Somatic mutations in cancer can result in neoantigens that serve as attractive targets for cancer immunotherapy. Most human cancers develop “spontaneously” and harbor fewer mutations than murine cancers induced with carcinogens. The murine fibrosarcoma Ag104A arose spontaneously in an aging C3H mouse and serves here as a model to analyze adoptive T cell therapy under conditions that closely resemble the clinical situation. A tumor-specific TCR (M2/3) was isolated from T cells of mice immunized with Ag104A cancer cells. To identify the target of M2/3, mutations in Ag104A were determined by whole exome sequencing and their expression was verified by RNA sequencing. The resulting 77 potential neoantigens were expressed as tandem minigenes and pulsed into target cells engineered with H-2k restriction elements. Subsequent analysis revealed that the specific M2/3 response was directed against an H-2Kk-presented peptide derived from mutant Trp53. The p53D253E mutation (mp53) was tested as target for neoantigen-specific TCR gene therapy. M2/3-engineered T cells were adoptively transferred into immunodeficient C3HxRag2-/- mice bearing established Ag104A tumors. The transferred T cells expanded in an antigen-specific manner and induced growth arrest of this otherwise aggressive and fast-progressing cancer followed by a phase of stable disease. However, all tumors relapsed 4-5 weeks after treatment. Analysis of the tumors that escaped mp53-specific TCR gene therapy showed that their populations were dominated by antigen-negative variants. Further analysis indicated that tumor escape was a result of low-frequency occurring mp53-negative variants due to intratumor heterogeneity. These findings suggest that neoantigen-specific TCR gene therapy is effective against a single target, since mp53-positive cancer cells were successfully eliminated in vivo. Interestingly, although the p53D253E mutation confers a growth advantage and higher tumorigenicity to Ag104A cancer cells and is supposed to be cancer-driving, it proved to be insufficient for eradication of established cancer when used alone as a target antigen of T cell therapy. Analysis of the Ag104A tumor model emphasizes intratumor heterogeneity as a critical parameter of neoantigen-specific TCR gene therapy that has to be addressed in clinical settings in order to make this form of immunotherapy successful for the treatment of human cancer.