Methods for profiling the t cell repertoire

A method of selecting a T cell receptor (TCR) or one or more subject-specific antigenic peptides 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) obtaining a biological sample from the human subject, wherein the biological sample comprises a plurality of T cells comprising T cells that are reactive against one or more subject-specific antigenic peptides;
• (b) obtaining at least 1,000 paired TCR-alpha and TCR-beta chain nucleic acid sequences, wherein each pair of the paired TCR-alpha and TCR-beta chain nucleic acid sequences are distinct and have originated from a single T cell of the biological sample of (a);
• (c) cloning CDR3 alpha and CDR3 beta sequences of each of the 1,000 paired TCR-alpha and TCR-beta chain nucleic acid sequences into a pre-assembled variable chain library of recombinant expression vectors to assemble a plurality of recombinant TCR expression vectors, wherein the recombinant expression vectors of the pre-assembled variable chain library 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);
• (d) introducing the plurality of recombinant TCR expression vectors comprising each of the at least 1,000 paired TCR-alpha and TCR-beta chain nucleic acid sequences into 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 of the plurality of T cells of the biological sample of (a);
• (e) assaying recombinant TCRs encoded by the recombinant expression vectors of (d) that are expressed by the reporter T cells, wherein assaying comprises
• (i) incubating the reporter T cells with antigen presenting cells (APCs) that (A) express an HLA 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;
• (ii) measuring activation of a reporter in the reporter T cells that is activated upon binding of the recombinant TCRs encoded by the recombinant expression vectors of (d) to HLA-peptide complexes of the APCs;
• and
• (f) selecting
• (i) at least one of the one or more subject-specific antigenic peptides that when in complex with the HLA encoded by the human subject's genome binds to at least one of the recombinant TCRs encoded by the recombinant expression vectors of (d) based on (e)(ii), or
• (ii) at least one of the recombinant TCRs encoded by the recombinant expression vectors of (d) that binds to the one or more subject-specific antigenic peptide based on (e)(ii).