3Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA. 4Data Science Institute, Columbia University, New York, NY, USA. 5These authors contributed equally: Andrew Redenti, Jongwon Im. ✉e-mail: td2506@columbia.edu; na2697@cumc.columbia.edu 454 | Nature | Vol 635 | 14 November 2024 Articlethe ability of adaptive immunity to recognize a neoantigen. Predicted neoantigens were selected from the set of tumour-specific mutations satisfying all criteria, notably encompassing numerous recovered, previously validated CT26 neoantigens15 (Extended Data Fig. 1a). We then sought to create a microbial system that could accommodate the production and delivery of diverse sets of neoantigens to lymphoid tissue and the tumour microenvironment (TME). For the purpose of assessing neoantigen production capacity, a prototype gene encoding a synthetic neoantigen construct (NeoAgp) was created by concatenat - ing long peptides encompassing linked CD4+ and CD8+ T cell mutant epitopes—previously shown as an optimal form for stimulating cellular immunity21—derived from CT26 neoantigens (Extended Data Fig. 1b and Extended Data Table 1). The construct was cloned into a stabilized plasmid22 under constitutive expression and transformed into EcN; however, both immunoblot and enzyme-linked immunosorbent assay (ELISA) assessment showed low production of the prototype construct by EcN across several tested promoters (Extended Data Fig. 1c). Given the dependency on antigen dosage for establishing an effec- tive and immunodominant antigen-specific immune response23–25, we developed a system for improved recombinant neoantigen con - struct production. The incorporation of 5-mer glycine-serine linkers between neoantigen long peptides in the prototype increased expres - sion roughly sixfold (Extended Data Fig. 1c,d). Conversely, expressing only minimal neoepitopes, decreasing the number of neoantigen long peptides in a construct or incorporating 10-mer glycine-serine or immunoprotease-sensitive linkers did not improve production (Extended Data Fig. 1e). To evaluate the capacity of constructs with 5-mer glycine-serine linkers to accommodate production of various neoantigens, and for eventual in vivo testing, we created three more constructs with unique neoantigens from the predicted set, selected on a spectrum of predicted affinity for MHC class I and MHC class II (MHCIa, MHCIIa, MHCI/IIv) (Extended Data Table 1). Neoantigens were grouped on the basis of high predicted affinity for MHC-I (MHCIa) or MHC-II (MHCIIa) or low–moderate affinity for MHC-I or MHC-II (MHCI/IIv). Prototype and new construct expression were evaluated in EcN versus BL21, a strain that harbours chromosomal deletions of the Lon (Δlon) and OmpT (ΔompT) proteases to facilitate recombinant protein produc - tion26. Unlike BL21, EcN also bears cryptic plasmids that can suppress the copy number of transformed recombinant plasmids27. Indeed, on average, BL21 produced tenfold higher levels of neoantigen construct relative to EcN (Extended Data Fig. 1f). Thus, to further enhance con - struct expression in EcN, we performed sequential synthetic modi - fications of the microbe. Removal of the EcN cryptic plasmids led to maintenance of roughly 30-fold higher levels of therapeutic plasmid DNA (EcNc) (Extended Data Fig. 2a), with successive deletion of the Lon protease (EcNcΔlon), OmpT protease (EcNcΔompT) or both proteases c b da eGFP Lat. A++ ++ –– –+ –+ –+ EcN EcNc/uni0394lon//uni0394ompTDilution100 10–1 10–2 10–3 EcN EcNc /uni0394lon//uni0394ompT Blood bactericidal EcNc /uni0394lon//uni0394ompTEcNNeoantigen selectionVector engineering Vector engineering LonOmpT +LLONeoantigen accumulation Complement sensitivityPhagocytosis Improved safety and antigen delivery rAntigen T cell activation Media