Probiotic neoantigen delivery vectors for precision cancer immunotherapy

Cell lines

The B16F10 melanoma (ATCC CRL-6475), CT26 colon carcinoma (ATCC CRL-2638) and 4T1 breast cancer (ATCC CRL-2539) authenticated cell lines were purchased directly from ATCC. CT26-Luc, B16F10-Luc and 4T1-Luc cells were lentivirally transduced with luciferase. Cells were confirmed mycoplasma free. Cells were cultured in incubators at 37 °C with atmosphere of humidified 5% CO₂. B16F10 and B16F10-Luc cells were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1× GlutaMax, 1% (vol/vol) MEM non-essential amino acids solution (Gibco-11140050) and 100 U ml^−1 penicillin–streptomycin. CT26, CT26-Luc, 4T1 and 4T1-Luc cells were grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% (vol/vol) FBS, 1× GlutaMax, 1% (vol/vol) MEM non-essential amino acids solution and 100 U ml^−1 penicillin–streptomycin. No commonly misidentified cell lines were used in this study.

Exome sequencing

Paired tumour and tail DNA from BALB/c mice bearing subcutaneous CT26 tumours or C57BL/6 mice bearing subcutaneous B16F10 tumours was extracted in triplicate (n = 3 mice per tumour line) using Qiagen DNeasy Blood & Tissue Minikit following the manufacturer’s instructions. Exome capture from mouse tumour and tail DNA triplicates was conducted using Agilent SureSelectXT All Exon kit for target enrichment DNA library preparation⁵⁶, according to the manufacturer’s instructions (Agilent). Genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument. Fragmented DNAs were cleaned, end-repaired and adenylated at the 3′ end. Adaptors were ligated to DNA fragments, and adaptor-ligated DNA fragments enriched with limited-cycle PCR. Adaptor-ligated DNA fragments were validated using Agilent TapeStation (Agilent) and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific) and Real-Time PCR (KAPA Biosystems). Sequencing libraries were clustered onto a lane of a flow cell. After clustering, the flow cell was loaded on an Illumina HiSeq4000 Instrument per the manufacturer’s instructions. Samples were sequenced using 2 × 150 bp paired end configuration. Image analysis and base calling was conducted by the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq2.17. Sequence reads were trimmed to remove adaptor sequences and nucleotides with poor quality using Trimmomatic v.0.39 (ref. ⁵⁷). Trimmed reads were aligned to the GRCm38 reference genome using the Illumina Dragen Bio-IT platform. Alignments were sorted and PCR or optical duplicates marked for generation of BAM files. Somatic single-nucleotide variants and insertion or deletion (indel) variants were called using Illumina Dragen⁵⁸ and GATK Mutect2 (ref. ⁵⁹). All variants from paired-normal tissue and murine variants from the dbSNP database⁶⁰ were removed during the process. VCF files were left aligned and normalized, with splitting of multiallelic sites into several sites using bcftools v.1.13 (ref. ⁶¹). Only tumour-specific variants called by both algorithms were used for further analysis.

RNA sequencing

Tumour RNA from BALB/c mice bearing subcutaneous CT26 tumours or C57BL/6 mice bearing subcutaneous B16F10 tumours was extracted in triplicate using Qiagen RNeasy Minikit as per the manufacturer’s instructions. Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies) and RNA integrity checked using Agilent TapeStation 2400 (Agilent). RNA sequencing libraries were prepared using the NEBNext Ultra RNA library Prep Kit for Illumina as per the manufacturer’s instructions (New England Biolabs). mRNAs were enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 min at 94 °C. First- and second-strand complementary DNAs (cDNAs) were synthesized subsequently. cDNA fragments were end-repaired and adenylated at 3′ ends, and universal adaptors ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. Sequencing libraries were validated on Agilent TapeStation (Agilent), and quantified using Qubit 2.0 Fluorometer (Invitrogen) and quantitative PCR (qPCR) (KAPA Biosystems). Library loading, sequencing and read trimming were done as described above. Trimmed reads were aligned to the mm10 reference using STAR aligner v.2.5.2b (ref. ⁶²). Unique gene hit counts were calculated using feature counts from Subread Package v.1.5.2. Unique reads that fell within exon regions were counted. The gene hit counts table was used for expression analysis using DESeq2 v.1.20.0 (ref. ⁶³).

Neoantigen prediction and selection

Mutation-specific RNA expression and allele fraction were added to somatic VCF files using Bam-readcount⁶⁴ and VAtools (http://vatools.org). Somatic VCFs were annotated with The Ensembl Variant Effect Predictor (VEP Ensembl v.104)⁶⁵. Only PASS variants from VCFs were considered. Annotated VCFs were analysed using pVacSeq for neoepitope discovery¹⁴. MHC-I affinities were predicted with NetMHCpan v.4.1 (ref. ⁶⁶) and NetMHC v.4.0 (ref. ⁶⁷), and MHC-II affinities were predicted with NetMHCIIpan v.4.1 (ref. ⁶⁸) and NNalign v.2.0 (ref. ⁶⁹). Exonic mutation-derived long peptides based on single-nucleotide polymorphisms (SNP) or indels predicted to generate mutant MHC-binding peptides were first filtered on the basis of the set of minimum criteria: (1) present in all tumour sample triplicates (DNA variant allele fraction ≥ 0.05) and none of the normal tissue triplicates, (2) non-synonymous mutation resulting from either SNP or indel, (3) confirmed exonic mutation transcription (RNA variant allele fraction ≥ 0.05) and gene expression by RNA sequencing in tumour sample triplicate (transcripts per million ≥ 1), (4) at least one predicted MHC-I or MHC-II binding epitope and (5) MHC-I or MHC-II differential binding affinity^17,20 (wild-type half-maximum inhibitory concentration (IC₅₀)/mutant IC₅₀) ≥ 1.2. Predicted neoantigens fulfilling all previous criteria were then prioritized for inclusion and selected according to the following hierarchy: (1) high predicted affinity (MHC-I or MHC-II IC₅₀ ≤ 500 nM), (2) moderate predicted affinity (MHC-I or MHC-II IC₅₀ 500–1,000 nM) and (3) low predicted affinity (MHC-I or MHC-II IC₅₀ 1,000–5,000 nM) (Extended Data Tables 1 and 2).

