A Framework for Comparing Mouse Neoantigen Immunogenicity

Cytotoxic CD8+ T cell responses targeting tumor neoantigens are critical for immunotherapy efficacy and are widely studied across different preclinical mouse tumor models. Defined neoantigens are commonly introduced to enable tracking of tumor-specific T cells; however, variation in neoantigen choice may yield immune phenotypes attributable to differences in neoantigen immunogenicity, complicating interpretation of tumor-intrinsic mechanisms. Here, we determined the relative immunogenicity of a set of 25 commonly used mouse tumor-derived and model neoantigens to facilitate comparison of neoantigens across studies. We found that <i>in silico</i> predicted major histocompatibility complex (MHC) binding affinity poorly stratified <i>in vivo</i> immunogenicity. In contrast, experimental measurement of peptide-MHC complex stability (K_off), more so than measured affinity (K_D), closely correlated with the relative magnitude of neoantigen-targeted vaccine responses <i>in vivo</i>. Thus, we report the relative stability of a known set of commonly used neoantigens as a reference and provide a simple method to benchmark novel neoantigens against this library. This framework will allow contextualization of the level of immunogenicity of newly identified neoantigens and aid in comparative interpretation of tumor-immune phenotypes across studies.