s.c., subcutaneous. CR, complete response. Nature | Vol 635 | 14 November 2024 | 457OVA (Extended Data Fig. 6h). As validation of immune memory forma - tion in tumour-bearing hosts, no tumour growth was observed on rechal - lenge of mice that had cleared CT26 tumours after EcNcΔlon /ΔompT /LLO+ nAg19 treatment (Extended Data Fig. 6i). Together, these experi - ments indicate generation of tumour neoantigen-specific T cells that are proficient in tumour cell killing and establish durable immune memory.Beyond induction of tumour antigen-specific T cell responses, treatment with EcNcΔlon /ΔompT /LLO+ nAg19 resulted in reduced frequen - cies of tumour-resident immunosuppressive PD-L1+Ly6G+ poly - morphonuclear cells and PD-L1+F4/80+ macrophages39,40 (Fig. 3f). Bacteria-treated groups further showed reduced numbers and frequencies of Foxp3+CD4+ regulatory T cells and MHC-IIloF4/80+ tumour-associated macrophages (Fig. 3g and Extended Data Fig. 6j), two cell populations known for their roles in inhibiting anti - tumour immunity41,42. Moreover, within TDLNs of the neoantigen therapeutic-treated group, myeloid immunophenotyping showed a reduction of PD-L1 on cDC1s and cDC2s populations (Extended Data Fig. 6k), which has been shown to facilitate antitumour immunity43. In summary, intravenously delivered microbial neoantigen thera - peutics sustain neoantigen production and availability in lymphoid tissue in vivo, recruit and activate dendritic cells, stimulate both neoantigen-specific and broad adaptive immunity, and reduce immu - nosuppression within the TME, shaping a more effective environment for productive antitumour immunity (Fig. 3h). Treatment of melanoma Neoantigens are generally unique to the individual tumour9, thus vac - cination platforms must be able to flexibly incorporate and deliver diverse sets of neoantigens on the basis of the unique mutations present in a particular tumour. To evaluate the suitability of our engineered microbial platform in this regard, we performed paired exome and transcriptome sequencing on a second, more aggressive tumour cell type (B16F10 melanoma) grown orthotopically in C57BL/6 mice and designed tumour-specific therapeutics (Fig. 4a). We applied an equivalent neoantigen prediction algorithm as performed for CT26 and identified numerous putative B16F10-specific neoantigens, includ - ing many that had previously been validated by others15 (Extended Data Fig. 7a). A set of seven constructs were devised from neoantigens of varying imputed MHC-I and MHC-II affinities, with each construct containing six unique predicted neoantigens (Extended Data Table 2) and confirmed to be robustly expressed by EcNcΔlon /ΔompT /LLO+ (Fig. 4a and Extended Data Fig.