Strains and plasmids

Plasmids were constructed using restriction-enzyme mediated and Gibson assembly cloning methods. Neoantigen construct iterations were designed and created as Geneblocks (IDT) encoding a constitutive promoter and 5′ untranslated region (UTR) containing selected ribosome-binding site, followed by coding region composed of mutant-residue containing long peptides connected in tandem or by various linkers as indicated. 5′ BamHI and 3′ XbaI restriction endonuclease sites were added to constructs. Coding sequences were codon optimized for E. coli. Constructs were cloned between BamHI and XbaI restriction sites on a stabilized p246-luxCDABE plasmid where luxCDABE had been cloned out²², and flanked by 3′ λ-phage transcription terminator, with high-copy pUC origin. For protein expression assessment studies, the codon sequence for a 6×-Histidine Tag (HisTag) was added immediately before the stop codon within the neoantigen construct coding sequence by PCR amplification of full construct plasmids with oligonucleotide containing 6×-HisTag sequence followed by kinase, ligase, DpnI enzyme mix protocol (NEB). Neoantigen construct plasmids were transformed into chemically competent E. coli DH5α or BL21(DE3) (New England Biolabs), or electrocompetent EcN parental strain or genetic derivatives. The parental EcN strain and all derivatives used in this study harbour an integrated luxCDABE cassette within the genome, which also contains an erythromycin resistance gene⁷⁰. Plasmid encoding constitutive LLO was constructed by cloning in the hok/sok stabilization system to pCG02-p15a backbone⁷¹, PCR amplification of backbone with SLC cloned out, and Gibson assembly of Geneblock encoding LLO under constitutive promoter and 5′ UTR containing selected ribosome-binding site. Constitutive LLO plasmids were transformed into electrocompetent EcN parental and genetic derivative strains. Strains were cultured in Luria-Bertani (LB) medium with antibiotics for plasmid retention (pUC:kanamycin 50 μg ml^−1, p15a:spectinomycin 50 μg ml^−1) in a 37 °C orbital incubator.

Construction of cryptic plasmid-cured EcN

EcN cryptic plasmids were cured with Cas9-mediated double-strand break, as described previously²⁷. Briefly, EcN was transformed with pFREE or pCryptDel4.8 to cure the cryptic plasmids pMUT1 or pMUT2, respectively. The transformants were grown overnight and diluted 1:1,000 the next day into fresh LB containing 0.2% rhamnose and 0.43 μM anhydrotetracycline. After 24 h of incubation, the culture was streaked onto LB plates without antibiotics and incubated overnight in a 30 °C incubator. Colonies were screened with colony PCR to verify the loss of cryptic plasmids.

Construction of genetic knockout strains

Genetic knockouts were performed using the lambda red recombination system⁷². In brief, EcNc was transformed with pKD46. Transformants were grown at 30 °C in LB with ampicillin and l-arabinose, then made electrocompetent. The chloramphenicol resistance cassette with corresponding overhangs for each target gene for deletion was prepared by PCR amplification of pKD3. Electroporation was performed using 100 μl of competent cells and 50–300 ng amplified DNA. After 2 h of recovery, cells were plated on LB agar containing chloramphenicol and incubated at 37 °C overnight. Target gene deletion was verified by colony PCR. For excision of the antibiotic resistance marker, pCP20 was transformed, and the transformants were plated on fresh LB plates containing ampicillin and incubated at 30 °C overnight. Selected colonies were then inoculated onto fresh LB plates without antibiotics and cultured at 43 °C overnight for induction of flippase and plasmid curing. Clones were subsequently screened for loss of antibiotic resistance.

qPCR for PCN

Copy number variant plasmids were constructed from a high-copy pUC-GFP²² plasmid. The plasmid backbone excluding the pUC origin was PCR-amplified and Gibson assembled with sc101*, p15A or ColE1 origin of replication insert. The respective inserts were prepared from PCR amplification of template plasmid pCG02_sc101*, pCG02_p15A or pTH05_ColE1. Plasmid copy number (PCN) was determined as reported previously²², in which the relative abundance of plasmid DNA compared to genomic DNA is measured by qPCR. Briefly, strains with the plasmid of interest were grown at 37 °C overnight in fresh LB with appropriate antibiotics. Cells were collected by centrifugation at 3,000g at 4 °C for 10 min, the supernatant removed and the cell pellet resuspended in distilled water for optical density measurement at 600 nm (OD₆₀₀) equal to 1. Resuspended cells were fivefold serially diluted. Samples were denatured at 95 °C for 10 min and 2 μl of each sample dilution was added into 18 μl of NEB Luna Universal qPCR Master Mix in each well of a 96-well plate. Then 25-fold diluted samples were used for the measurement of crossing point values: the cycle number when amplified sample fluorescence exceeds the background. Fivefold diluted samples were used for generation of the standard curve for PCR efficiency (E). E was defined from the slope (S) of each standard curve with the equation E = 5(−1/S) and PCN was determined with the equation PCN = (E_G^CTG)/(E_P^CTP), where respective values for genomic DNA are denoted by a subscript G and plasmid DNA by subscript P.

