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Background Overcoming the immunosuppressive tumor microenvironment remains a major challenge in cancer immunotherapy. While adoptive cell therapies and cancer vaccines show promise, their efficacy is often limited by poor antigen presentation and insufficient T cell activation in situ. Here, we introduce SyntiGen, a next-generation live microbial therapy that mimics antigen-presenting cells (APCs). Engineered Escherichia coli (E. coli) are surface-modified to present peptide-major histocompatibility complex class I (pMHC-I) and co-stimulatory ligands, enabling direct T cell priming within the tumor microenvironment (figure 1A).Methods We screened multiple outer membrane scaffolds in E. coli to optimize surface display of a single-chain trimer MHC-I complex (H-2Kb) loaded with the model epitope OVA257–264 (SIINFEKL). Constructs co-expressing pMHC and co-stimulatory ligands (e.g., murine CD80 or OX40L) were evaluated for surface expression by flow cytometry. Functional responses were assessed using OT-I CD8+ T cells in vitro. Tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry to evaluate T cell proliferation and activation. In vivo efficacy was tested in immunocompetent C57BL/6 mice bearing subcutaneous B16F10-OVA or Panc02 tumors, treated intravenously with engineered bacteria or TILs primed by SyntiGen.Results Among tested constructs, YiaT181-based E. coli displaying pMHC-SIINFEKL induced the strongest antigen-specific T cell activation in vitro, as indicated by elevated Ki67, CD25, and CD69 expression. Co-expression of mCD80 (SyntiGen) further enhanced T cell proliferation and effector function. In B16F10-OVA tumor-bearing mice, intravenous administration of SyntiGen significantly expanded SIINFEKL-specific CD8+ T cells, led to tumor regression, and achieved complete responses in ~34% of mice (figure 1B-D). ELISA analysis confirmed no evidence of systemic toxicity or cytokine release syndrome. In the Panc02 model, SyntiGen strains displaying pancreatic cancer-associated neoepitopes robustly activated TILs and lymph node-resident T cells ex vivo (figure 1 E, F). Adoptive transfer of these SyntiGen-primed TILs without lymphocytes significantly delayed tumor progression in vivo (figure 1G, H).Conclusions SyntiGen is a novel live bacterial immunotherapy platform that mimics natural APCs to activate T cells within the tumor microenvironment. By co-displaying pMHC and co-stimulatory ligands, E. coli-based SyntiGen enhances local T cell activation and infiltration, resulting in potent anti-tumor responses. Furthermore, SyntiGen displaying tumor-associated neoepitopes effectively activates TILs ex vivo, which can be leveraged therapeutically. These findings support SyntiGen as a promising strategy for personalized and localized cancer immunotherapy.Acknowledgements This work was supported by Bridge grant between Koch Institute and DFCI. The cartoons were created using BioRender (https://biorender.com/). Figures were generated by GraphPad Prism (version 10.1.1).Ethics Approval All animal studies and procedures were performed following federal, state and local guidelines for institutional animal care and approved by the Institutional Animal Care and Use Committee at DFCI.Abstract 1031 Figure 1