Identification of immunogenic neoantigens from intron retention in colorectal cancer.

Intron retention (IR) is an underexplored source of alternative neoantigens in cancer. Unlike mutation-derived neoantigens, IR-derived neoantigens can arise even in malignancies with low tumour mutational burden (TMB), such as microsatellite-stable colorectal cancer (MSS-CRC), making them a potentially important source of tumour antigens. Here, we provide experimental evidence that IR-derived neoantigens are expressed in tumours and are recognized by T cells, with approximately 30% of predicted epitopes eliciting measurable CD8+ T cell responses. We applied a bioinformatics pipeline to RNA sequencing data from 23 CRC patients, identifying 49 patient-specific and 24 shared IR-derived neoantigens, the latter present in ~ 30% of the cohort. Notably, most shared neoantigens exhibited high binding affinity to the predominant HLA alleles, highlighting their potential as broadly targetable vaccine candidates. Together, these findings establish IR-derived neoantigens as a validated and clinically relevant class of tumour antigens, expanding opportunities for personalized and off-the-shelf immunotherapies in CRC and other low-TMB cancers.