Immunoblot and ELISA

For immunoblot and ELISA, a C-terminal 6×-HisTag was attached to each neoantigen construct. Strains expressing neoantigen construct with C-terminal 6×-HisTag were grown overnight in LB media with appropriate antibiotics. Equalization of OD₆₀₀ measurement to match colony-forming units (CFU) per ml (CFU ml^−1) between all cultures was done before all sample processing. CFU-matched cultures were centrifuged at 3,000g at 4 °C for 10 min. For immunoblot, samples were resuspended in B-PER lysis reagent (ThermoFisher Scientific) containing 250 U ml^−1 benzonase nuclease (Millipore Sigma) and 1 U ml^−1 rLysozyme (Millipore Sigma) and placed on an orbital shaker for 15 min at room temperature. Samples were centrifuged at 10,000g for 20 min at 4 °C to separate soluble and insoluble fractions or total lysate used directly. Processed samples were mixed with SDS-loading buffer with 5 mM dithiothreitol, boiled and subject to immunoblot analysis. For relative quantification of immunoblot chemiluminescent intensity, target protein bands on the same blot were normalized to the loading control band DnaK for the same sample. DnaK loading controls were always run on the same gel as target proteins. Normalized values were divided to provide relative intensity values. Mouse anti-6×His (αTHE) was purchased from GenScript, mouse anti-DnaK was purchased from Abcam (8E2/2). αTHE and 8E2/2 antibodies were used at 1:5,000 dilution.

For HisTag ELISA, samples were resuspended in ice-cold PBS containing HALT protease inhibitor cocktail (ThermoFisher Scientific). Samples were sonicated on ice for 2 min total time. Sonicated samples were centrifuged at 10,000g for 20 min at 4 °C. Soluble sample fractions were analysed using GenScript HisTag ELISA Detection Kit as per the manufacturer’s instructions.

For ex vivo immunoblot analysis, BALB/c mice bearing established hind-flank CT26 tumours were injected intravenously with the EcNc^{Δlon/ΔompT/LLO+} nAg^19-His strain cocktail that contains all three neoantigen constructs, in which each construct (MHCIa, MHCIIa and MHCI/II^v) contained a C-terminal 6×-HisTag. Then 48 h after treatment, tumours and TDLNs were extracted from mice and placed in B-PER lysis reagent (ThermoFisher Scientific) with 250 U ml^−1 benzonase nuclease (Millipore Sigma), and homogenized using a gentleMACS tissue dissociator (Miltenyi Biotec, C-tubes). Tissue homogenate was sonicated on ice for 3 min. Sonicated samples were centrifuged at 10,000g for 20 min at 4 °C to separate soluble and insoluble fractions, and fractions subsequently resuspended and diluted in lysis buffer. Sample fractions were mixed with SDS-loading buffer with 5 mM dithiothreitol, boiled and subject to immunoblot analysis.

For ex vivo IL-12p70 ELISA analysis, BALB/c mice bearing established hind-flank CT26 tumour were injected intratumourally with PBS, EcNc^{Δlon/ΔompT} or EcNc^{Δlon/ΔompT/LLO+}. Then 4–24 h after treatment, tumours were extracted and placed in ice-cold PBS containing HALT protease inhibitor cocktail without dimethylsulfoxide (DMSO) (ThermoFisher Scientific). Tumours were homogenized using a gentleMACS tissue dissociator (Miltenyi Biotec, C-tubes), and centrifuged at 3,000g for 10 min at 4 °C. The supernatant was then collected and centrifuged at 10,000g for 20 min at 4 °C to separate soluble and insoluble fractions. IL-12p70 in soluble sample fractions was analysed using the Mouse IL-12p70 Quantikine ELISA Kit (R&D systems) according to the manufacturer’s instructions.

Blood bactericidal assay

EcN wild-type or EcNc^{Δlon/ΔompT} were cultured overnight in LB media without antibiotics. Cultures were centrifuged at 3,000g for 10 min, resuspended in 1 ml of ice-cold sterile PBS and normalized to OD₆₀₀ = 1. Then 50 μl of OD₆₀₀ = 1 microbe suspension was added to 1 ml of single donor human whole blood (Innovative Research) in triplicate and incubated in a 37 °C stationary incubator. After 2 h of incubation, a sample was taken from each blood–microbe mixture and serial dilution was prepared in PBS. Dilutions were plated on LB agar with erythromycin (25 μg ml^−1). After incubation overnight at 37 °C, colonies were quantified by spot-forming assay and CFU ml^−1 blood was calculated.

Biofilm assay

Biofilm formation assays were conducted as described previously⁷³. Briefly, EcN wild-type, cryptic plasmid-cured (EcNc), Lon knockout (EcNc^Δlon), OmpT knockout (EcNc^ΔompT) or double protease knockout (EcNc^{Δlon/ΔompT}) were cultured for 48 h in LB media with 25 μg ml^−1 erythromycin in borosilicate glass tubes in a 30 °C stationary incubator, with tube caps wrapped with parafilm to prevent evaporation. At 48 h, cultures were discarded and borosilicate tubes were washed three times with PBS. Tubes were inverted and allowed to dry for 6 h. Biofilms left on borosilicate tubes were stained with 0.1% (vol/vol) crystal violet for 15 min. Crystal violet stain was discarded and tubes washed three times with PBS, then inverted and allowed to dry overnight. Crystal violet-stained biofilms were dissolved with 95% ethanol and transferred to 96-well plates for measurement of absorbance at 590 nm.

