Extended Data Fig. 3 | Characterization of intratumoral treatment with microbial tumor neoantigen vaccines. a–g, BALB/c mice with established hind-flank CT26 tumors were treated when average tumor volumes were 150–200mm3. a,b, Mice received a single intratumoral injection of EcN WT, EcNcΔlon /ΔompT, or EcNcΔlon /ΔompT /LLO+. a, Representative image of tumors colonized by microbes with a genome-integrated luminescence cassette. b , Average radiance of microbe colonized tumors in designated ( n = 4 mice for EcN WT 96 h, 5 for all other groups). c , Mice received intratumoral injections of PBS or EcN WT. Tumor growth curves ( n = 5 mice per group, ns = P > 0.05, two-way ANOVA with Tukey’s multiple comparisons test). d , Mice received intratumoral injections of EcN WT without therapeutic (NC), expressing construct MHCIa, MHCIIa, or MHCI/IIv, or EcN nAg19. Tumor growth curves ( n = 5 mice per group, ns = P > 0.05, two-way ANOVA with Tukey’s multiple comparisons test). e ,f, Mice received a single intratumoral injection of PBS, EcNcΔlon /ΔompT NC, EcNcΔlon /ΔompT nAg19, or EcNcΔlon /ΔompT /LLO+ expressing construct MHCIa, MHCIIa, or MHCI/IIv, or EcNcΔlon /ΔompT /LLO+ nAg19. e, Tumor growth curves ( n = 5 mice for PBS, 7 for other groups). f, Tumor growth rate ( n = 5 mice for PBS and EcNcΔlon /ΔompT NC, 7 for other groups) for designated groups. g, Individual tumor trajectories after intratumoral treatment with PBS or indicated microbial therapeutic. b –f, Data are mean ± s.e.m. Article Extended Data Fig. 4 | Comparative profile of intratumoral treatment with engineered microbial neoantigen vaccines. a–d, BALB/c mice with established hind-flank CT26 tumors were treated when average tumor volumes were 150–200mm3. a,b, Mice received intratumoral injection of wildtype EcN, EcNcΔlon /ΔompT or EcNcΔlon /ΔompT /LLO+ either without neoantigen expression (NC) or the strain mixture nAg19 on day 0. a , Relative body weight of CT26 tumor- bearing mice ( n = 5 mice for PBS, EcN NC, and EcN nAg19, 7 for other groups, **P = 0.0034, **** P < 0.0001, ns = P > 0.05, two-way ANOVA with Tukey’s multiple comparisons test). b –d, Mice received intratumoral injection on day 0 and 8 (n = 7 mice for EcNcΔlon /ΔompT NC and EcNcΔlon /ΔompT /LLO+ nAg19, 8 for other groups). b, Tumor growth curves (** P = 0.0020, **** P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test). c , Kaplan-Meier survival curves for CT26 tumor-bearing mice (** P = 0.0061, ** P = 0.0076, Log-rank Mantel-Cox test). d, Individual tumor trajectories after intratumoral treatment with indicated microbial strain. e ,f, BALB/c mice were implanted with CT26 tumors on both hind flanks. When average tumor volumes were 100–150mm3 mice received an intratumoral injection of PBS, EcNcΔlon /ΔompT /LLO+ (NC), or EcNcΔlon /ΔompT /LLO+ nAg19 into a single tumor. e, Tumor growth curves ( n = 6 mice for EcNcΔlon /ΔompT /LLO+ nAg19, 5 for other groups, ** P = 0.0014, **** P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test). f , CFU g−1 of tumor ( n = 4 mice for EcNcΔlon /ΔompT /LLO+ nAg19, 5 for all other groups), LOD 1 ×103 CFU g−1. a,b,e,f, Data are mean ± s.e.m. Extended Data Fig. 5 | Intravenous treatment with engineered microbial therapeutics in primary and metastatic solid tumors. a–c, BALB/c mice with established hind-flank CT26 tumors were intravenously injected with indicated microbial therapeutics when average tumor volumes were 150–200 mm3 (n = 9 mice for EcNcΔlon /ΔompT /LLO+ nAg19, 8 for other groups). a , Kaplan-Meier survival curves for CT26-tumor-bearing mice treated with indicated therapeutic (**** P < 0.0001, ** P = 0.0021, Log-rank Mantel-Cox test). b , Relative body weight of mice after intravenous treatment with indicated microbial therapeutic. c, Individual tumor trajectories