Granted Patent B2

US 12,351,872 · App. 17/579,900 · Granted Jul 8, 2025

Inventors: Mark Klinger (Seattle, WA); Malek Faham (Seattle, WA)

Assignee: Adaptive Biotechnologies Corporation

C12Q1/6881C12Q2600/106C12Q2600/158C12Q2600/16

Abstract

Claims (28)

1. A method for identifying clonotypes of antigen-specific T cells in a sample comprising T cells, wherein each clonotype comprises the nucleotide sequences of a TCRα segment and a TCRβ segment that are a pair of sequences specific to a T cell, the method comprising the steps of:

(a) exposing the sample to a plurality of antigens in a plurality of different reaction mixtures, wherein each antigen of said plurality of antigens is present in a predetermined unique subplurality of the plurality of reaction mixtures;

(b) selecting antigen-specific T cells from each of the plurality of reaction mixtures;

(c) sequencing nucleic acids encoding the TCRα segment and the TCRβ segment from:

(i) the antigen-specific T cells selected in step (b) from each of the plurality of reaction mixtures, wherein the sequencing produces a plurality of sequence reads for each reaction mixture from which clonotypes and clonotype frequencies are determined, and

(ii) T cells obtained from the sample prior to step (a), wherein the sequencing produces a plurality of sequence reads from which clonotypes and clonotype frequencies are determined;

(d) for each reaction mixture, comparing the clonotype frequencies determined in step (c) (i) with the clonotype frequencies determined in step (c) (ii); and

(e) for each of the plurality of antigens, identifying a clonotype of an antigen-specific T cell that has an increased frequency in each of the reaction mixtures in which the antigen was present and does not have an increased frequency in each of the reaction mixtures in which the antigen was absent.

2. The method of claim 1 , wherein the sample comprises at least 1000 T cells.

3. The method of claim 2 , wherein the sequencing is performed by high-throughput sequencing.

4. The method of claim 3 , wherein the high-throughput sequencing comprises a step of spatially isolating individual molecules of the nucleic acids on a solid surface and sequencing the isolated individual molecules in parallel to provide the sequence reads.

5. The method of claim 3 , wherein the plurality of antigens comprises a plurality of proteins or a plurality of peptides.

6. The method of claim 5 , wherein the antigen-specific T cells are selected based on interaction with an antigen in the reaction mixture.

7. The method of claim 6 , wherein the T cells in the sample are from a population of individuals.

8. The method of claim 1 , wherein the sequencing is performed by high-throughput sequencing.

9. The method of claim 8 , wherein the high-throughput sequencing comprises a step of spatially isolating individual molecules of the nucleic acids on a solid surface and sequencing the isolated individual molecules in parallel to provide the sequence reads.

10. The method of claim 1 , wherein the plurality of antigens comprises a plurality of proteins or a plurality of peptides.

11. The method of claim 1 , wherein the antigen-specific T cells are selected based on interaction with an antigen in the reaction mixture.

12. The method of claim 1 , wherein the T cells in the sample are from a population of individuals.

13. The method of claim 1 , wherein the sample is a tissue sample.

14. The method of claim 13 , wherein the tissue sample is a blood sample or a sample of peripheral blood mononuclear cells derived from peripheral blood.

15. The method of claim 13 , wherein the plurality of antigens are presented by gene-modified cells expressing the antigens.

16. The method of claim 14 , wherein the plurality of antigens are presented by gene-modified cells expressing the antigens.

17. A method of producing a patient-specific immunotherapeutic reagent comprising:

(a) identifying clonotypes of antigen-specific T cells in a sample comprising T cells, wherein each clonotype comprises the nucleotide sequences of a TCRα segment and a TCRβ segment that are a pair of sequences specific to a T cell, and wherein the method comprises the steps of claim 1 ; and

(b) producing a patient-specific immunotherapeutic reagent comprising a TCRα segment and a TCRβ segment that are encoded by one of the clonotypes identified in step (a).

18. The method of claim 17 , wherein the patient-specific immunotherapeutic reagent is a reconstituted T cell.

19. The method of claim 17 , wherein the patient has cancer and wherein the antigen-specific T cells are cancer antigen-specific T cells.

Assignments (4)

Continuity (3)

References Cited (400)