Phagocytosis assay

Bacterial phagocytosis assays were adapted from previous work⁷⁴. Culture and isolation of murine BMDMs was performed as described previously⁷⁵. Bulk femoral bone marrow cells from BALB/c or C57BL/6 mice were cultured on 15 cm non-treated cell culture Petri dishes in RPMI with 20% FBS, 25 ng ml^−1 M-CSF (R&D Systems) and 100 U ml^−1 penicillin–streptomycin. Media was replaced with fresh media after 4 days of culture. After 7 days of culture, plates were washed with PBS and adherent macrophages were dissociated using trypsin-EDTA. Macrophages were washed in PBS, resuspended at a density of 2 × 10⁵ cells per ml in media and 1 ml transferred to each well of 24-well plates. The 24-well plates were incubated overnight in a 37 °C incubator with humidified 5% CO₂. EcN wild-type or EcNc^{Δlon/ΔompT}, with or without a constitutive green-fluorescent protein (GFP)-expressing plasmid were cultured overnight in LB media with appropriate antibiotics. Bacterial cultures were centrifuged at 3,000g for 10 min, washed three times with sterile PBS and resuspended at a density of 4 × 10⁸ bacteria per ml in sterile PBS. Media from wells containing adherent macrophages was aspirated, wells washed three times with PBS and 1 ml of RPMI with 5% mouse serum added to each well. Latrunculin A was added at a concentration of 1 μM to selected wells to inhibit phagocytosis. Next, 2 × 10⁷ CFU of microbes were added to each well with each condition tested in triplicate. Microbial strains were incubated with BMDMs for 30 min in a 37 °C incubator at 20 rpm. After 30 min, media was aspirated and wells were washed six times with sterile ice-cold PBS. Adherent macrophages were dissociated using non-enzymatic cell dissociation buffer (Gibco), resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% FBS, 2 mM EDTA and 0.09% sodium azide) and analysed by flow cytometry.

In vitro BMDM activation

BMDMs were cultured as described above for phagocytosis assays. BMDMs were washed in PBS, resuspended at a density of 2 × 10⁵ ml^−1 in media and 1 ml transferred to each well of 24-well plates. The 24-well plates were incubated overnight in a 37 °C incubator with humidified 5% CO₂. Wild-type EcN or EcNc^{Δlon/ΔompT} with constitutive expression of OVA from a pUC origin plasmid were cultured overnight in LB media with appropriate antibiotics. Cultures were centrifuged at 3,000g for 10 min, washed three times with PBS and resuspended at a density of 4 × 10⁸ bacteria ml^−1 in sterile PBS. Media from wells containing macrophages was aspirated, wells were washed three times with PBS and 1 ml of RPMI with 5% mouse serum was added to each well. Next, 1 × 10⁷ live microbes were added to each well, with each condition replicated in triplicate. Live microbial strains were incubated with BMDMs for 6 h in a 37 °C incubator. After 6 h, media was aspirated and wells were washed six times with sterile ice-cold PBS. Adherent macrophages were dissociated using non-enzymatic cell dissociation buffer (Gibco), resuspended in FACS buffer and analysed by flow cytometry. DRAQ7 cell viability reagent was used to exclude dead cells (diluted 1:1,000 in FACS buffer). Extracellular antibodies for BMDM activation panel included CD80 (catalogue no. 16-10A1, Biolegend), MHC-II (catalogue no. M5/114.15.2, Biolegend), PD-L1 (catalogue no. 10F.9G2, Biolegend) and H2K^b-SIINFEKL (catalogue no. 25-D1.16, Biolegend), each used at 1:200 dilution.

In vitro BMDC stimulation

BMDC isolation and culture from mouse bone marrow was adapted from previous methods⁷⁶. BMDCs from C57BL/6 mice were cultured on 15 cm non-treated cell culture Petri dishes in RPMI with 20% FBS, 20 ng ml^−1 GM-CSF (Biolegend) and 100 U ml^−1 penicillin–streptomycin. Every 1–2 days for the first 4 days, plates were gently washed and non-adherent granulocytes removed by aspirating 50% of the culture media with subsequent replacement of fresh media. On day 4, media was aspirated completely and replaced with fresh culture media with 20 ng ml^−1 GM-CSF. On day 6, BMDC plates were washed with PBS and loosely adherent and non-adherent cells collected. Cells were centrifuged at 300g for 5 min, resuspended in fresh culture media and replated on 15 cm non-treated cell culture Petri dishes. On days 7–8, plates were washed with PBS and loosely adherent and non-adherent cells were collected. Cells were centrifuged at 300g for 5 min, resuspended in fresh culture media at a density of 2.5 × 10⁵ ml^−1 and 200 μl was transferred to 96-well plates and incubated overnight in a 37 °C incubator. The next day, media from wells containing BMDCs was aspirated, and 1 ml of RPMI with 5% mouse serum was added to each well. BMDCs were pulsed with live bacteria at an multiplicity of infection of 10 for 2 h. Plates were centrifuged at 300g for 5 min, media aspirated and replaced with fresh RPMI with 10% FBS, 10 μg ml^−1 gentamicin and 100 U ml^−1 penicillin–streptomycin. Gentamicin concentration was increased to 40 μg ml^−1 after 2–4 h. Plates were incubated for 5–48 h in a 37 °C incubator, at which time the supernatant was assessed for IL-12p70 using the Mouse IL-12p70 Quantikine ELISA Kit (R&D systems) according to the manufacturer’s instructions.

