Abstract
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.
Data availability
The RNA sequencing data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE292858. The data are publicly available at [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE292858](https:/ddec1-0-en-ctp.trendmicro.com/wis/clicktime/v1/query? url=https%3a%2f%2fwww.ncbi.nlm.nih.gov%2fgeo%2fquery%2facc.cgi%3facc%3dGSE292858&umid=4a364a60-d0f2-4f71-9881-a4526044ec57&rct=1743129791&auth=8d3ccd473d52f326e51c0f75cb32c9541898e5d5-acb7683a3c72dd10f31d7c7a9537684199862f27).
Code availability
The bioinformatics pipeline code can be found in a GitHub Repository (https://github.com/kingofburg/IRANP).
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Acknowledgements
We thank Shyam Prabhakar for providing access to the RNA and DNA sequencing data previously published in Joanito et al., Nature Genetics (2022). We thank Teo Hong Kai and Liu Beijia from our laboratory for providing the HLA-A*02:06 and HLA-A*02:01 aAPCs and GZMB reporter cell lines, respectively, for the immunology experiments. We thank Dr. Renyi for cloning the HLA allele-specific coding sequences into the expression plasmid. We are grateful to the patients, their families, and the medical teams involved in the clinical trial. We also extend our thanks to the Singapore Health Sciences Authority for providing healthy donor blood samples. Computational work for this study was performed in part using resources and support provided by the Bioinformatics Core Facility, Yong Loo Lin School of Medicine, National University of Singapore.
Funding
This work was supported by grants from the National Research Foundation (NRF-NRFF12-2020-0007), the Ministry of Education (T2EP30123-0038), the Singapore Ministry of Health’s National Medical Research Council (NMRC) (OF-IRG23jul-0080), the NMRC Open Fund-Large Collaborative Grant (“OF-LCG”) (MOH-001573), NRF Competitive Research Programme (CRP28) and the Institute for Health Innovation & Technology (iHealthtech). T.M. was supported by NUS Research Scholarship from the Singapore Ministry of Education.
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T.M and G.C conceived the study. T.M conducted all the RT-PCR and immunology experiments. B.K.B.R performed all the bioinformatics analysis. C.Z.M.C did the haplotyping experiments. M.C.K assisted T.M with immunology experiments. S. B. E., W.C, K.L., I.J.T., B.L., I.B.H.T., K. K. T. and Y.C.K provided clinical samples. T.M, B.K.B.R and G.C wrote the manuscript. T.K.T. contributed to bioinformatics guidance and manuscript preparation. G.C supervised the entire study and edited the manuscript.
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All the experiments involving human PBMCs and clinical samples were approved by Institutional Review Board of National University of Singapore (NUS) (Approval #LH-20-026E) and National Healthcare Group Domain Specific Review Board (Approval #2020/01343). Written informed consent was obtained from all participants before their participation in the study. All methods were performed in accordance with the relevant guidelines and regulations.
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Manoharan, T., Kee, B.B.R., Cheng, C.Z.M. et al. Identification of immunogenic neoantigens from intron retention in colorectal cancer. Sci Rep (2026). https://doi.org/10.1038/s41598-026-43687-2
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DOI: https://doi.org/10.1038/s41598-026-43687-2
