US 12,435,372 B2
Methods for profiling the T-cell-receptor repertoire
Nir Hacohen, Brookline, MA (US); Catherine Ju-Ying Wu, Brookline, MA (US); Edward F. Fritsch, Concord, MA (US); and Ute E. Burkhardt, Belmont, MA (US)
Assigned to The Broad Institute, Inc., Cambridge, MA (US); Dana-Farber Cancer Institute, Inc., Boston, MA (US); and The General Hospital Corporation, Boston, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); Dana-Farber Cancer Institute, Inc., Boston, MA (US); and The General Hospital Corporation, Boston, MA (US)
Filed on Mar. 30, 2021, as Appl. No. 17/217,864.
Application 17/217,864 is a continuation of application No. 15/537,785, granted, now 10,993,997, previously published as PCT/US2015/067154, filed on Dec. 21, 2015.
Claims priority of provisional application 62/094,859, filed on Dec. 19, 2014.
Prior Publication US 2021/0379168 A1, Dec. 9, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. A61K 39/00 (2006.01); A61K 35/17 (2025.01); A61K 40/11 (2025.01); A61K 40/32 (2025.01); A61K 40/42 (2025.01); A61K 40/46 (2025.01); C12N 15/10 (2006.01); C12Q 1/6881 (2018.01); G01N 33/569 (2006.01)
CPC C12Q 1/6881 (2013.01) [A61K 39/0011 (2013.01); A61K 40/11 (2025.01); A61K 40/32 (2025.01); A61K 40/42 (2025.01); A61K 40/4201 (2025.01); A61K 40/46 (2025.01); C12N 15/1093 (2013.01); G01N 33/56972 (2013.01); A61K 2039/515 (2013.01); A61K 2039/572 (2013.01); C12Q 2600/158 (2013.01); C12Q 2600/16 (2013.01); G01N 2500/10 (2013.01)] 24 Claims
OG exemplary drawing
 
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).