Two proteins that often carry a mutation (red dot) that drives cancer (KRASG12V and RAC2P29L) are chosen for further exploration (A). An algorithm predicts multiple potential spliced peptides encompassing the mutations for each protein (B). A second algorithm identifies a small number of putative spliced peptides predicted to bind to HLA-A2 on the surface of target cells (C). In vitro, the proteasome does actually generate a predicted spliced peptide carrying the mutation for RAC2 but not for KRAS (D). Exposing mice to the predicted spliced peptides generates killer T cells that identify the peptides with high affinity (E). The T cell receptors that bind to the spliced peptides are successfully transferred to human immune cells called lymphocytes (F). These ‘transformed’ cells efficiently recognize tumor cells pulsed with the synthetic spliced peptides (G). However, different tumor cell lines that express the mutant proteins (but are not artificially equipped with the spliced peptides) are not recognized by the transformed human immune cells. This suggests that, despite the algorithm’s prediction, these peptides are not produced (or are not produced in large enough numbers) in actual cells (H).