OT-I and OT-II T cell stimulation and proliferation

BMDCs were cultured as above, resuspended at a density of 2.5 × 10⁵ ml^−1 and 5 × 10⁴ BMDCs transferred to 96-well plates and incubated overnight in a 37 °C incubator. The next day, media from wells containing BMDCs was aspirated, and 1 ml of RPMI with 5% mouse serum was added to each well. BMDCs were pulsed with 2 × 10⁶ CFU of the respective bacterial strain for 2.5 h, plates were centrifuged at 300g for 5 min, media aspirated and replaced with fresh RPMI with 10% FBS and 10 μg ml^−1 gentamicin and 100 U ml^−1 penicillin–streptomycin. Gentamicin concentration was increased to 40 μg ml^−1 after 2–4 h. Spleens from naive OT-I and OT-II mice were extracted, filtered through 100 µm cell strainers and washed in complete RPMI (RPMI-1640 supplemented with 10% (vol/vol) FBS, 1× GlutaMax, 1% (vol/vol) MEM non-essential amino acids solution (Gibco-11140050) and 100 U ml^−1 penicillin–streptomycin). OT-I and OT-II T cells were isolated from single-cell suspensions of spleens from the respective transgenic mouse using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Purified OT-I and OT-II T cells were resuspended in T cell media (complete RPMI supplemented with 50 μM β-mercaptoethanol) at a density of 5 × 10⁵ ml^−1 and 5 × 10⁴ T cells incubated with 5 × 10⁴ BMDCs pulsed with the respective microbial strains. For cytokine secretion assessment, T cells were incubated with BMDCs for 24 h, at which time supernatant was assessed for IFNγ and IL-2 using Mouse IFNgamma Quantikine ELISA Kit and Mouse IL-2 Quantikine ELISA Kit (R&D systems) according to the manufacturer’s instructions.

Carboxyfluorescein succinimidyl ester (CFSE) proliferation assays were conducted as previously described⁷⁷. Here, 1 × 10⁷ OT-I or OT-II T cells were resuspended in 1 ml of room temperature PBS, and 1 μl of 5 mM CFSE (Biolegend) was added. T cells were incubated in CFSE solution for 5 min at room temperature protected from light, after which time staining was quenched by adding ten times the staining volume of cell culture media. T cells were centrifuged at 300g for 5 min, resuspended in T cell media at a density of 5 × 10⁵ ml^−1 and incubated for an extra 10 min at room temperature. Then 5 × 10⁴ T cells were incubated with 5 × 10⁴ BMDCs pulsed with the respective live microbial strains. At 48 h, 50% of the media from each well was gently aspirated so as to not disturb any cells, and replaced with fresh T cell media. At 72–96 h, OT-I and OT-II T cells were collected and analysed by flow cytometry. DRAQ7 cell viability reagent was used to exclude dead cells (diluted 1:1,000 in FACS buffer). Extracellular antibody staining for CFSE assays included antimouse CD3 (catalogue no. 17A2, Biolegend), used at 1:200 dilution.

Listeriolysin haemolytic activity assay

Sheep red blood cell (RBC) lysis by bacterial lysate was performed as described previously⁷⁸. Briefly, bacteria were grown overnight in fresh LB containing appropriate antibiotics. Cultures were centrifuged at 3,000g for 10 min, supernatants discarded and the cell pellet resuspended to OD₆₀₀ = 8 in 0.1% (w/w) bovine serum albumin (BSA) in sterile PBS titrated to pH of 5.25 with 1 M HCl. Bacteria were sonicated for 2 min. After sonication, the soluble fraction was isolated by centrifugation at 10,000g at 4 °C for 20 min. Sheep RBCs were washed three times with PBS and resuspended at a final concentration of 6 × 10⁸ ml^−1 in 0.1% (w/w) BSA in PBS titrated to pH of 5.25. Equal parts of bacterial lysate soluble fraction and sheep RBC suspension were mixed and incubated for 15 min at 37 °C. After incubation RBC mixtures were centrifuged at 1,000g for 1 min at 4 °C and supernatant absorbance at 541 nm was then measured to quantify RBC lysis.

Listeriolysin cytosolic access assay

BMDMs were cultured as described above for phagocytosis assays. BMDMs were washed in PBS, resuspended at a density of 2.5 × 10⁵ ml^−1 in media, 100 ml transferred to wells of an eight-well Lab-Tek Chamber Slide system (ThermoFisher) and incubated overnight in a 37 °C incubator. The next day, media from wells containing BMDMs was aspirated, and 1 ml of complete RPMI without antibiotics was added to each well. BMDMs were then pulsed with 1.25 × 10⁶ CFU of the respective bacterial strain for 60 min. After the designated time media from each well was aspirated, wells were washed four times with PBS and media was replaced with fresh RPMI with 10% FBS and 40 μg ml^−1 gentamicin and incubated in a stationary 37 °C incubator. After either 30 or 60 min of more incubation, media was aspirated and wells washed four times with ice-cold PBS. Then 100 ml of 100% methanol at −80 °C was then added to each well for fixation and allowed to incubate at room temperature for 10 min. Methanol was then removed, 100 ml of ice-cold PBS added to each well and slides incubated at 4 °C. Cells were permeabilized with 0.5% Triton X in PBS for 10 min. Blocking solution in 10% heat-inactivated horse serum and 3% BSA was added to each well for 30 min. After washing three times, primary antibodies in 1% heat-inactivated horse serum and 1% BSA were incubated overnight at 4 °C in a humidified chamber. The next day, slides were washed with PBS three times for 10 min each and secondary antibodies were applied for 1 h at room temperature in the dark. DAPI (4′6-diamidino-2-phenylindole) was applied as part of the secondary antibody cocktail for nuclear staining. Slides were washed in PBS three times before mounting coverslips with DAKO gel and stored at 4 °C until immunofluorescence analysis. Anti-ovalbumin (catalogue no. EPR27117-90, Abcam) and anti-CD11b (catalogue no. M1/70, Abcam) primary antibodies were used for staining, both at 1:200 dilution.

