A multi-epitope mRNA vaccine for effective immunotherapy in esophageal squamous cell carcinoma.

e16007 Background: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with limited treatment options, particularly in patients who develop resistance to immune checkpoint inhibitors (ICIs). The development of cancer vaccines capable of inducing robust and durable T cell responses represents a promising strategy to overcome these challenges. Here, we describe a universal, multi-epitope mRNA-lipid nanoparticle (LNP) vaccine targeting tumor-associated antigens (TAAs) commonly expressed in ESCC and investigate its immunogenicity, antitumor efficacy, and potential synergy with PD-1 blockade in preclinical models. Methods: TAAs were identified through transcriptomic analysis of The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets, and validated in ESCC patient samples. The mRNA vaccine was optimized for stability and antigen presentation and tested in HLA-A2.1 and HLA-A11.1 transgenic mouse models. Antigen-specific immune responses were assessed using ELISPOT and flow cytometry. Therapeutic efficacy was evaluated in prophylactic and therapeutic tumor models with or without PD-1 blockade. Tumor-infiltrating immune cells were characterized using immunohistochemistry and single-cell RNA sequencing. Results: In both HLA-A2.1 and HLA-A11.1 transgenic mice, the vaccine induced measurable immune responses for most antigen fragments, with ELISPOT assays showing significant IFN-γ secretion in response to peptide pools (p < 0.001 vs. control). In prophylactic models, tumor incidence was reduced by 100% (p < 0.001). In therapeutic models, tumor growth was significantly suppressed, and median survival increased to 44.5 days compared to 31 days in the control group (p < 0.05). Combination therapy with PD-1 blockade further enhanced tumor control, achieving a tumor reduction rate of 85%, and increased the infiltration of cytotoxic CD8+ T cells by 3.4-fold. Single-cell RNA sequencing revealed clonal expansion of T cells and reprogramming of the tumor microenvironment toward a more immunogenic state. Conclusions: This multi-epitope mRNA-LNP vaccine induces robust, durable T cell responses and effectively suppresses tumor growth in ESCC models. Its combination with PD-1 blockade further enhances antitumor efficacy by overcoming immune resistance and reshaping the tumor microenvironment. These findings provide a strong rationale for advancing this vaccine strategy into clinical trials to improve outcomes in patients with ESCC.