EcNEcNc/uni0394lon//uni0394ompT EcNc/uni0394lon//uni0394ompT/LLO+OT-I T cells 05001,0001,5002,000IL-2 (pg ml–1) OVA– –+ –+ –+ OVA– –+ –+ –+02,0004,0006,0008,00010,000IFN/uni03B3 (pg ml–1) Expression ExpressionEcNc/uni0394lon//uni0394ompTOVA EcNc/uni0394lon//uni0394ompT/LLO+ OVAMediaf 0204060IL-12p70 (pg ml–1)BMDC CFSEOVA + + + – Expression0102030 ****Percentage of GFP+ BMDM EcN EcNc 20-MHCIIakDa/uni0394lon /uni0394ompT /uni0394lon//uni0394ompT DnaK75-MHCIIa 020406080100Relative expression (intensity)5-mer GS NeoAg LP Stop NeoaNeobNeocNeodNeoeNeonRBS (His6) LOD 102103104105106CFU ml–1 ******** ******** ****** **/uni0394Cryptic plasmids Fig. 1 | Engineering live microbial tumour neoantigen vaccines. a, Design of microbial tumour neoantigen vaccines, with a Circos plot of CT26 mutanome. b, Top, an optimized synthetic neoantigen construct schematic. Middle, relative immunoblot chemiluminescent intensity of neoantigen construct MHCIIa expressed from EcN versus derivative strains ( n = 3 biological replicates per group). Bottom, a representative immunoblot of neoantigen construct MHCIIa expression in a designated strain. c , Percentage of GFP+ BMDMs after incubation with EcN or EcNcΔlon /ΔompT expressing constitutive GFP ( n = 3 biological replicates per group, **** P < 0.0001, two-sided unpaired Student’s t -test); Lat. A, latrunculin A. d , Left, a representative image of EcN or EcNcΔlon /ΔompT spotted on an LB agar plate after incubation in human blood. Right, a microbial burden in colony-forming units (CFU) per ml (CFU ml−1) (n = 3 biological replicates per group, ** P = 0.0039, two-sided unpaired Student’s t -test with Welch’s correction). Limit of detection (LOD) was 2 × 102 CFU ml−1. e, IL-12p70 quantification in culture supernatants of pulsed BMDCs ( n = 3 biological replicates per group, ** P = 0.0018, **** P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). f , Naive OT-I T cells were incubated with pulsed BMDCs. Left, IFNγ quantification of supernatants of OT-I cultures ( n = 3 biological replicates per group, **** P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). Middle, IL-2 quantification of supernatants of OT-I cultures ( n = 3 biological replicates per group, **** P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). Right, a representative histogram of carboxyfluorescein succinimidyl ester (CFSE) dilution-stimulated OT-I T cells. b–f, Data are mean ± s.e.m. Gel source data are in Supplementary Figs. 1 and 2. Nature | Vol 635 | 14 November 2024 | 455(EcNcΔlon/ ΔompT) allowing up to 80-fold increased production of synthetic neoantigen constructs relative to the parental EcN strain (Fig. 1b and Extended Data Fig. 2b,c). As the Lon protease has been connected to capsule and biofilm regu - lation28,29, and OmpT with the degradation of complement30, we tested the susceptibility of the engineered vector EcNcΔlon/ ΔompT to phagocytosis and blood clearance, as well as for its proficiency in biofilm formation. Notably, EcNcΔlon/Δ ompT was fourfold more susceptible to phagocytosis by bone marrow-derived macrophages (BMDMs) relative to EcN (Fig. 1c). Incubation in human blood further revealed a 1000-fold greater sensi - tivity to blood clearance for EcNcΔlon/Δ ompT versus EcN (Fig. 1d). Moreover, EcNcΔlon/Δ ompT was significantly attenuated in biofilm formation, a major mechanism of microbial resistance to immunity and antimicrobial agents in humans31 (Extended Data Fig. 2d). As an antitumour vaccine, the microbial platform must also facilitate presentation of recombinant antigens and activation of APCs. To evalu - ate the system in this capacity, the model antigen ovalbumin (OVA) was expressed in the cytosol of EcNcΔlon/Δ ompT using a strategy analogous to that used for synthetic neoantigen constructs. BMDMs pulsed