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.