Implementation of neoantigen vaccines in pilot clinical trial. (A) Trial overview and timeline for patient FLNA-04 (see supplemental Figures 11 and 12 for more detailed versions). (B) The table lists all variants that were predicted to result in high-quality neoantigen vaccine candidates along with corresponding HLA allele, selection criteria, long peptide sequence submitted, synthesis success status, and vaccination pool (supplemental Tables 11 and 12). Short epitope sequences that binding predictions were based on are bolded within the long peptide sequences submitted column. (C) Peptide stabilization of selected candidate neoantigens. Various concentrations of peptides were incubated overnight with ICP-47 expressing (TAP deficient) B-cell line expressing HLA A∗68:01 heavy chain, washed and then stained with W6/32 APC. Mean fluorescence intensity (MFI) of cells pulsed with decreasing peptide concentrations were compared with no peptide pulsed control cells to validate predicted peptide stabilization of MHC class I molecule. (D) PBMCs from C6 apheresis were pulsed with vaccinating peptides and cultured for 12 days in vitro and then challenged with predicted short peptides overnight on IFN-γ–coated ELISPOT plates. (E) Bar graph shows triplicate value SPUs per million PBMCs from D12 culture. (F) An example positive antigen-specific CTL enrichment detected by peptide loaded tetramers stained with tetramer PE and tetramer APC gated on live, CD3/CD8DP CTL from D12 CTL. (G) Antigen-specific CTL from D12 cultures were challenged with artificial APCs pulsed with specific peptides, incubated for 6 hours, and then IFN-γ or TNF–expressing CD3/CD8 DP cells were detected by FACS. APC, antigen-presenting cells; CTL, cytotoxic T lymphocyte; D12, day 12; EOT, end of treatment; FACS, fluorescence-activated cell sorting; MT, mutant; PE, phycoerythrin; PET/CT, positron emission tomography-computed tomography; TAP, transporter associated with antigen processing; TNF, tumor necrosis factor; Tx, treatment.