Determining antigen-specific t-cells

• US 10,066,265 B2
• Filed: 04/01/2014
• Issued: 09/04/2018
• Est. Priority Date: 04/01/2014
• Status: Active Grant

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Adaptive Biotechnologies Corp.
• Inventors: Klinger, Mark, Faham, Malek
• Primary Examiner(s): Ault, Addison D
• Application Number:
  US14/242,520
• Publication Number:
  US 20150275296A1
• Time in Patent Office: 1,617 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The invention is directed to methods for determining antigen-specific T cells. In some embodiments, methods of the invention may be implemented by the steps of reacting under interaction conditions one or more antigens with T cells in a plurality of subsets of a tissue sample, such as peripheral blood; sorting antigen-interacting T cells from other T cells; separately sequencing for each subset recombined nucleic acid encoding a segment of a TCR chain from a sample of T cells prior to exposure to antigen and from a sample of T cells isolated based on their interaction with antigen, thereby forming a clonotype profile for the former sample and the latter sample for each subset; and identifying as antigen-specific T cells those T cells associated with a clonotype whose frequency increases in the latter sample relative to its frequency in the former sample.

Claims

1. A method for determining the sequence of one or more T-cell receptor (TCR) chain(s), or a portion thereof, specific for one or more antigens of interest in a sample from one or more individuals and comprising T cells specific for a plurality of antigens, the method comprising the steps of:

(a) sequencing recombined nucleic acids encoding one or more TCR chain(s), or a portion thereof, from a first portion of the sample to generate a first multiplicity of sequence reads obtained from T cells prior to antigen exposure of the sample, wherein the sequencing is high-throughput sequencing;

(b) partitioning a second portion of the sample comprising T cells into a plurality of reaction mixtures and exposing each reaction mixture of the plurality of reaction mixtures to a plurality of antigens, wherein each antigen of said plurality of antigens is present in a unique and predetermined subplurality of the plurality of reaction mixtures;

(c) for each reaction mixture in the plurality of reaction mixtures, separating T cells that interact with one or more antigens in the plurality of antigens from the reaction mixture to obtain a subset of antigen-specific T cells, wherein each of the subsets of antigen-specific T cells corresponds to one reaction mixture in the plurality of reaction mixtures;

(d) for each of the subsets of antigen-specific T cells separated in step (c), sequencing recombined nucleic acids encoding one or more TCR chain(s), or a portion thereof, to generate a multiplicity of sequence reads obtained from each of the subsets of antigen-specific T cells, wherein the sequencing is high-throughput sequencing;

(e) for each reaction mixture in the plurality of reaction mixtures, identifying a plurality of antigen-specific TCR chains, or portion thereof, by comparing the multiplicity of sequence reads obtained from each of the subsets of antigen-specific T cells in step (d) to the first multiplicity of sequence reads obtained from unstimulated T cells in step (a),

wherein the frequency of the sequence reads for the antigen-specific TCR chains, or portion thereof, is increased in the multiplicity of sequence reads obtained from the subsets of antigen-specific T cells compared to the frequency of sequence reads for the antigen-specific TCR chains, or portion thereof, in the first multiplicity of sequence reads obtained from unstimulated T cells; and

(f) for each of the one or more antigens of interest, identifying one or more TCR chains, or portion thereof, specific for the antigen of interest from the one or more TCR chains, or a portion thereof, identified in step (e), wherein the frequency of the sequence reads for the one or more TCR chains, or portion thereof, specific for the antigen of interest is increased in the multiplicity of sequence reads obtained from each of the subsets of antigen-specific T cells in which the antigen of interest was present in the corresponding reaction mixture.

2. The method of claim 1 wherein the high throughput sequencing includes isolating individual molecules of said recombined nucleic acids on a solid surface and sequencing the isolated individual molecules to provide said multiplicity of sequence reads.

3. The method of claim 2, further comprising coalescing the multiplicity of sequence reads obtained from the subsets of antigen-specific T cells to identify each of said antigen-specific TCR chains or portions thereof.

4. The method of claim 1 wherein said step of separating is implemented with an antigen reagent capable of forming a complex with one or more T cells specific for one or more of the plurality of antigens or a binding compound specific for an activation marker present on one or more T cells specific for one or more of the plurality of antigens.

5. The method of claim 4 wherein said antigen reagent is a peptide-MHC multimer complex wherein the peptide of the complex is derived from said antigen of interest.

6. The method of claim 1 wherein said sample comprises peripheral blood or is derived from peripheral blood.

7. The method of claim 1 wherein the sample is from a subject with cancer.

8. The method of claim 1 wherein the number of antigen present in the plurality of antigens is between 2 to 100 antigens.

9. The method of claim 8, wherein the number of antigen present in the plurality of antigens is at least 4.

11. The method of claim 1, wherein the number of antigen present in the plurality of antigens is between 2 to 1000 antigens.

10. A method for determining the sequence of one or more T-cell receptor (TCR) chain(s), or a portion thereof specific for one or more antigens of interest in a sample derived from peripheral blood from one or more individuals and comprising T cells specific for a plurality of antigens, the method comprising the steps of:

(a) sequencing recombined nucleic acids encoding one or more TCR chain(s), or a portion thereof, from a first portion of the sample to generate a first multiplicity of sequence reads obtained from T cells prior to antigen exposure of the sample, wherein the sequencing is high-throughput sequencing;

(b) partitioning a second portion of the sample comprising T cells into a plurality of reaction mixtures and exposing each reaction mixture of the plurality of reaction mixtures to a plurality of antigens, wherein each antigen of said plurality of antigens is present in a unique and predetermined subplurality of the plurality of reaction mixtures, and wherein the plurality of antigen comprises at least 4 antigens;

(c) for each reaction mixture in the plurality of reaction mixtures, separating T cells that interact with one or more antigens in the plurality of antigens from the reaction mixture to obtain a subset of antigen-specific T cells, wherein each of the subsets of antigen-specific T cells corresponds to one reaction mixture in the plurality of reaction mixtures;

(d) for each of the subsets of antigen-specific T cells separated in step (c), sequencing recombined nucleic acids encoding one or more TCR chain(s), or a portion thereof, to generate a multiplicity of sequence reads obtained from each of the subsets of antigen-specific T cells, wherein the sequencing is high-throughput sequencing;

(e) for each reaction mixture in the plurality of reaction mixtures, identifying a plurality of antigen-specific TCR chains, or a portion thereof, by comparing the multiplicity of sequence reads obtained from each of the subsets of antigen-specific T cells in step (d) to the first multiplicity of sequence reads obtained from unstimulated T cells in step (a),

wherein the frequency of the sequence reads for the antigen-specific TCR chains, or a portion thereof, is increased in the multiplicity of sequence reads obtained from the subsets of antigen-specific T cells compared to the frequency of sequence reads for the antigen-specific TCR chains, or a portion thereof, in the first multiplicity of sequence reads obtained from unstimulated T cells; and

(f) for each of the one or more antigens of interest, identifying one or more TCR chains, or a portion thereof, specific for the antigen of interest from the one or more TCR chains, or a portion thereof, identified in step (e), wherein the frequency of the sequence reads for the one or more TCR chains, or a portion thereof, specific for the antigen of interest is increased in the multiplicity of sequence reads obtained from each of the subsets of antigen-specific T cells in which the antigen of interest was present in the corresponding reaction mixture.