Spatiotemporally programmed probiotic vaccines potentiate the efficacy of single-epitope neoantigen immunotherapy for solid tumors

Neoantigen vaccines represent a highly promising strategy for precision cancer immunotherapy due to their tumor specificity and personalized nature. However, single neoantigens peptide often suffer from limited immunogenicity, which restricts their ability to induce robust and durable T cell responses and thus hampers clinical application. Here, we developed a multilayered synergistic platform focusing on single neoantigens peptides by integrating nano‐delivery, in situ immune modulation, and controlled release. The DSPE-PEG-peptide nanovaccine was engineered to actively target tumor-draining lymph nodes (TDLNs), enhancing dendritic cells (DCs) uptake and cross-presentation and consequently strengthening antigen-specific T cell activation and memory formation. In parallel, an engineered probiotics vaccine was generated by covalently coupling an OX40 agonist (αOX40) and the TLR7/8 agonist R848 onto the probiotics, enabling local remodeling of the immunosuppressive tumor microenvironment, amplification of innate signaling, and promotion of DC maturation and T cell infiltration. Both vaccine components were subsequently co-encapsulated within a thermosensitive Poloxamer 407 hydrogel, providing spatial retention and sustained release to maintain continuous immune stimulation. In vitro and in vivo studies demonstrated that this composite system, while preserving the antigenic specificity of single peptides, markedly enhanced DC maturation, strengthened CD8⁺ memory T cell responses, reprogrammed the tumor immune microenvironment, and elicited potent antitumor effects with favorable safety across multiple tumor models. Our findings provide key mechanistic insights and a versatile strategy to enhance the potency of single neoantigen vaccines, paving the way for their clinical translation in personalized cancer immunotherapy.