Identification of neoantigen-reactive t cell receptors

A method for identifying a neoantigen-reactive TCR, comprising:

• i. obtaining single-cell gene expression profiles from a population of tumor infiltrating lymphocytes (TIL) isolated from a patient sample,
• ii. performing bioinformatics analyses on the single cell gene expression data to identify TCR clonotypes of interests,
• iii. creating recombinant TCR sequences;
• iv. preparing a reporter T cell comprising a TCR expression cassette encoding a TCR sequence reconstructed from paired TCR α and β chain sequences identified from the clonotype of interest in step ii,
• v. preparing a tandem minigene (TMG) expression vector;
• vi. analyzing the patient sequencing data to identify class I and class II HLA alleles and preparing HLA expression vectors comprising the class I HLA and class II HLA allele sequences;
• vii. preparing an antigen presenting cell (APC) comprising transfecting the TMG expression vector and one or more HLA expression vectors into a cell wherein each transfection condition comprises a TMG and one or two HLA types;
• viii. co-culturing the reporter T cell in step iii with the APC of step vii,
• ix. identifying a positive reporter activity in the reporter T cell to identify a neoantigen-reactive TCR.

A method of identifying a neoantigen-reactive T cell receptor (TCR), comprising:

• i) obtaining TCR α and β chain sequences from tumor infiltrating lymphocytes (TILs) isolated from a patient sample;
• ii) obtaining neoantigen sequences comprising somatic mutations present in the tumor sample, and the germline HLA typing of the patient sample;
• iii) co-culturing
• a) a reporter T cell expressing a TCR sequence reconstructed from the TCR α and 3 chain sequences obtained in step i), and
• b) an antigen presenting cell (APC) that expresses a neoantigen sequence and a matched human leukocyte antigen (HLA) sequence obtained in step ii);
• and
• iv) evaluating the reporter activity in the reporter T cell to identify a neoantigen-reactive TCR.

A recombinant vector comprising a polycistronic expression cassette, wherein the polycistronic expression cassette comprises a transcriptional regulatory element operably linked to a polycistronic polynucleotide that comprises:

• a. a first polynucleotide sequence that encodes a T cell receptor (TCR) alpha chain comprising an alpha chain variable (Vα) region and an alpha chain constant (Cα) region;
• b. a second polynucleotide sequence that comprises a first 2A element;
• c. a third polynucleotide sequence that encodes a TCR beta chain comprising a beta chain variable (Vβ) region and a beta chain constant (Cβ) region;
• d. a fourth polynucleotide sequence that comprises a second 2A element;
• and
• e. a fifth polynucleotide sequence that encodes a fusion protein that comprises IL-15, or a functional fragment or functional variant thereof, and IL-15Rα, or a functional fragment or functional variant thereof.