Granted Patent B2

US 12,435,372 · App. 17/217,864 · Granted Oct 7, 2025

Inventors: Nir Hacohen (Brookline, MA); Catherine Ju-Ying Wu (Brookline, MA); Edward F. Fritsch (Concord, MA); Ute E. Burkhardt (Belmont, MA)

Assignees: The Broad Institute, Inc.; Dana-Farber Cancer Institute, Inc.; The General Hospital Corporation

C12Q1/6881A61K39/0011A61K40/11A61K40/32A61K40/42A61K40/4201A61K40/46C12N15/1093G01N33/56972A61K2039/515A61K2039/572C12Q2600/158C12Q2600/16G01N2500/10

Abstract

Claims (32)

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.

Assignments (4)

Continuity (3)

References Cited (400)