2. Nature | Vol 635 | 14 November 2024 | 459Treatment with EcNcΔlon/Δ ompT /LLO+ nAg42 significantly extended survival, with 60% of mice surviving to 55 days with no detectable metastases, whereas all control treated mice had died by day 27 (Fig. 5h). Again, treatment was well tolerated, with no significant weight change rela - tive to PBS (Extended Data Fig. 8i). These data demonstrate that the microbial tumour neoantigen vaccination system stimulates produc- tive antitumour immunity in vivo after intravenous administration in established, systemic metastatic melanoma. Discussion Through microbial engineering, we couple the tumour-homing and immunostimulatory nature of bacteria with precise instructions for coordinated adaptive immunity towards tumour neoantigens, achieving a platform capable of mediating control and eradication of advanced solid tumours. Bioinformatic-based identification of neoantigens that are both immunogenic and able to generate T cells capable of tumour cell killing remains a challenge. Predicted MHC-binding affinity is a frequently used criterion in neoantigen identification, as analyses of MHC-I bind - ing epitopes from viruses showed that immunogenic epitopes usually possess less than 500 nM affinity, but most show less than 50–200 nM affinity45. Whereas many immunogenic tumour neoantigens similarly show strong MHC-binding affinity (less than 50–500 nM)12,20, only a small fraction of predicted strong binding tumour neoepitopes are immunogenic20, and studies have revealed presentation, immunogenicity and TIL reactivity for neoantigens with low bind - ing affinity (greater than 500 nM)17,46,47. A detailed analysis of MHC-I binding neoepitopes from human tumours showed that of the 10.5% of neoepitopes with more than 500 nM affinity, 50% contained cysteine residues, suggesting that low-affinity neoantigens may pos - sess distinct characteristics48. Further criteria have been proposed to aid neoantigen identification, including differential binding affin - ity of the neoepitope versus corresponding wild-type epitope17,20, variant allele fraction of the mutation14 and expression level of the mutation-containing gene15; however, no standardized prediction algorithm has been identified20. Although we recognize the neoan - tigen prediction methods we use may have limitations, in this study, we used a combination of prediction criteria and primarily selected high-affinity neoantigens, with the incorporation of some low-affinity neoantigens. We found that antigen sets encompassing both predicted MHC-I and MHC-II binding neoantigens mediated antitumour efficacy. Indeed, enhanced frequencies of neoantigen-specific CD4+ and CD8+ T cells were observed, MHC-I and MHC-II binding neoantigens were targeted by TILs in immunized mice, and both CD4+ and CD8+ T cells were required for efficacy of the microbial vaccination system in vivo. This agrees with the critical role of both CD4+ and CD8+ T cells in effective antitumour immunity, and the expanding set of verified MHC-I- and MHC-II-dependent neoantigens recognized across tumours12,15 ,18,46,47. Together, these results support the targeting of both MHC-I and MHC-II binding tumour neoantigens in antitumour immunotherapies.nAg42EcNc/uni0394lon//uni0394ompT LLO+ OVAPBS B16F10 s.c. i.v.aTumour PMA-Ionomycin NSIFN/uni03B3+ of Foxp3– CD4+ (%) 0102030 CD4+ T cellsNSIFN/uni03B3+ of CD8+ (%) 010203040 CD8+ T cells Granzyme-B+ of Foxp3–CD4+ (%) CD4+ T cells020406080Granzyme-B+ of CD8+ (%)NS CD8+ T cells NS 020406080Granzyme-B+ of NK1.1+ (%) NK cellse f Time (days post-engraftment)05.0 × 1081.0 × 1091.5 × 1092.0 × 1092.5 × 109 B16F10-Luc mets i.v. NS ***nAg42EcNc/uni0394lon//uni0394ompT LLO+ OVAPBS 01 02 03 0Mean total /f_lux (photons s–1)05101520NSb Regulatory B cells0246MFI TIM-1 of CD19+ (×103) 012345TIM1+CD19+ of CD45+ (%)NSc 051015MHC-II–Ly6C+ MDSC of CD45+ (%) NS MDSCFoxp3+ of CD4+ (%) Regulatory T cells020406080d g Time (days post-engraftment)h Radiance (p s–1 cm–2 sr–1)Day 22 PBS EcNc /uni0394lon//uni0394ompT/LLO+OVA nAg42EcNc/uni0394lon//uni0394ompT LLO+ **OVA nAg42PBS 01 02 03 04 05 0020406080100 Survival (%) ×107 M2 M3 M4 M5 M10.51.01.52.02.5******** ******** ******* * ******** ***** ** Fig. 5 | Microbial neoantigen vectors restructure the tumour immune microenvironment and suppress established metastatic melanoma. a–e, Mice with orthotopic B16F10 received an intravenous treatment