after intravenous treatment with the indicated microbial therapeutic. d , BALB/c mice with established hind-flank CT26 tumors were intravenously injected with EcNcΔlon /ΔompT or EcNcΔlon /ΔompT /LLO+. Upper: representative luminescent signature of tumors colonized with EcNcΔlon /ΔompT (LLO−) or EcNcΔlon /ΔompT /LLO+ (LLO + ), 48 h post-injection. Lower: average radiance of colonized tumors ( n = 7 mice per group). e –f, BALB/c mice with established hind-flank CT26 tumors were intravenously injected with the indicated microbial therapeutics or PBS, or subcutaneously injected with nAg19-SLP , when average tumor volumes were 100–120 mm3. e, Kaplan-Meier survival curves for CT26 tumor-bearing mice treated with indicated therapeutic (n = 8 mice per group, ** P = 0.0055, ** P = 0.0018, * P = 0.0210, Log-rank Mantel-Cox test). f , Individual tumor trajectories ( n = 8 mice per group) after intravenous treatment with the indicated therapeutic. g , Established hind-flank CT26 tumors were intravenously injected with the indicated microbial therapeutics or PBS, or subcutaneously injected with nAg19- S L P, when average tumor volume was 160 mm3. Tumor growth curves ( n = 5 mice for PBS, 8 mice for other groups; **** P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test). h –k, BALB/c mice were injected intravenously with CT26-Luc cells. h , Upper-left: In vivo, or Upper-right: ex vivo bioluminescent images of mice ( n = 3) lungs 96 h post-intravenous injection of CT26-Luc cells. Lower: Histology of metastatic lung foci 96-hours post-intravenous injection of CT26-Luc cells. i– k, Every 3–5 days mice ( n = 5 per group) received intravenous injection of PBS, EcNcΔlon /ΔompT /LLO+ without therapeutic (NC), or EcNcΔlon /ΔompT /LLO+ nAg19 starting 4 days after CT26-Luc engraftment. i , Microbial tissue burden quantified as CFU g−1, LOD 4 ×102 CFU g−1 (n = 3 mice). j , Relative body weight of mice ( n = 5 per group) after intravenous treatment with indicated therapeutic. k, Individual lung metastases luminescence trajectories ( n = 5 mice per group). b,d,g,i,j Data are mean ± s.e.m. Article Extended Data Fig. 6 | See next page for caption. Extended Data Fig. 6 | Modulation of anti-tumor immunity by engineered microbial tumor neoantigen vaccines. a–f,h–i, BALB/c mice with established hind-flank CT26 tumors received intravenous injections of indicated therapeutic or control. a , 2 days or b –g, 8 days after treatment, tumors and TDLNs were extracted. a , Frequency of cDC2 in TDLNs ( n = 9 mice per group, **P = 0.0042, ** P = 0.0099, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). b –c, Lymphocytes from TDLNs were stimulated ex vivo with PMA and ionomycin in the presence of brefeldin A. b , Left: Frequency of IFN-γ+ Foxp3−CD4+ post-stimulation ( n = 3 mice per group, * P = 0.0313, * P = 0.0246, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). Right: Frequency of TNF-α+ Foxp3−CD4+ T cells post-stimulation ( n = 3 mice per group, *P = 0.0445, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). c, Left: Frequency of IFN-γ+ CD8+ post-stimulation ( n = 3 mice per group, *P = 0.0257, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). Right: Frequency of TNF-α+ CD8+ T cells post-stimulation ( n = 3 mice per group, ** P = 0.0017, *** P = 0.0008, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). d , TILs were stimulated with individual 29-mer neoepitope-containing long peptides. Number IFN-γ spots ( n = 8 mice per group, * P = 0.0439, * P = 0.0364, * P = 0.0281, * P = 0.0200, Kruskal-Wallis test with Dunn’s multiple comparisons test). Data are mean ± s.e.m. of background (medium control) subtracted responses. e,f, TILs were stimulated ex vivo with PMA and ionomycin in the presence of brefeldin A. e , Left: Frequency of IFN-γ+ Foxp3−CD4+ post-stimulation ( n = 9 mice per group, * P = 0.0461, ** P = 0.0014, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Right: frequency of IFN-γ+ CD8+ T cells post-stimulation ( n = 9 mice per group, *P = 0.0486, ** P = 0.0040, **** P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). f , Frequency of IFN-γ+ B220+ B cells post-stimulation ( n = 9 mice per group, * P = 0.0351, ** P = 0.0010, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). g , Left: Percentage Ki-67+ of Foxp3−CD4+ T cells in tumors ( n = 9 mice per group, * P = 0.0183, *** P = 0.0008, one-way ANOVA with Dunnett’s multiple comparisons test). Right: Percentage Ki-67+ of CD8+ T cells in tumors ( n = 9 mice per group, * P = 0.0453, ns = P > 0.05, One-way ANOVA with Dunnett’s multiple comparisons test). h , Naïve, tumor-free BALB/c mice were vaccinated intravenously with the designated treatment. CT26 was engrafted on a single hind-flank after the final vaccination. Tumor growth curves ( n = 8 mice per group, **** P < 0.0001, two-way ANOVA with Šídák’s multiple comparisons test). i , BALB/c mice with established CT26 tumors were treated intravenously with EcNcΔlon/ΔompT/LLO+ nAg19 (as in Fig. 2e). Mice that had cleared tumors, and age-matched naïve mice, were subcutaneously rechallenged with CT26 tumor cells. Tumor growth curves ( n = 5 for naïve mice, 6 for nAg19). j, Left: Frequency of FoxP3+CD4+ regulatory T cells in tumors ( n = 9 mice per group, * P = 0.0491, ** P = 0.0072, one-way ANOVA with Holm-Šídák’s multiple comparisons test), Right: Frequency of MHCIIloF4/80+CD11b+ macrophages in tumors ( n = 8 mice for PBS, 9 for other groups, * P = 0.0173, **P = 0.0057, one-way ANOVA with Dunnett’s multiple comparisons test). k, Left: Percentage PD-L1+ of cDC1 in TDLN ( n = 5 mice per group, ** P = 0.0074, ns = P > 0.05, one-way ANOVA with Dunnett’s multiple comparisons test), Right: Percentage PD-L1+ of cDC2 in TDLN ( n = 5 mice per group, * P = 0.0103, *P = 0.0244, one-way ANOVA with Dunnett’s multiple comparisons test). a–k, Data are mean ± s.e.m. Article Extended Data Fig. 7 | Assessment of engineered microbial neoantigen therapeutics in B16F10 melanoma. a, Percentage of predicted B16F10 neoantigens containing mutant-epitope(s) with ≤500 nM MHC-I affinity (MHC-I), MHC-II affinity (MHC-II), both MHC-I and MHC-II affinity (Shared), or no epitope meeting affinity criteria (Neither). Previously validated neoantigens within the set are labeled. b , Immunoblot of B16F10 neoantigen construct expression in EcNcΔlon /ΔompT /LLO+. c–j, C57BL/6 mice with established hind-flank B16F10 melanoma tumors were treated 9 days after tumor engraftment. c–e, Every 3–5 days, mice received an intravenous injection of PBS, EcNcΔlon /ΔompT /LLO+ OVA, or the 7-strain combination EcNcΔlon /ΔompT /LLO+ nAg42. c, Individual tumor trajectories after intravenous treatment with indicated therapeutic. d, Relative body weight of B16F10-tumor bearing mice ( n = 5 mice for PBS, 7 for other groups, ns = P > 0.05, two-way ANOVA with Tukey’s multiple comparisons test). e , Individual tumor trajectories after intravenous treatment with indicated therapeutic and intraperitoneal treatment with indicated monoclonal antibody. f –j, On day 9 and 12 post-engraftment, B16F10 tumor- bearing mice received an intravenous injection of PBS, EcNcΔlon /ΔompT /LLO+ OVA, or EcNcΔlon /ΔompT /LLO+ nAg42. Flowcytometric analysis of TILs was performed 8 days after treatment initiation ( n = 8 mice for nAg42, 7 for other groups). f , Left: Frequency of CD103+XCR1+ cDC1 in tumors (* P = 0.0132, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Right: Frequency of CD301b+ cDC2 in tumors (* P = 0.0162, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). g , Frequency of Foxp3−CD4+ T cells in tumors (*** P = 0.0001, **** P < 0.0001, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). h , Frequency of CD8+ cytotoxic T cells in tumors (**P = 0.0097, *** P = 0.0002, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). i , Left: Number of NK1.1+ NK cells per mg tumor (*P = 0.0243, * P = 0.0224, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Right: Frequency of NK1.1+ NK cells in tumors (* P = 0.0189, *P = 0.0389, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). j, Left: Number of MHCII+CD64+Ly6c+ monocytes per mg tumor (**P = 0.0041, ** P = 0.0073, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Right: Frequency of MHCII+CD64+Ly6c+ monocytes in tumors (* P = 0.0230, * P = 0.0495, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). d ,f–j, Data are mean ± s.e.m. Gel source data in Supplementary Fig. 2. Extended Data Fig. 8 | Mechanisms of anti-tumor immunity and metastases suppression in melanoma. a–e, On days 9 and 12 post-engraftment, B16F10 tumor-bearing mice received intravenous injections of PBS, EcNcΔlon /ΔompT /LLO+ OVA, or EcNcΔlon /ΔompT /LLO+ nAg42. Flow cytometric analysis was performed 8 days after treatment initiation ( n = 8 mice for nAg42, 7 for other groups). a , Left: Percentage CD69+ of Foxp3−CD4+ T cells in tumors (* P = 0.0228, ** P = 0.0092, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Right: Percentage CD69+ of CD8+ T cells in tumors (** P = 0.0021, **** P < 0.0001, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). b , Left: Percentage Ki-67+ of Foxp3−CD4+ T cells in tumors (* P = 0.0188, ** P = 0.0020, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Middle: Percentage Ki-67+ of CD8+ T cells in tumors (** P = 0.0048, ** P = 0.0086, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Right: Percentage Ki-67+ of NK1.1+ NK cells in tumors (* P = 0.0366, ** P = 0.0070, **** P < 0.0001, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). c, Left: Representative histogram of TIM-1 expression on CD19+ B cells. Right: Percentage Ki-67+ of CD19+B cells in tumors (** P = 0.0015, **** P < 0.0001, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). d, Frequency of MHC-IIloF4/80+ macrophages in tumors (* P = 0.0130, ns = P > 0.05, one-way ANOVA with Dunnett’s multiple comparisons test). e , Left: MHC-II MFI of CD64+Ly6c+ monocytes in tumors (* P = 0.0171, ** P = 0.0041, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Right: MHC-II MFI of CD301b+ cDC2 in tumors (** P = 0.0090, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). f –i, C57BL/6 mice were injected intravenously with B16F10-Luc cells. f , Upper-left: In vivo, or Upper-right: ex vivo bioluminescent images of mice ( n = 3) lungs 48-hours post-intravenous injection of B16F10-Luc cells. Lower: Histology of metastatic lung foci 48-hours post-intravenous injection of B16F10-Luc cells. g –i, Mice received intravenous injection of either PBS, EcNcΔlon /ΔompT /LLO+ OVA or nAg42 every 3–5 days starting 2 days post intravenous injection of B16F10-Luc cells. g, Images of systemic metastases luminescence in each mouse in all groups over treatment course. h, Individual systemic metastases luminescence trajectories ( n = 5 mice per group). i, Relative body weight of mice ( n = 5 per group) after intravenous treatment with indicated therapeutic. a –e,i, Data are mean ± s.e.m. ArticleExtended Data Table 1 | CT26 peptides in neoantigen constructs For each therapeutic construct 6–7 predicted neoantigens were included. All 3 groups (MHCIa, MHCIIa, MHCI/IIv) in combination represent nAg19. 5 antigens were included in the production optimization prototype construct (NeoAgp). Extended Data Table 2 | B16F10 peptides in neoantigen constructs In each construct, 6 predicted neoantigens were included. All 7 groups in combination represent nAg42. α ε ε β α μ μ μ