Animal experiments

All animal experiments were approved by the Institutional Animal Care and Use Committee (Columbia University, protocol AABQ5551). The 6–7-week-old female BALB/c, C57BL/6 and B6(Cg)-Tyrc-2J/J (Jackson Laboratories) mice were kept in accordance with all rules for animal research at Columbia University. Mice were housed in a facility with a 12 h light–dark cycle, and provided unrestricted access to both food and water. The housing facility was maintained at 21–24 °C, and kept at 40–60% humidity. Sample size was determined on the basis of our previous studies and/or pilot experiments. For subcutaneous tumour models: 5 × 10⁶ CT26 cells in 100 μl of sterile PBS were inoculated subcutaneously on the hind flank of BALB/c mice, or 5 × 10⁵ B16F10 melanoma cells in 100 μl of sterile PBS subcutaneously on the hind flank (orthotopic) of C57BL/6 mice using a 26G needle on a 1 cc syringe. CT26 tumours were allowed to establish as indicated for each experiment, and mice were distributed between groups to equate the average starting tumour volume before treatment. B16F10 orthotopic tumours were allowed to establish for 9 days, and initial average tumour volume equated between groups before treatment. Tumour dimensions were measured unblinded with a calliper every 1–3 days for calculating tumour volumes using the equation (a²â€‰Ã— b)/2 (a is width, b is length, where width is the smaller dimension). Group tumour sizes were computed as mean ± s.e.m. Body weight was measured each time tumour measurements were taken. Animals were euthanized when any of the following criteria were met: tumour burden greater than 2 cm in the largest dimension for any subcutaneous tumour, greater than 20% body weight loss, as otherwise recommended by veterinary staff or when showing clinical signs of impaired health. To examine the requirement of individual T cell populations for the efficacy of the microbial neoantigen vaccines, mice were injected intraperitoneally with 200 μg (in 100 μl of InVivoPure pH 6.5 buffer, BioXcell) of antimouse CD4 (clone GK1.5, BioXcell), 200 μg (in 100 μl of InVivoPure pH 7.0 buffer, BioXcell) of antimouse CD8β (clone Ly-3.2, BioXcell) or 200 μg (in 100 μl of InVivoPure pH 7.0 buffer, BioXcell) of IgG1 isotype control (clone HRPN, BioXcell) beginning 2 days before the initiation of therapeutic treatment and every 2–3 days thereafter until study endpoint.

In prophylactic vaccination studies, BALB/c mice received an intravenous injection of either EcNc^{Δlon/ΔompT/LLO+} OVA or EcNc^{Δlon/ΔompT/LLO+} nAg¹⁹ every 3–5 days for a total of four injections. Four days after the final injection, 1 × 10⁶ CT26 cells in 100 μl of sterile PBS were inoculated subcutaneously on the hind flank. In rechallenge studies, BALB/c mice that had cleared subcutaneous CT26 tumours on a single hind flank were engrafted with 1 × 10⁶ CT26 cells on the opposite hind flank 100 days after tumour clearance. Age-matched naive BALB/c mice were engrafted with 1 × 10⁶ of the same CT26 cells on a single hind flank as controls.

For therapeutic studies in systemic metastases models, 5 × 10⁵ CT26-Luc cells or 1.5 × 10⁵ B16F10-Luc cells were injected in 100 μl of sterile PBS through the lateral tail vein with a 27G needle on 1 cc syringe. Metastases were allowed to establish for 4 days in Balb/C mice before treatment for CT26-Luc, and for 2 days in C57BL/6 albino mice (B6(Cg)-Tyrc-2J/J) for B16F10-Luc. Mice were randomly distributed between groups after metastases engraftment and before treatment. For in vivo luminescence tracking of metastases burden, mice were injected intraperitoneally with 125 μl of aqueous solution of d-Luciferin (50 mg ml^−1) 6 min before imaging, and placed under isoflurane anaesthesia for imaging using an in vivo imaging system (IVIS), with exposure time set to 6 min. Total flux from the lungs (CT26-Luc) or body (B16F10-Luc) was used to quantify tumour burden. For evaluation of lung metastases burden at the timepoint of treatment initiation, mice were injected intraperitoneally with 250 μl of aqueous solution of d-Luciferin (50 mg ml^−1) 6 min before imaging and placed under isoflurane anaesthesia for imaging using an IVIS with exposure time set to 10 min. After in vivo IVIS analysis, mice were then re-injected with 100 μl of aqueous solution of d-luciferin (50 mg ml^−1) and lungs were extracted for ex vivo IVIS imaging with exposure time set to 2 min.

No formal blinding was done for in vivo experiments. For all animal experiments, intratumoural treatments were injected directly into the tumour core with care to not allow leakage of any therapeutic solution. Intravenous treatments were injected through the lateral tail vein, with care not to allow leakage of any therapeutic solution.

SLP vaccination

The formulation and administration of SLP vaccines was adapted from previous studies^15,79,80. Each dose contained either 20 μg of each 29-mer CT26 neoantigen peptide (19 neoantigens, 380 μg of total peptide per dose) and 50 μg of poly I:C in 200 μl of 10% DMSO/90% PBS (vol/vol) in Fig. 2e and Extended Data Fig. 5e,f, or 25 μg of each 29-mer CT26 neoantigen peptide (19 neoantigens, 475 μg total peptide per dose) and 100 μg of poly I:C in 200 μl of 10% DMSO /90% PBS (vol/vol) in Extended Data Fig. 5g. Therapeutic SLP vaccinations were administered subcutaneously to BALB/c mice with established hind-flank CT26 tumours on the contralateral hind flank using a 29G needle. SLP vaccine groups were treated on the same days as microbial therapeutic groups.

Ex vivo lung histology

Explanted lungs from BALB/C mice bearing CT26-Luc metastases or C57BL/6 albino mice (B6(Cg)-Tyrc-2J/J) bearing B16F10-Luc metastases were washed three times in PBS and placed in 10% formalin. After at least 24 h of fixation, lungs were transferred to 70% ethanol and subsequently embedded in paraffin. Then 50 μm consecutive sections were stained with haematoxylin and eosin. Lung sections were analysed for the presence of tumour foci.

