Compositions and methods for identifying functional anti-tumor t cell responses

A method of functionally evaluating a candidate tumor antigen for the ability to induce a T cell response in a subject comprising:

• performing next generation sequencing on pre-treatment tumor and matched normal samples;
• identifying somatic mutations in matched tumor and normal samples;
• wherein (1) distinct paired reads contained the mutation in the tumor;
• (2) fraction of distinct paired reads containing a particular mutation in the tumor comprise at least 10% of the total distinct read pairs and (3) mismatched bases are not present in >1% of the reads in the matched normal sample nor databases of common germline variants derived from dbSNP and (4) position of the mutation is covered in both the tumor and normal sample;
• inputting exome data into a neoantigen prediction pipeline to generate candidate tumor mutation associated neoantigens (MANAs), wherein the neoantigen prediction pipeline assesses immunogenicity of a candidate tumor MANA antigen by analyzing exome data combined with the subject's HLA class I haplotype utilizing a neoantigen prediction platform for evaluating binding of somatic peptides to class I HLA, antigen processing, self-similarity, MHC presentation, and gene expression to generate candidate antigens;
• culturing autologous T cells from the subject with the candidate tumor MANA antigen, said candidate tumor MANA antigen comprising a peptide or a protein, in order to stimulate expansion of the T cells;
• isolating deoxyribonucleic acid (DNA) from bulk T cells from the culture;
• amplifying and sequencing T cell receptor-β (TCR-β) complementarity-determining region 3 (CDR3) DNA to determine frequencies of Vβ CDR23 clonotypes in the bulk T cells;
• comparing the frequencies of the Vβ CDR3 clonotypes in the bulk T cells to Vβ CDR3 clonality in T cells cultured in the absence of the candidate tumor MANA antigen and/or in uncultured T cells to identify antigen specific Vβ CDR3 clonotypes;
• determining that the candidate tumor MANA antigen has the ability to induce a T cell response if the frequency of antigen specific Vβ CDR3 clonotypes identified in the bulk T cells is higher than the frequency in the T cells cultured in the absence of the candidate tumor MANA antigen and/or uncultured T cells;
• and
• incorporating the candidate tumor MANA antigen determined to have the ability to induce a T cell response into an immunogenic treatment composition.