and TCRβ chains are selected from rearranged DNA sequences or RNA sequences.

4. The method of claim 2 , wherein the population of T-cells is obtained from the tumor by protein marker expression based capture.

5. The method of claim 1 , wherein the T-cell barcoding in one or both steps (b) and (k) is performed by a method comprising:
(a) contacting the plurality of T-cells with a mixture of primers comprising a gene-specific sequence and a barcode oligonucleotide annealing region;
(b) contacting the plurality of T-cells with barcode oligonucleotides complementary to the barcode oligonucleotide annealing region in the primer; and
(c) contacting the plurality of T-cells with additional barcode oligonucleotides in each of one or more rounds of split pool synthesis wherein the barcoded oligonucleotide in each round comprises an annealing region complementary to the annealing region of the barcode oligonucleotide from the previous round, thereby assembling cell-specific barcodes on each primer in each of the plurality of T-cells.

6. The method of claim 1 , wherein the barcoding of nucleic acids in step (b) is performed by a method comprising:
(a) partitioning the population of T-cells containing nucleic acids into a plurality of first partitions containing a single cell;
(b) mixing the cell-containing partitions with a plurality of second partitions each containing multiple copies of barcoded oligonucleotide primers comprising gene-specific sequence and a barcode, wherein the barcodes are the same within each partition but differ among partitions;
(c) fusing the first and second partitions; and
(d) forming amplicons with barcoded oligonucleotide primers thereby barcoding the nucleic acids.

7. The method of claim 1 , wherein the barcoding in step (b) and sequencing in step (c) are performed by a method comprising:
(a) partitioning the population of T-cells into a plurality of partitions containing groups of cells;
(b) ligating barcoded adaptors to nucleic acids in each partition;
(c) sequencing the TCRA and TCRB in the adapted nucleic acid;
(d) determining TCRA and TCRB gene pairs base on frequency of co-occurrence in partitions.

8. The method of claim 1 , wherein the sequencing of barcoded TCR genes is sequencing the CDR3 hypervariable regions of the genes.

9. The method of claim 1 , wherein identifying the TCR genes is identifying the CDR3 hypervariable regions of the genes.

10. The method of claim 1 , wherein the tumor neoantigen genes are identified by sequencing of the nucleic acids in the tumor cell and identifying genes with non-silent mutations as tumor neoantigen genes.

11. The method of claim 1 , wherein the tumor neoantigen genes are identified by screening peptide arrays with the patient's serum.

12. The method of claim 1 , wherein the T-cell of step (e) lacks endogenous TCR gene expression.

13. The method of claim 1 , wherein the genes in step (e) are introduced as transcriptionally active DNA fragments.

14. The method of claim 1 , wherein the identified neoantigens are further selected by the ability to bind the MHC-1 molecule.

15. The method of claim 1 , wherein the further barcoding the neoantigen and the TCR genes and the barcoded MHC-in the neoantigen-TCR complex comprises compartmentalizing each complex into a reaction volume containing a cell-specific barcode.

Assignees

• Roche Sequencing Solutions Inc
• Ventana Med Syst Inc

Inventors

• Alexander Nelson R
• Barhoumi Aoune
• Berka Jan
• Chen Rui
• Gallegos Lisa L
• Guettouche Toumy
• Kim Seoyoung
• O'Huallachain Maeve E
• Ozturk Sedide
• Patel Jigar
• Rubelt Florian
• Stanislaw Stacey

Classifications

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title

• C12Q1/6881 Primary

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1034

  Isolating an individual clone by screening libraries · CPC title

• G01N33/56977

  HLA or MHC typing · CPC title