Figure 1.

Establishment of a pipeline to discover and validate KRAS neoantigens

Overview of the cell-based systematic pipeline established to discover and validate KRAS neoantigens using targeted MS (top) and an in vitro T cell stimulation protocol (middle). A375 cells were engineered to simultaneously express four KRAS^G12C/D/R/V mutations and an HLA-I molecule of interest. After purifying HLA-I peptides, targeted MS was used to detect individual KRAS neoantigens (lines with a colored box) in the presence of endogenous peptides that are also presented by the transduced HLA-I molecules (gray lines). An in vitro stimulation protocol was used to elicit naive T cell responses against the detected KRAS neoantigens. For a subset of the detected neoantigens (dashed arrows represent expanded pipeline), a cytotoxicity assay was performed to show that induced T cells can kill tumor cell lines that naturally express the KRAS^G12V mutation. Cytotoxicity assays were also used to demonstrate that cloned TCRs can kill tumor cells that naturally present the KRAS^G12V neoantigen.