Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

Abstract T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR specific for a tumor epitope, is a form of adoptive cell therapy (ACT) that has demonstrated promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, but also attractive targets for TCR-T cell therapy. However, MHC class I-restricted TCRs specifically targeting G12-mutant KRAS epitopes in the context of tumors expressing HLA-A2, the most common human HLA-A allele, have remained elusive despite evidence that an epitope encompassing the mutation can bind HLA-A2 and induce T cell responses. We report that post-translational modifications of the protein on this epitope may allow tumor cells to evade immunologic pressure from TCR-T cells. A lysine side chain-methylated KRAS G12V peptide, rather than unmodified epitope, may be presented in HLA-A2 by tumor cells and impact recognition by TCRs. Using a novel computationally guided approach to design TCRs, we developed by mutagenesis TCRs that recognize this methylated peptide, enhancing tumor recognition and destruction. Additionally, we identified TCRs with similar functional activity in normal repertoires from rare primary T cells by stimulation with modified peptide, clonal expansion, and selection. Mechanistically, a gene knockout screen to identify mechanism(s) by which tumor cells methylate or demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these TCRs. These findings highlight the role of post-translational modifications in immune evasion and suggest that identifying and targeting such modifications should facilitate development of more effective TCR-T cell therapies.