Microbial administration for in vivo experiments

For therapeutic administration, bacterial strains were grown overnight in fresh LB media containing the appropriate antibiotics. Overnight cultures were centrifuged at 3,000g at 4 °C for 10 min and washed three times with ice-cold sterile PBS. Microbes were delivered intratumourally at a concentration of 5 × 10⁸ CFU ml^−1 in sterile PBS, with 20 μl of injected using a 1 cc syringe with a 29G needle. For intravenous treatment, 100 μl of microbes were delivered at a concentration of 1 × 10⁸ CFU ml^−1 in sterile PBS, through the lateral tail vein using a 1 cc syringe with a 29G needle.

Biodistribution and in vivo bacterial dynamics

For biodistribution experiments, BALB/c mice bearing established hind-flank CT26 or lung metastatic CT26-Luc tumours were injected intravenously with 100 μl of 1 × 10⁸ CFU ml^−1 EcNc^{Δlon/ΔompT/LLO+}. Then 96–120 h after a single i.v. injection for hind-flank tumours or at endpoint for lung metastases, tumours or tumour-bearing lungs and other organs were extracted from mice, weighed and homogenized using a gentleMACS tissue dissociator (Miltenyi Biotec, C-tubes). Homogenates were serially diluted in sterile PBS and plated on LB agar plates at 37 °C overnight. Colonies were quantified per organ and computed as CFU per gram of tissue (CFU g^−1). For tracking bacterial colonization of subcutaneous tumours by microbial luminescence, tumour-bearing mice treated intratumourally or intravenously with wild-type EcN parental strain or genetic derivates were imaged using IVIS at various time points. For abscopal experiments, treated and untreated tumours were harvested 14 days after a single intratumoural bacterial injection.

Ex vivo T cell killing assay

For B16F10-Luc specific killing, naive tumour-free C57BL/6 mice were injected intravenously every 4 days with PBS, EcNc^{Δlon/ΔompT/LLO+} OVA or nAg⁴² for a total of four doses. Five days after the final dose spleens from treated mice were extracted, filtered through 100 µm cell strainers and washed in complete RPMI. T cells were isolated from single-cell suspensions of spleens from the respective mouse using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Purified T cells were resuspended in T cell media for use in the specific lysis assay.

The luciferase-based killing assay was adapted from previous methods⁸¹. B16F10-Luc target cells were grown for 48 h in the presence of 100 U ml^−1 murine IFNγ. Target cells were gathered and plated at 1 × 10⁴ cells per well in a 96-well plate. After 12 h, T cells were added to each well to achieve designated effector-to-target ratios (10:1, 20:1 or 40:1). After 42 h of co-incubation, 50 U ml^−1 IL-2 was added to all wells.

For CT26-Luc versus 4T1-Luc luciferase-based specific killing assay: BALB/c mice with established hind-flank CT26 tumours were treated intravenously with EcNc^{Δlon/ΔompT/LLO+} nAg¹⁹ on days 0 and 3. On day 8, tumours were extracted and mechanically homogenized, followed by digestion with collagenase A (1 mg ml^−1, Roche) in isolation buffer (RPMI-1640 with 5% FBS, 1% l-glutamine, 1% penicillin–streptomycin and 10 mM HEPES) with gentamicin (40 μg ml^−1) for 1 h at 37 °C on a shaker platform at 150 rpm. Tumour homogenates were filtered through 100 µm cell strainers and washed in T cell media. Tumour-infiltrating T cells were isolated from single-cell suspensions of tumours from mice using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Purified T cells were resuspended in T cell media.

CT26-Luc or 4T1-Luc target cells were grown for 12 h in the presence of 100 U ml^−1 murine IFNγ. Target cells were gathered and plated at 1 × 10⁴ cells per well in a 96-well plate. After 12 h, T cells were added to each well to achieve designated effector-to-target ratios (5:1 or 10:1), with 50 U ml^−1 IL-2 added to all wells.

Luminescence from each well was quantified after addition of One-Glo Luciferase Assay System (Promega), as per the manufacturer’s instructions, after 24–96 h of coculture. Minimum lysis wells contained only the respective luciferase-expressing tumour target cells. In maximum lysis wells, 20 μl of media was replaced with 20 μl of 3% Triton X-100 60 min before luminescence readout. Specific lysis (%) was calculated using the luminescence values of the respective conditions with the following formula: 100 − (100 × ((sample − maximum lysis)/(minimum lysis − maximum lysis))).

IFNγ ELISpot

BALB/c mice with established hind-flank CT26 tumours were treated intravenously with EcNc^{Δlon/ΔompT/LLO+} nAg¹⁹ on day 0 and 3. On day 8, tumours were extracted and mechanically homogenized, followed by digestion with collagenase A (1 mg ml^−1, Roche) in isolation buffer (RPMI-1640 with 5% FBS, 1% l-glutamine, 1% penicillin–streptomycin and 10 mM HEPES) with gentamicin (40 μg ml^−1) for 1 h at 37 °C on a shaker platform at 150 rpm. Tumour homogenates were filtered through 100 µm cell strainers and washed in RPMI containing CTL-Wash Supplement (Immunospot) and 1% l-glutamine. Splenocytes from naive BALB/c mice were isolated in the same way, without digestion. T cells were isolated from single-cell suspensions of tumours from mice using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Purified T cells were resuspended in CTL-Test Medium supplemented with 1% l-glutamine for use in the enzyme-linked immunosorbent spot (ELISpot) assay.

Mouse IFNγ Single-Color ELISpot plates and kits were purchased from Immunospot. ELISpot plates were prepared as per manufacturer’s instructions. Here, 5 × 10⁵ naive splenocytes were plated with 2 × 10⁴ TILs per well in 200 μl CTL-Test Medium supplemented with 1% l-glutamine and gentamicin (30 μg ml^−1). Then 29-mer synthetic neoantigen or negative control (OVA) peptides were added to each well at a final concentration of 5 μg ml^−1. Cells were stimulated overnight in a stationary 37 °C incubator with atmosphere of humidified 5% CO₂. After incubation, plates were developed as per the manufacturer’s protocol and spots quantified using a CTL Immunospot S6 Universal machine and CTL ImmunoSpot software v.7.0.24.0.

