Personalized Neoantigen Vaccine plus Regorafenib Increases Rgs2⁺CD8⁺ T Cells Infiltration and Reprograms the Tumor Microenvironment in Microsatellite Stable Colorectal Cancer Liver Metastases

Abstract Microsatellite stable colorectal cancer liver metastases (MSS‐CRLM) resist immune checkpoint inhibitors due to their immunosuppressive tumor microenvironment (TME) and low mutation burden (TMB). A personalized neoantigen vaccine, Neo‐CRCVAS, using whole‐exome and RNA sequencing of murine MSS‐CRC cells, comprising 7 immunogenic neoantigen peptides with Poly(I:C) is developed and combined with regorafenib as a novel therapy (RegoNeo). In MSS‐CRLM mouse models, RegoNeo significantly enhanced tumor regression and survival while establishing durable immune memory. Single‐cell RNA and TCR sequencing revealed that RegoNeo expanded a distinct Rgs2⁺CD8⁺ T cell population with strong cytotoxic activity and TCR clonal expansion. These Rgs2⁺CD8⁺ T cells, enriched for neoantigen‐specific T cells, demonstrated potent tumor‐killing capabilities in both mouse models and patient‐derived organoids. The findings establish RegoNeo as a promising personalized immunotherapy that reprograms the immunosuppressive tumor microenvironment by increasing Rgs2⁺CD8⁺ T cell infiltration, highlighting both the treatment approach and this specific T cell subset as potential therapeutic targets for MSS‐CRLM patients.