Methods and compositions for detecting single T cell receptor affinity and sequence

Patent metadata

Field	Value
Publication number	US-11162945-B2
Application number	US-201716092607-A
Country	US
Kind code	B2
Filing date	Apr 6, 2017
Priority date	Apr 11, 2016
Publication date	Nov 2, 2021
Grant date	Nov 2, 2021

Abstract

Provided herein are methods for determining the T cell receptor affinity and sequence of antigen-specific T cells using a micropipette adhesion assay and single cell paired TCRα/TCRβ sequencing. Further provided are methods for the treatment of viral infections or cancers by adoptive transfer of high affinity functional T cells.

First claim

What is claimed is:

1. A method for measuring T cell receptor (TCR) affinity and obtaining TCR sequence comprising:
(a) obtaining a population of antigen-specific T cells from a starting population of T cells using pMHC-tagged streptamers;
(b) individually performing a micropipette adhesion assay on each of said antigen-specific T cells, thereby measuring the two-dimensional (2D) TCR affinity to a peptide-major histocompatibility complex (pMHC); and
(c) sequencing the TCR of each of said antigen-specific T cells by paired TCRα/TCRβ sequencing, thereby obtaining the sequence of said TCR.

2. The method of claim 1 , wherein antigen-specific T cell isolation comprises:
(a) staining the starting population of T cells with pMHC-tagged streptamers;
(b) sorting for the streptamer-bound T cells; and
(c) dissociating the streptamers from the T cells, thereby obtaining a population of antigen-specific T cells.

3. The method of claim 2 , wherein dissociating comprises incubating the streptamer-bound T cells in a dissociation buffer comprising biotin and/or sodium azide.

4. The method of claim 3 , wherein the streptamer-bound T cells are incubated for about 15 minutes to about 90 minutes.

5. The method of claim 2 , wherein antigen-specific T cell isolation is at 3° C. to 5° C.

6. The method of claim 1 , wherein step (a) comprises in vitro clonal expansion of an antigen-specific T cell by co-culturing a population of polyclonal T cells with antigen presenting cells loaded with antigen peptide and/or activating a population of polyclonal T cells with CD3 and CD28.

7. The method of claim 1 , wherein the starting population of T cells is a peripheral blood sample comprising CD8 + cytotoxic T Lymphocytes (CTLs) or CD4 + T cells obtained from a patient sample or healthy blood donor sample.

8. The method of claim 1 , wherein the population of antigen-specific T cells comprises less than 200 T cells or less than 100 T cells.

9. The method of claim 1 , wherein the antigen is a tumor associated self-antigen, tumor neo-antigen, or viral antigen.

10. The method of claim 9 , wherein the viral antigen is HCV.

11. The method of claim 1 , wherein step (b) comprises contacting the antigen-specific T cells with red blood cells coated with agonist pMHC and measuring adhesion frequency.

12. The method of claim 11 , wherein the contacting is for 2 seconds to 6 seconds or 3 seconds to 5 seconds.

13. The method of claim 11 , further comprising measuring the TCR and pMHC site density and calculating 2D affinity from adhesion probability and site density.

14. The method of claim 13 , further comprising estimating that variance associated with the 2D TCR affinity measurement comprising:
(a) measuring the standard deviation of TCR site density fluctuation between T cells by measuring the adhesion frequencies of one red blood cell (RBC) coated with anti-TCR antibody against multiple T cells;
(b) measuring the standard deviation of pMHC site density fluctuation between RBCs by measuring the adhesion frequencies of one antigen-specific T cell against multiple RBCs coated with pMHC; and
(c) measuring the standard deviation of TCR site density fluctuation on the surface of one T cell by measuring the adhesion frequencies of one RBC coated with anti-TCR antibody against multiple areas of one T cell.

15. The method of claim 11 , wherein the adhesion frequency is between 30% and 80%.

16. The method of claim 1 , wherein step (c) comprises transferring the antigen-specific T cells individually by micropipette into lysis buffer for reverse transcription, PCR amplification of TCRα/TCRβ, and next-generation sequencing.

17. The method of claim 1 , wherein the paired TCRα/TCRβ sequencing is performed on single antigen-specific T cells.

18. The method of claim 16 , further comprising separating reads according to cell barcodes and clustering reads based on sequence similarity.

19. The method of claim 1 , wherein the TCR affinity is obtained and TCR sequence is amplified in 15 hours to 48 hours or 20 hours to 24 hours.

20. A method of monitoring the TCR repertoire of a subject comprising obtaining TCR affinity and sequence according to claim 1 .

21. The method of claim 16 , wherein the PCR amplification of TCRα/TCRβ comprises primers sequences selected from Table 1 and/or Table 2.

22. The method of claim 1 , wherein the antigen is an antigen associated with a disease.

Assignees

• Univ Texas

Inventors

• Jiang Ning
• Zhang Shuqi
• Ma Keyue
• He Chenfeng

Classifications

• C07K14/7051 Primary

  T-cell receptor (TcR)-CD3 complex · CPC title

• A61K39/385

  Haptens or antigens, bound to carriers · CPC title

• G01N33/555

  Red blood cell · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• G01N33/56972

  White blood cells · CPC title