with EcNcΔlon/Δ ompT OVA, but not EcN-OVA, presented the H2Kb-SIINFEKL com - plex, indicating efficient processing and cross-presentation of recombi - nant antigens from EcNcΔlon/Δ ompT (Extended Data Fig. 2e). Furthermore, pulsed BMDMs upregulated MHC class II and CD80, and downregulated PD-L1, demonstrating effective APC activation by EcNcΔlon/Δ ompT express - ing a recombinant antigen (Extended Data Fig. 2e,f). To refine the immune program orchestrated by APCs, we reasoned that constitutive coexpression of LLO—a pH-dependent pore-forming pro - tein derived from Listeria that permeabilizes the phagolysosomal mem - brane—would facilitate cytosolic delivery of encoded neoantigens for presentation to CD8+ T cells32, enhance IL-12 production33,34 and promote induction of TH1 immunity35. Of note, engineered microbes produced functional LLO, and LLO expression did not affect viability of APCs incu - bated with LLO-expressing strains (EcNcΔlon/Δ ompT /LLO+) or the coexpression of neoantigen constructs (Extended Data Fig. 2g,h). Immunofluores - cence microscopy of BMDMs co-incubated with either live EcNcΔlon /ΔompT OVA or EcNcΔlon/Δ ompT /LLO+ OVA confirmed that LLO coexpression ena - bled recombinant antigen escape into the cytosol (Extended Data Fig. 2i). Bone marrow-derived dendritic cells (BMDCs) pulsed with live EcNcΔlon/Δ ompT /LLO+ OVA secreted threefold higher levels of IL-12p70 compared to those pulsed with EcNcΔlon/Δ ompT OVA (Fig. 1e), indicating greater TH1 instruction by APCs. Moreover, BMDCs pulsed with live EcNcΔlon/Δ ompT /LLO+ OVA mediated superior activation of naive OT-I and OT-II T cells, with 2–2.5-fold increased secretion of interferon-γ (IFNγ) and interleukin-2 (IL-2) from both T cell types relative to EcNcΔlon/Δ ompT OVA, and marked proliferation of both OT-I and OT-II T cells (Fig. 1f and Extended Data Fig. 2j). Conversely, BMDCs pulsed with wild-type EcN- OVA induced no measurable proliferation of either T cell type, 13–15-fold lower secretion of IL-2 and IFNγ from OT-I T cells, and no detectable cytokine secretion from OT-II T cells (Fig. 1f and Extended Data Fig. 2j). Taken together, these data suggest recombinant antigens expressed in EcNcΔlon/Δ ompT /LLO+ lead to potent antigen-specific activation of both naive cytotoxic and helper T cells, with incorporation of LLO facilitating both enhanced presentation to CD8+ T cells and TH1-type immunity. Overall, synthetic neoantigen construct optimization and genetic engineering achieved a microbial platform (EcNcΔlon/Δ ompT /LLO+) capable of robust production across diverse sets of tumour neoantigens, which was attenuated in immune-resistance mechanisms, effectively taken up by and proficient in activating APCs, and able to drive potent activa - tion of T cells specific for encoded recombinant antigens to support enhanced cellular immunity. Treatment of colorectal cancer To assess the in vivo efficacy of the engineered system, BALB/c mice bearing advanced CT26 tumours on a single hind flank received an intratumoural injection of EcN wild-type, EcNcΔlon/Δ ompT or EcNcΔlon/Δ ompT /LLO+ strains. These strains were tested either without any neoantigen plasmid (NC), expressing a single neoantigen construct (MHCIa, MHCIIa, MHCI/ IIv), or as a combination of the three neoantigen construct-expressing strains in equal parts: a microbial antitumour vaccine delivering 19 total unique neoantigens (nAg19). Notably, no difference in tumour colonization efficiency was observed for EcNcΔlon/Δ ompT strains compared to wild-type EcN (Extended Data Fig. 3a,b). Whereas intratumoural treatment with wild-type EcN expressing any neoantigen construct did not demonstrate therapeutic benefit (Extended Data Fig. 3c,d), a single intratumoural injection of EcNcΔlon/Δ ompT /LLO+ nAg19 