Abstract The mutational repertoire of cancers creates the epitopes that make cancers immunogenic. Here, through a comprehensive genomic, bioinformatic and immunological analyses, we uncover over a hundred new neo-epitopes in the Meth A fibrosarcoma of BALB/c mice. Interestingly, the predicted affinities of neo-epitopes for MHC I have no bearing on protective anti-tumor immunogenicity; instead, the numerical difference of such affinities between the mutated and un-mutated sequences, named the Differential Agretopic Index (DAI), is a significant although imperfect predictor. The mutated Transportin 3 (Tnpo3)-derived epitope, the highest ranking (by DAI) epitope of Meth A is naturally presented by Meth A cells. Immunization with whole Meth A cells induces mutated Tnpo3 epitope-specific CD8+ T-cell responses. Immunization with mutated Tnpo3 peptide elicits CD8+ T-cell responses that recognize Meth A cells ex vivo, as well as significant protection from a tumor challenge. This tumor immunity is further enhanced by combination of immunization with mutant Tnpo3 with a Toll-like receptor 9 (TLR9) ligand or anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocking antibody.