on day 9 and 12 post-engraftment. a , Left, experimental schematic. Middle, the frequency of IFNγ+Foxp3−CD4+ post-stimulation (* P = 0.0335, ** P = 0.0040; NS, P > 0.05). Right, the frequency of IFNγ+CD8+ T cells (**** P < 0.0001; NS, P > 0.05). b, Left, the frequency of Granzyme-B+Foxp3−CD4+ (** P = 0.0024, ** P = 0.0041; NS, P > 0.05). Right, the frequency of Granzyme-B+ CD8+ T cells (* P = 0.0495, **P = 0.0014; NS, P > 0.05). c, Frequency of Granzyme-B+NK1.1+ NK cells (**** P < 0.0001; NS, P > 0.05). d, Left, the median fluorescence intensity (MFI) of TIM-1 on CD19+ B cells (* P = 0.0457, ** P = 0.0029, **** P < 0.0001). Right, the frequency of TIM-1+CD19+ B cells of CD45+ cells (* P = 0.0442; NS, P > 0.05). e, Left, the frequency of Foxp3+CD4+ T cells of CD4+ cells (** P = 0.0035, ** P = 0.0038). Right, the frequency of MHCII−Ly6c+ MDSCs of CD45+ cells (* P = 0.0440; NS, P > 0.05). f–h, C57BL/6 mice ( n = 5 mice per group) with 2-day established B16F10-Luc systemic metastases. Intravenous treatment every 3–5 days. f, Representative systemic metastases luminescence. g , Mean total flux from systemic metastases (*** P > 0.0003; NS, P > 0.05, two-way ANOVA with Dunnett’s multiple comparisons test). h, Kaplan–Meier survival curve for mice with B16F10-Luc systemic metastases (** P = 0.0015, log-rank Mantel–Cox test). a–e, One-way ANOVA with a –c, Tukey’s, d , left, Tukey’s or right, Dunnett’s and e, Dunnett’s multiple comparisons test. a–e, n = 8 mice for nAg42, n = 7 for other groups. a–e,g, Data are mean ± s.e.m. 460 | Nature | Vol 635 | 14 November 2024 ArticleWe show that across distinct tumour models and genetic back - grounds, the antitumour effect of vaccination is accompanied by broad modulation of the immune compartment within the TME. The coordinated regulation of APCs, reduction of immunosuppressive myeloid, regulatory T and B cell populations, and activation of NK cells and CD4+ and CD8+ T cells together indicate the advantage of precisely engineered microbial platforms as next-generation antitumour vac - cines that align several arms of immunity13. In agreement with previous reports regarding synthetic peptide, adenoviral and messenger RNA (mRNA) neoantigen vaccines49–51, we did not observe suppression of established CT26 tumour growth on SLP vaccination. By contrast, engi - neered microbial vaccines significantly reduced growth and achieved a portion of complete responses. This is consistent with a model whereby microbial vectors enable direct modulation of innate and adaptive immunity and sustained neoantigen delivery within the TME to promote effective therapeutic vaccination of established tumours. The unique ability of microbial vaccines to directly remodel the TME may promote synergy with other forms of immunotherapy. Adoptive T cell therapy (ACT) has resulted in regression of advanced malignancy in a subset of patients, although solid tumours often show resistance52. Previous murine studies demonstrated that target-antigen vaccination amplifies ACT efficacy in solid tumours when targeting a carcinoem - bryonic antigen (claudin-6) with chimeric antigen receptor T (CAR-T) cell therapy53. However, in a human solid tumour trial the effect of combining claudin-6 vaccination with CAR-T cell therapy was unclear compared with CAR-T cell monotherapy54. Solid tumour resistance mechanisms to T cell-mediated immunotherapies often occur within the TME: restricted antigen availability, reduced APC infiltration and function, antitumour immune cell exclusion, upregulation of immu - noinhibitory ligands and enrichment of immunosuppressive popula- tions55. As the microbial neoantigen vectors locally increase neoantigen density, recruit and activate dendritic cells and CD4+ and CD8+ T cells, and reduce immunosuppressive populations and ligands within the TME, combination with ACT may oppose these resistance mechanisms and provide synergistic benefit. Through extra programming of the microbial vectors and rational incorporation of other immunotherapeutics, this system may achieve reliable eradication of established solid tumours and metastases through precision cancer immunotherapy using living antitumour vaccines. Online content Any methods, additional references, Nature Portfolio reporting summa - ries, source data, extended data, supplementary information, acknowl - edgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-024-08033-4 .