provided strong antitumour efficacy, with a complete response observed for three out of seven tumours and the combination nAg19 more effec - tive than each construct alone (Fig. 2a and Extended Data Fig. 3e–g). Moreover, treatment with EcNcΔlon/Δ ompT and EcNcΔlon/Δ ompT /LLO+ strains was well-tolerated, with significantly attenuated body weight change compared to wild-type EcN, and no significant body weight differ - ences compared to PBS treatment over the course of observation (Extended Data Fig. 4a). Direct comparisons of intratumoural treat - ment with EcNcΔlon/Δ ompT /LLO+ nAg19 versus EcNcΔlon/Δ ompT nAg19 showed that the inclusion of LLO significantly enhanced tumour control and extended survival (Fig. 2a and Extended Data Fig. 4b–d). Assessment of intratumoural IL-12p70 levels demonstrated that inclusion of LLO also significantly enhanced IL-12p70 levels in tumours, suggestive of enhanced TH1 instruction in vivo (Fig. 2b). Thus, the combination of all synthetic modifications (EcNcΔlon/Δ ompT /LLO+ nAg19) synergized to produce a microbial antitumour vaccine with favourable toxicity profile and strong therapeutic effect in vivo. To evaluate the induction of systemic antitumour immunity after treatment with the microbial neoantigen vaccines, mice with established CT26 tumours on both hind flanks were treated with an injection of microbes into a single tumour. Whereas treatment with EcNcΔlon/Δ ompT /LLO+ without neoantigen expression (NC) did not suppress tumour growth, a single injection of EcNcΔlon/Δ ompT /LLO+ nAg19 induced tumour control and complete regression of two out of six treated and untreated tumours (Extended Data Fig. 4e). Microbial quantification from tumours 14 days after injection showed that microbes colonized treated tumours at high densities, with no bacteria able to be cultured from untreated tumours (Extended Data Fig. 4f). This demonstrates that the engineered neoantigen vaccines stimulate systemic antitu - mour immunity capable of eliminating distant tumours. We next evaluated the efficacy of our microbial antitumour vaccina - tion platform following intravenous administration, the preferred route of administration as to circumvent dependence on tumour accessibil - ity. Similar to intratumoural treatment, intravenous administration of EcNcΔlon /ΔompT /LLO+ nAg19 to mice with advanced CT26 tumours provided potent antitumour efficacy and survival benefit with minimal body weight alteration (Fig. 2b and Extended Data Fig. 5a–c). After intra - venous injection, the engineered microbes persisted at high density within tumours and were cleared rapidly from all other surveyed organs (Fig. 2c and Extended Data Fig. 5d). Given the potent efficacy observed with intravenous treatment, we compared intravenous treatment with the microbial vaccination system to a standard antitumour vaccination strategy using synthetic long peptide (SLP) vaccination12. Mice bearing established hind-flank CT26 tumours received subcutaneous injections of SLP vaccination containing the 19 neoantigens (nAg19-SLP), or intravenous injections of PBS, EcNcΔlon/Δ ompT /LLO+ expressing the strong irrelevant xenoantigen OVA or EcNcΔlon/Δ ompT /LLO+ nAg19. Compared to SLP vaccination, treatment with EcNcΔlon/Δ ompT /LLO+ nAg19 significantly reduced tumour growth and extended survival, with complete regression of two of eight tumours in the EcNcΔlon/Δ ompT /LLO+ nAg19-treated group (Fig. 2e and Extended Data Fig. 5e,f). Increasing the dose of neoantigen SLPs and adjuvant did not enhance the efficacy of SLP vaccination in further comparison trials (Extended Data Fig. 5g). 