Methods for profiling the T-cell-receptor repertoire

Patent metadata

Field	Value
Publication number	US-12435372-B2
Application number	US-202117217864-A
Country	US
Kind code	B2
Filing date	Mar 30, 2021
Priority date	Dec 19, 2014
Publication date	Oct 7, 2025
Grant date	Oct 7, 2025

Abstract

The present disclosure relates to methods for profiling subject specific and personalized T cell receptor (TCR) repertoires using a single-cell sequencing method. More particularly, disclosed are methods for determining binding of T cell receptors to subject specific neoantigens. In addition, the techniques herein may identify the antigenic targets of T cell receptors in the context of tumor neoantigens. Moreover, the present disclosure enables the discovery of T cell targets in numerous diseases, with implications for understanding the basic mechanisms of the mammalian immune response and for developing antigen-specific diagnostic markers and therapies. Finally, cloned TCRs can be used to formulate personalized immunotherapies for those inflicted with a disease, such as cancer.

First claim

What is claimed is:

1. A method of selecting a T cell receptor (TCR) or a subject-specific peptide targeted by a TCR for preparing a personal pharmaceutical composition for administration to a human subject with a disease or condition, the method comprising:
(a) cloning CDR3 alpha and CDR3 beta sequences of each of at least 1,000 paired TCR-alpha and TCR-beta chain nucleic acid sequences into a pre-assembled library of recombinant expression vectors, thereby assembling a plurality of recombinant TCR expression vectors, wherein each pair of the at least 1,000 paired TCR-alpha and TCR-beta chain nucleic acid sequences originated from a single T cell of a biological sample comprising a plurality of T cells from the human subject, wherein the plurality of T-cells comprises T-cells that are reactive against one or more subject-specific peptides, wherein the pre-assembled variable chain library of recombinant expression vectors comprises:
(i) a first set of recombinant expression vectors, each recombinant expression vector of the first set comprising a different TCR-alpha variable fragment and a constant beta (V alpha-C beta), and
(ii) a second set of recombinant expression vectors, each recombinant expression vector of the second set comprising a different TCR-beta variable fragment and a constant alpha (V beta-C alpha);

(b) expressing recombinant TCRs encoded by the plurality of recombinant TCR expression vectors in reporter T cells, wherein the at least 1,000 paired TCR-alpha and TCR-beta chain nucleic acid sequences comprise paired TCR-alpha and TCR-beta chain nucleic acid sequences that occur at a frequency of 0.3% or less in the plurality of T cells of the biological sample;

and
(c) selecting
(i) a recombinant TCR expressed in (b) that binds to a subject-specific peptide: MHC complex; or
(ii) a subject-specific peptide of a subject-specific peptide: MHC complex that binds to a recombinant TCR expressed in (b).

2. The method of claim 1 , wherein the method comprises assaying binding of the recombinant TCRs expressed in the reporter T cells to subject-specific peptide: MHC complexes.

3. The method of claim 2 , wherein assaying comprises incubating the reporter T cells with antigen presenting cells (APCs) that (A) express an MHC encoded by the human subject's genome and (B) present one or more subject-specific antigenic peptides expressed by diseased cells of the human subject.

4. The method of claim 3 , wherein assaying further comprises measuring activation of a reporter in the reporter T cells that is activated upon binding of a recombinant TCR expressed in the reporter T cells to a subject-specific peptide: MHC complex of the APCs.

5. The method of claim 1 , wherein peripheral blood mononuclear cells (PBMCs) in the biological sample have been stimulated with a subject-specific peptide prior to (a), wherein the PBMCs comprise the single T cell.

6. The method of claim 1 , wherein the method further comprises determining the sequences encoding at the least 1,000 paired TCR-alpha and TCR-beta chains by high-throughput sequencing.

7. The method of claim 6 , wherein high-throughput sequencing comprises sequencing polynucleotides from single T cells.

8. The method of claim 7 , wherein sequencing comprises sequencing polynucleotides from single T cells that have not been expanded ex vivo.

9. The method of claim 1 , wherein the reporter T cells express at least one activation marker selected from the group consisting of CD25, CD54, CD69, CD38, CD45RO, CD49d, CD40L, CD137, IFN-y, IL-2, IL-4, CD107a, and CD134.

10. The method of claim 1 , wherein expressing recombinant TCRs comprises expressing recombinant soluble TCRs (sTCRs).

11. The method of claim 1 , wherein the human subject has cancer, an autoimmune disease or an infection.

12. The method of claim 11 , wherein the human subject has cancer, and wherein the one or more subject-specific antigenic peptides are expressed by cancer cells of the human subject and are not encoded by non-cancer cells of the human subject.

13. The method of claim 1 , wherein the method further comprises sequencing the whole genome or whole exome of cancer cells and non-cancer cells of the human subject and identifying the peptides that comprise a mutation that is encoded by cancer cells of the human subject and not encoded by non-cancer cells of the human subject.

14. The method of claim 1 , wherein the biological sample from the human subject comprises a tumor biopsy, tumor infiltrating lymphocytes (TILs) or peripheral blood.

15. The method of claim 1 , wherein the reporter T cells are a cell line.

16. The method of claim 15 , wherein the reporter T cells comprise a fluorescent reporter gene.

17. The method of claim 1 , wherein the reporter T cells are deficient for endogenous TCRs and comprise a reporter gene that is activated upon binding of a TCR to an antigenic peptide.

18. The method of claim 1 , wherein each of the 1,000 paired TCR-alpha and TCR-beta chain sequences comprise a unique barcode sequence.

19. The method of claim 1 , wherein the method comprises selecting a subject-specific peptide of a subject-specific peptide: MHC complex that binds to a recombinant TCR expressed in (b).

20. The method of claim 19 , wherein the method further comprises preparing a personal pharmaceutical composition comprising the selected subject-specific peptide.

21. The method of claim 20 , further comprising administering the pharmaceutical composition to the human subject.

22. The method of claim 1 , wherein the method comprises selecting a recombinant TCR expressed in (b) that binds to a subject-specific peptide: MHC complex.

23. The method of claim 22 , wherein the method further comprises preparing a personal pharmaceutical composition comprising the selected recombinant TCR or a T cell comprising a polynucleotide encoding the selected recombinant TCR.

24. The method of claim 23 , further comprising administering the pharmaceutical composition to the human subject.

Assignees

• Broad Inst Inc
• Dana Farber Cancer Inst Inc
• Massachusetts Gen Hospital

Inventors

• Hacohen Nir
• Wu Catherine Ju-Ying
• Fritsch Edward F
• Burkhardt Ute E

Classifications

• G01N2500/10

  involving cells · CPC title

• G01N33/56972

  White blood cells · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2600/158

  Expression markers · CPC title

• A61K39/0011

  Cancer antigens · CPC title