Nature | Vol 635 | 14 November 2024 | 453 ArticleProbiotic neoantigen delivery vectors for precision cancer immunotherapy Andrew Redenti1,2,5, Jongwon Im2,5, Benjamin Redenti1, Fangda Li1, Mathieu Rouanne1, Zeren Sheng2, William Sun2, Candice R. Gurbatri2, Shunyu Huang2, Meghna Komaranchath2, YoungUk Jang2, Jaeseung Hahn2, Edward R. Ballister1,2, Rosa L. Vincent2, Ana Vardoshivilli2, Tal Danino2,3,4 ✉ & Nicholas Arpaia1,3 ✉ Microbial systems have been synthetically engineered to deploy therapeutic payloads in vivo1,2. With emerging evidence that bacteria naturally home in on tumours3,4 and modulate antitumour immunity5,6, one promising application is the development of bacterial vectors as precision cancer vaccines2,7. Here we engineered probiotic Escherichia coli Nissle 1917 as an antitumour vaccination platform optimized for enhanced production and cytosolic delivery of neoepitope-containing peptide arrays, with increased susceptibility to blood clearance and phagocytosis. These features enhance both safety and immunogenicity, achieving a system that drives potent and specific T cell-mediated anticancer immunity that effectively controls or eliminates tumour growth and extends survival in advanced murine primary and metastatic solid tumours. We demonstrate that the elicited antitumour immune response involves recruitment and activation of dendritic cells, extensive priming and activation of neoantigen-specific CD4+ and CD8+ T cells, broader activation of both T and natural killer cells, and a reduction of tumour-infiltrating immunosuppressive myeloid and regulatory T and B cell populations. Taken together, this work leverages the advantages of living medicines to deliver arrays of tumour-specific neoantigen- derived epitopes within the optimal context to induce specific, effective and durable systemic antitumour immunity. Bacteria support activation of both innate and adaptive immunity through their inherent foreignness and immunostimulatory prop - erties8. These features, coupled with the ease to synthetically engi - neer bacteria for safe delivery of immunomodulatory compounds, make them ideal vectors to augment and direct antitumour immune responses1,2. Tumour neoantigens are attractive immunotherapeutic payloads for delivery; these antigenic species are not present in other tissues, pose minimal risk for inducing autoimmunity, and are theo - retically excluded from central immunologic tolerance mechanisms9. So far, a variety of tumour neoantigen vaccines have demonstrated promising immunologic responses and survival benefit in clinical trials, although benefit remains limited to only a subset of patients10–12. In this regard, programming bacteria with genetic directives to release high levels of identified tumour neoantigens thereby provides a system to precisely instruct neoantigen targeting in situ. Here we describe new microbial immunotherapy vectors that stimulate effective and durable tumour antigen-specific immunity and inhibit immunosuppressive mechanisms that may otherwise limit traditional neoantigen vaccines13. Engineering microbial cancer vaccines To enable effective cancer vaccination, we developed an engineered bacterial system in probiotic Escherichia coli Nissle 1917 (EcN) to enhance expression, delivery and immune-targeting of arrays of tumour exonic mutation-derived epitopes highly expressed by tumour cells and predicted to bind major histocompatibility complex (MHC) class I and II (Fig. 1a). This system incorporates several key design ele- ments that enhance therapeutic use: (1) optimization of synthetic neoantigen construct form with (2) removal of cryptic plasmids and deletion of Lon and OmpT proteases to increase neoantigen accumula - tion, (3) increased susceptibility to phagocytosis for enhanced uptake by antigen-presenting cells (APCs) and presentation of MHC class II-restricted antigens, (4) expression of listeriolysin O (LLO) to induce cytosolic entry for presentation of recombinant encoded neoantigens by MHC class I molecules and T helper 1 cell (TH1)-type immunity and (5) improved safety for systemic administration due to reduced survival in the blood and biofilm formation. To assemble a repertoire of neoantigens, we conducted exome and transcriptome sequencing of subcutaneous CT26 tumours. Neoantigens were predicted from highly expressed tumour-specific mutations using established methods14,15, with selection criteria inclu - sive of putative neoantigens across a spectrum of MHC affinity16,17. Given the importance of both MHC class I and MHC class II binding epitopes in antitumour immunity15,18,19, we integrated a measure of wild-type-to-mutant MHC affinity ratio—termed agretopicity17,20—for both epitope types derived from a given mutation, to help estimate https://doi.org/10.1038/s41586-024-08033-4 Received: 28 September 2023 Accepted: 6 September 2024 Published online: 16 October 2024 Open access Check for updates 1Department of Microbiology & Immunology, Vagelos College of Physicians and Surgeons of Columbia University, New York, NY, USA. 2Department of Biomedical Engineering, Columbia University, New York, NY, USA.