456 | Nature | Vol 635 | 14 November 2024 ArticleHaving observed robust efficacy in subcutaneous tumours, we then assessed therapeutic efficacy against established metastatic disease. CT26 carcinoma cells with genomically integrated fire - fly luciferase (CT26-Luc) were injected intravenously, which form rapidly progressive lung metastases traceable by biolumines- cence imaging (Extended Data Fig. 5h). Intravenous treatment with PBS, EcNcΔlon/ ΔompT /LLO+ without neoantigen expression (NC) or EcNcΔlon/Δ ompT /LLO+ nAg19 was initiated 4 days after engraftment. We found that engineered microbes colonized metastases-bearing lungs and were not detectable in other tissues (Extended Data Fig. 5i). Micro - bially treated groups again demonstrated minimal body weight fluc - tuation, similarly to mice treated with PBS (Extended Data Fig. 5j). Treatment with EcNcΔlon/Δ ompT /LLO+ nAg19 strongly restrained metastatic growth, with 100% of neoantigen therapeutic-treated mice surviving to 45 days after engraftment, whereas both control groups had com - pletely succumbed to disease (Fig. 2f–h and Extended Data Fig. 5k). This demonstrates both safety and efficacy of intravenously adminis- tered EcNcΔlon/Δ ompT /LLO+ nAg19 in the setting of aggressive, established metastatic disease. Mechanisms of antitumour immunity As the engineered microbial neoantigen vaccines are strong immu - nostimulants and persist within the TME, we reasoned that sustained intratumoural neoantigen production and reduced immunosup - pression would facilitate enhanced activation of adaptive immu - nity to mediate the observed tumour control. To confirm in situ delivery of encoded neoantigens, we intravenously administered EcNcΔlon/Δ ompT /LLO+ nAg19-His (wherein all three neoantigen constructs con - tain a C-terminal 6×His-tag) and performed immunoblots of tumour and tumour-draining lymph node (TDLN) lysates 2 days following treat - ment. We observed three His-tagged protein species corresponding to the three encoded neoantigen constructs (Fig. 3a). Immunophenotyp - ing of TDLNs 2 days following intravenous treatment showed signifi - cantly higher frequencies of cDC2s in TDLNs of microbial vector-treated mice (Extended Data Fig. 6a). Enhanced frequencies of both CD80+ and CD86+ cDC1s and cDC2s (Fig. 3b,c ) were also observed, demonstrating that intravenously delivered microbial vectors recruit and activate dendritic cells within the TDLN. Consistent with delivered neoanti - gens enhancing T cell activation, ex vivo restimulation of lymphocytes isolated from TDLNs at 8 days post-treatment with phorbol myristate acetate (PMA) and ionomycin showed increased production of IFNγ and TNF by conventional CD4+ (Foxp3−CD4+) and CD8+ T cells in mice treated with EcNcΔlon/Δ ompT /LLO+ nAg19 versus those treated with PBS or control bacteria (EcNcΔlon/Δ ompT) (Extended Data Fig. 6b,c). Next, to assess the ability for engineered neoantigen therapeutics to drive neoantigen-specific immunity, tumour-infiltrating lympho - cytes (TILs) were isolated at 8 days post-treatment and restimulated ex vivo with a pool of synthetic peptides representing the 19 bacteri - ally encoded tumour neoantigens. Flow-cytometric analysis showed increased frequencies of IFNγ secreting conventional Foxp3−CD4+ and CD8+ TILs, demonstrating that treatment with EcNcΔlon/Δ ompT /LLO+ nAg19 enhanced encoded neoantigen-specific immunity (Fig. 3d). Analysis of TIL reactivity ex vivo demonstrated that several predicted MHC-I and MHC-II binding neoantigens from each neoantigen construct were targeted in EcNcΔlon/Δ ompT /LLO+ nAg19-treated mice (Extended Data Fig. 6d). Furthermore, purified TILs from EcNcΔlon/Δ ompT /LLO+ nAg19-treated mice were co-incubated with either CT26-Luc or irrelevant tumour cell targets (4T1-Luc) of the same MHC haplotype, demonstrating CT26 tumour cell-specific recognition and killing by generated TILs (Fig. 3e). Compared to peptide stimulation, restimulation