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 EcNc^{Δlon/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, EcNc^{Δlon/ΔompT/LLO+} OVA, or the 7-strain combination EcNc^{Δlon/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, EcNc^{Δlon/ΔompT/LLO+} OVA, or EcNc^{Δlon/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.

Source Data