Low neoantigen expression and poor T cell priming underlie early immune escape in colorectal cancer

Immune evasion is a hallmark of cancer, and therapies that restore immune surveillance have proven highly effective in cancers with high tumor mutation burden (TMB) (e.g. microsatellite instable (MSI) colorectal cancer (CRC)). Whether low TMB cancers, which are largely refractory to immunotherapy, harbor T cell neoantigens capable of engaging adaptive immunity remains unclear. Here, we show that tumors from all patients with microsatellite stable (MSS) CRC express potentially actionable clonal neoantigens despite low TMB. Unexpectedly, genes encoding these neoantigens are broadly expressed at lower levels relative to those in MSI CRC, suggesting a potential role of antigen expression in tumor immune surveillance. To investigate this, we developed a versatile platform for functional interrogation of neoantigens with variable expression and applied it to novel preclinical colonoscopy-guided mouse models of CRC. While high expression of multiple high-affinity Major Histocompatibility Complex I (MHC-I)-restricted neoantigens universally resulted in tumor rejection, low expression resulted in poor T cell cross priming and tumor progression. Strikingly, experimental or therapeutic rescue of priming rendered T cells fully capable of controlling tumors with low neoantigen expression. These findings underscore a critical role of neoantigen expression levels in immune evasion and suggest that poor expression or presentation may be a general feature of neoantigens acquired in tumorigenesis. Finally, poorly expressed neoantigens, commonly excluded in tumor vaccine pipelines, may hold untapped therapeutic potential.