with PMA and ionomycin showed even greater levels of IFNγ secreting Foxp3−CD4+ and CD8+ TILs in EcNcΔlon/Δ ompT /LLO+ nAg19-treated tumours and IFNγ producing B220+ B cells36,37, suggestive of epitope spreading and expanded immune activation38 (Extended Data Fig. 6e,f). Further - more, we observed increased frequencies of proliferating CD4+ and CD8+ tumour-infiltrating T cells in mice treated with EcNcΔlon/Δ ompT /LLO+ nAg19 in comparison to treatment with EcNcΔlon/Δ ompT or PBS (Extended Data Fig. 6g). To establish whether microbial neoantigen vaccine treat - ment generates tumour neoantigen-specific immune memory, we prophylactically vaccinated naive mice with EcNcΔlon /ΔompT /LLO+ OVA or nAg19 and grafted CT26 tumours post-vaccination. Tumour growth in mice prophylactically treated with EcNcΔlon/Δ ompT /LLO+ nAg19 was sig - nificantly reduced compared to those treated with EcNcΔlon/Δ ompT /LLO+ fd e 2.0 × 107 EcNc/uni0394lon//uni0394ompTPBS nAg19NCLLO+ 05 10 15 20 25 3005.0 × 1061.0 × 1071.5 × 107 Time (days post-engraftment)Mean total /f_lux (photons s–1) NS Time (days post-engraftment)Survival (%) EcNc/uni0394lon//uni0394ompTPBS NC nAg19LLO+ 01 02 03 04 05 0020406080100ab c EcNc/uni0394lon//uni0394ompT nAg19NC CT26 s.c. i.v. 05 10 15 2005001,0001,5002,0002,500 Time (days post-treatment)Tumour volume (mm3)EcNc/uni0394lon//uni0394ompT/LLO+ TumourTDLN BrainHeartLungsLiverSpleenKidneysBladder1021041061081010CFU g–1 LOD CT26 s.c. i.t. CR: 3/7PBS EcNc/uni0394lon//uni0394ompT LLO+NC nAg19NC LLO+ NCnAg19 05 10 1505001,0001,5002,0002,500 Time (days post-treatment)Tumour volume (mm3) CR: 2/9 ghEcNc/uni0394lon//uni0394ompT EcNc/uni0394lon//uni0394ompT/LLO+PBSNS 0200400600800IL-12p70 (pg per g tumour) CT26 s.c. i.v. 05 10 15 2005001,0001,5002,000 Time (days post-treatment)Tumour volume (mm3) EcNc/uni0394lon//uni0394ompT LLO+ OVA nAg19nAg19 - SLP PBS CR: 2/9 CT26-Luc lung met i.v. PBS EcNc/uni0394lon//uni0394ompT/LLO+NC nAg19Day 22 1.5 1.0 0.5 ×106Radiance (p s–1 cm–2 sr–1) M1 M2 M3 M4 M5**************************** ****** **** NS NSNS **** Fig. 2 | Efficacy of microbial tumour neoantigen vaccines in primary and metastatic colorectal carcinoma. a–e, BALB/c mice with hind-flank CT26 tumours treated when average tumour volumes were 120–200 mm3. a, Intratumoural (i.t.) treatment on day 0. Tumour growth curves ( n = 5 mice for PBS and EcNcΔlon /ΔompT NC, n = 7 for other groups, * P = 0.0469, ** P = 0.0096, **** P < 0.0001; NS, P > 0.05, two-way ANOVA with Tukey’s multiple comparisons test). b, Intratumoural treatment. Intratumoural IL-12p70 levels ( n = 6 mice per group, *** P = 0.0004, **** P < 0.0001; NS, P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). c , Intravenous (i.v.) treatment on day 0 and day 8. Tumour growth curves ( n = 9 mice for nAg19, n = 8 for other groups, **** P < 0.0001; NS, P > 0.05, two-way ANOVA with Tukey’s multiple comparisons test). d , Mice (n = 5) received intravenous injection of EcNcΔlon /ΔompT /LLO+. Microbial tissue burden in CFU per gram (CFU g−1), LOD = 1 × 103 CFU g−1. e, Intravenous treatment of microbial therapies or PBS, or subcutaneous injections of SLP vaccine every 3–6 days. Tumour growth curves ( n = 8 mice per group, **** P < 0.0001; NS, P > 0.05, two-way ANOVA with Tukey’s multiple comparisons test), f –h, BALB/c mice with 4 day established CT26-Luc lung metastases. Intravenous treatment every 3–5 days. f , Representative lung metastases luminescence. g , Mean total flux from lung metastases ( n = 5 mice per group, **** P > 0.0001; NS, P > 0.05, two-way ANOVA with Dunnett’s multiple comparisons test). h , Kaplan–Meier survival curve for mice with CT26-Luc lung metastases ( n = 5 mice per group, **P = 0.0017, ** P = 0.0018, log-rank Mantel–Cox test). a –e,g, Data are mean ± s.e.m.