Nonpathogenic E. coli engineered to surface display cytokines as a new platform for immunotherapy

Abstract

Given the safety, tumor tropism, and ease of genetic manipulation in non-pathogenic Escherichia coli (E. coli), we designed a novel approach to deliver biologics to overcome poor trafficking and exhaustion of immune cells in the tumor microenvironment, via the surface display of key immune-activating cytokines on the outer membrane of E. coli K-12 DH5α. Bacteria expressing murine decoy-resistant IL18 mutein (DR18) induced robust CD8⁺ T and NK cell-dependent immune responses leading to dramatic tumor control, extending survival, and curing a significant proportion of immune-competent mice with colorectal carcinoma and melanoma. The engineered bacteria demonstrated tumor tropism, while the abscopal and recall responses suggested epitope spreading and induction of immunologic memory. E. coli K-12 DH5α engineered to display human DR18 potently activated mesothelin-targeting CAR NK cells and safely enhanced their trafficking into the tumors, leading to improved control and survival in xenograft mice bearing mesothelioma tumor cells, otherwise resistant to NK cells. Gene expression analysis of the bacteria-primed CAR NK cells showed enhanced TNFα signaling via NFkB and upregulation of multiple activation markers. Our novel live bacteria-based immunotherapeutic platform safely and effectively induces potent anti-tumor responses in otherwise hard-to-treat solid tumors, motivating further evaluation of this approach in the clinic.

Extended Data Fig. 1 |

a, Representative plasmid map includes OmpA (EcOmpA) scaffold and mDR18 (mIL18-CS2).

Extended Data Fig. 2 |. Murine decoy-resistant IL18 displayed by bacteria inhibit tumor growth in an immune-competent syngeneic mouse model (MC38).

a-e, Mean tumor growth in mice bearing MC38 tumors after being treated with YiaT232, YiaT181, OmpA, Neae and C-IgAP displaying nothing, mDR18 or mIL15. Data are representative of one biological independent experiment, with n = 6 tumors per group. Two-way ANOVA test. Data represent means ± SEM (a-e).

Extended Data Fig. 3 |. The display levels of mDR18 are significantly increased by switching the replication origin in the plasmid.

a, The schematic figure shows an increase in the surface display levels of mIL18-CS2 (mDR18) by increasing the copy number of plasmids. b, Representative flow cytometry histograms of mDR18 levels on the surface of bacteria.

Extended Data Fig. 4 |. OmpA-mDR18 inhibits tumor growth in an immune-competent syngeneic melanoma mouse model (B16F10).

a, C57BL/6 mice were subcutaneously (s.c.) engrafted with 0.5 × 10⁶ B16F10 cells, starting on day 7 (tumor size reaches 40 – 70 mm³), mice were treated with PBS (n = 10), OmpA (0.5 × 10⁹ CFU, n = 10) or OmpA-mDR18 (0.5 × 10⁹ CFU, n = 10) (i.t.) five times (day 7, day 11 and day 14, day 18 and day 21). b, c, Mean tumor growth (b) and Kaplan–Meier survival curves (c) for mice bearing B16F10 tumors after treatment. d, e, Mean tumor growth (d) and Kaplan–Meier survival curves (e) of cured C57BL/6 mice (n = 3) obtained from mice treated with OmpA-mDR18 and naïve C57BL/6 (gender and age-matched with cured mice) were subcutaneously (s.c.) engrafted with 0.5 × 10⁶ B16F10 cells on the other side of flanks. Two-way ANOVA test for tumor growth curve (b, d) and Mantel-Cox test for survival curve (c, e). Data are representative of two independent experiments, with n = 10 mice per group (b, c). Data represent means ± SEM (b, d).

