Tetramer based approach for efficient identification and isolation of neo-antigen specific CD8 T cells from peripheral blood (PBL) of patients with metastatic cancers

Methods The strategy utilized whole exome sequencing data to identify somatic non-synonymous mutations and then insilico algorithms to predict minimal epitopes encoding mutated amino acids for each patient specific HLA-allele. CD8-enriched PBL from each patient were stained with tetramers generated in house by a UV-exchangeable technique as previously described for A*02:01, A*03:01, A*11:01, B*07:02, and a commercial tetramer was acquired for B*57:01. Based on the initial staining frequency (+tetramer T cells recognizing 7 unique neo-antigens from the PBL of 4 patients (ranging from 1 to 4 per patient). We enriched the frequencies of CD8tetramer cells from 0.5 to >85%, 0.3 to >65% and 0.01 to 3% from the PBL of patients with colorectal (3971-A*02:01), lung (4014B*57:01), and ovarian (4067-B*07:02) cancers respectively, using individual tetramers. Populations reactive with three HLA-A*11:0-restricted and one HLA-A*03:01-restricted neoantigens were also isolated from the PBL of lung cancer patients 4014 and 4037, respectively, using a pooled tetramer approach. Results Overall, the isolated T cells recognized mutated epitopes when co-cultured with autologous CD14 monocytes pulsed with mutated peptides in the context of appropriate MHC-I alleles including HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-B*07:02, and HLA-B*57:01, with reactivity detected using IFN-g ELISA. Using single cell PCR, we could clone the TCRs reactive with an HLA-*02:01presented colon cancer neoantigen and an HLA-B*57:01presented lung cancer neoantigen. Evaluation of PBL retrovirally-transduced with these TCRs demonstrated that they bound to tetramers and secreted IFN-g when cocultured with CD14 monocytes pulsed with appropriate mutated peptides.