Flow cytometry immunophenotyping

For CT26 flow-cytometric immunophenotyping, BALB/c mice with hind-flank CT26 tumours received intravenous treatment with the indicated microbial therapeutic or PBS on day 0. Two or 8 days after treatment, TDLNs and/or tumours were extracted. For B16F10 flow-cytometric immunophenotyping, C57BL/6 mice with hind-flank, orthotopic B16F10 tumours received intravenous treatment with the indicated microbial therapeutic or PBS on day 0 and 3. Eight days after treatment, tumours were extracted. Lymphoid and myeloid immune subsets were isolated from tumour tissue by mechanical homogenization of tumour or TDLN tissue, followed by digestion with collagenase A (1 mg ml^−1, Roche) and DNase I (0.5 µg ml^−1, Roche) in isolation buffer (RPMI-1640 with 5% FBS, 1% l-glutamine, 1% penicillin–streptomycin and 10 mM HEPES) for 1 h at 37 °C for tumours or 30 min at 37 °C for TDLNs, on a shaker platform at 150 rpm. For ex vivo lymphocyte stimulation with PMA and ionomycin, TDLNs were not digested beforehand. Tumour and TDLN homogenates were filtered through 100 µm cell strainers and washed in isolation buffer. To measure overall cytokine production by T cells, cells were stimulated for 3 h with PMA (50 ng ml^−1, Sigma-Aldrich) and ionomycin (1 nM, Calbiochem) in the presence of brefeldin A (1 μg ml^−1). To measure neoantigen-specific cytokine production by T cells, cells were stimulated for 5 h with pools of peptides (2 μg ml^−1) representing the neoantigens encoded in therapeutic strains in the presence of brefeldin A (1 μg ml^−1). Cells were stained in FACS buffer. Ghost Dye cell viability reagent was used to exclude dead cells (diluted 1:1,000 in PBS). Extracellular antibodies for lymphoid immunophenotyping included: CD4 (RM4-5, Biolegend), NKp46 (29A1.4, BD Biosciences), NK1.1 (PK136, Biolegend), CD45 (30-F11, BD Biosciences), B220 (RA3-6B2, BD Biosciences), CD19 (6D5, Biolegend), CD8a (53-6.7, Biolegend), TIM-1 (RMT1-4, BD Biosciences) and CD69 (H1.2F3, BD Biosciences). After extracellular staining, cells were washed with FACS buffer, and fixed using the FOXP3/transcription factor staining buffer set (Tonbo), as per the manufacturer’s instructions. Intracellular antibodies for lymphoid immunophenotyping included: Foxp3 (FJK-16s, Thermo), CD3ε (145-2C11, Biolegend), TCRβ (H57-507, BD Biosciences), Ki-67 (SolA15, Thermo), Granzyme-B (QA16A02, Biolegend), TNF (MP6-XT22, Biolegend) and IFNγ (XMG1.2, Biolegend). For myeloid immunophenotyping, extracellular antibodies included: Ly6C (HK1.4, Biolegend), I-A/I-E (M5/114.15.2, BD Biosciences), XCR1 (ZET, Biolegend), CD11b (M1/70, Biolegend), CD103 (2E7, Biolegend), CD45 (30-F11, BD Biosciences), F4/80 (BM8, Biolegend), CD11c (HL3, BD Biosciences), CD172a/SIRPα (P84, Biolegend), Ly6G (1A8, Biolegend and BD Biosciences), PD-L1 (10 F.9G2, Biolegend), CD301b (URA-1, Biolegend), CD3 (145-2C11, Biolegend), CD19 (1D3, Biolegend), NK1.1 (PK136, Biolegend), NKp46 (29A1.4, Biolegend) CD64 (X54-5/7.1, Biolegend), CD80 (16-10A1, Biolegend) and CD86 (GL-1, BD Biosciences). All antibodies for flow cytometry were used at a 1:200 dilution. After staining, cells were washed and resuspended with FACS buffer for flow cytometry analysis using a BD LSRFortessa or Cytek Aurora cell analyser. FACS Diva or SpectroFlo software was used for data acquisition. Collected flow cytometry data were analysed using FlowJo.

Synthetic peptides

Synthetic peptides representing neoantigens for lymphocyte restimulation assays and vaccination were synthesized by and purchased from Peptide v.2.0. All peptides were above or equal to 95% purity.

Statistics and reproducibility

Statistical analyses and P value calculations were performed using GraphPad Prism v.9 and v.10. For each experiment, the particular statistical analysis is detailed in the respective figure legend. A two-tailed unpaired Student’s t-test, one-way analysis of variance (ANOVA) or two-way ANOVA with appropriate post hoc test was used for data that were roughly normally distributed. For analysis of Kaplan–Meier survival experiments, the log-rank (Mantel–Cox) test was used. All analyses were two-tailed. For all statistical analyses, NS denotes not significant, which is P > 0.05.

For Fig. 4a and Extended Data Fig. 7b, immunoblot data are representative of four independent experiments. In Extended Data Figs. 1b and 2h, immunoblot data are representative of three independent experiments. Immunofluorescence data in Extended Data Fig. 2i are representative of three independent experiments. Histology data in Extended Data Figs. 5h and 8f are representative of three independent experiments. All other results in the paper were replicated at least two to three times in independent experiments.

Biological materials availability

Reasonable requests for biological materials used in this study will be promptly reviewed by Columbia Technology Ventures to verify whether the request is subject to any intellectual property or confidentiality obligations. Any materials that can be shared will be released through a material transfer agreement.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.