Extended Data Fig. 5 |. Treatment with the engineered bacteria induces immunological memory leading to enhanced recall responses.

a, b, C57BL/6 mice (n = 5) cured from MC38 upon treatment with OmpA-mDR18 and naïve C57BL/6 (gender and age-matched with the cured mice) subcutaneously (s.c.) engrafted with 0.5 × 10⁶ MC38 cells and then monitored for tumor growth and survival. Mean tumor growth (a) and Kaplan– Meier survival curves (b) for mice injected with 0.5 × 10⁶ MC38 cells on Day 0. Two-way ANOVA test for tumor growth curve (a) and Mantel-Cox test for survival curve (b). Data are representative of one independent experiment, with n = 5 mice per group (a, b). Data represent means ± SEM (a).

Extended Data Fig. 6 |. The concentration of key cytokines and chemokines and body weight in immune-competent mice upon treatment with OmpA-mDR18 and other controls.

a, The concentration of Eotaxin, granulocyte colony stimulating factor (G-CSF), interleukin-1α (IL1α), tumor necrosis factor alpha (TNF-α), interleukin-5 (IL5), interleukin-13 (IL13), leukemia inhibitory factor (LIF), C-X-C motif chemokine ligand 5 (CXCL5) and vascular endothelial growth factor (VEGF) in the plasma isolated from blood collected from submandibular vein. *p < 0.05; **p < 0.01; ****p < 0.0001, one-way ANOVA test for multi-comparison and unpaired t-test for single comparison (a). b, Body weight of mice mentioned in Fig. 3d. Data represent means ± SEM (a, b).

Extended Data Fig. 7 |. The concentration of key cytokines and chemokines in immune-competent mice upon treatment with OmpA-mDR18 and other controls.

a, The concentration of C-X-C Motif Chemokine Ligand 10 (CXCL10), Chemokine ligand 3 (CCL3), Chemokine ligand 4 (CCL4), interleukin-12 p40 (IL12p40), interleukin-12 p70 (IL12p70), interleukin-10 (IL10), interleukin-17 (IL17), C-X-C Motif Chemokine Ligand 1 (CXCL1), Macrophage colony-stimulating factor (M-CSF), C-X-C Motif Chemokine Ligand 9 (CXCL9), C-X-C Motif Chemokine Ligand 2 (CXCL2), and Chemokine ligand 5 (CCL5) in the plasma isolated from blood collected from submandibular vein. Unpaired t-test. Data represent means ± SEM (a).

Extended Data Fig. 8 |. OmpA-mDR18 rapidly promote tumor-infiltrating NK and CD8⁺ T cells in MC38 model.

a, C57BL/6 mice were subcutaneously (s.c.) engrafted with 0.5 × 10⁶ MC38 cells. When tumor size reached 150 – 200 mm³ on day 10, mice were treated with PBS (n = 5), OmpA-mDR18 (0.5 × 10⁹ CFU, n = 5), OmpA (0.5 × 10⁹ CFU, n = 5) or mDR18 (4mg/kg, n = 5) intratumorally (i.t.). Tumor tissues were harvested for further analysis for tumor-infiltrating immune cells (CD8⁺ T cells, CD4⁺ T cells, NK cells, NKT cells, macrophages, monocytes, granulocytes, and mononuclear myeloid-derived suppressor cells, M-MDSCs) 1 day after treatment by flow cytometer. b, The percentage of key cell types (as a proportion of live cells): CD8⁺ T cells (CD45⁺CD3⁺CD8⁺), CD4⁺ T cells (CD45⁺CD3⁺CD4⁺), NK cells (CD45⁺CD3⁻NK1.1⁺), T like NK cells (NKT, CD45⁺CD3⁺NK1.1⁺), granulocytes (CD45⁺CD11b⁺Ly6C⁺ Ly6G⁺), monocytes (CD45⁺CD11b⁺Ly6C^lowLy6G⁻), macrophages (CD45⁺CD11b⁺F4/80⁺) and M-MDSCs (CD45⁺CD11b⁺Ly6C^highLy6G⁻) in tumor microenvironment were shown in the bar graph. Unpaired t-test. c, Summary of the data showing the percentage of CD8⁺ T cells, NK cells, and granulocytes as a proportion of CD45⁺ cells blood cells in mice bearing MC38 on day 1 after being treated with bacteria (i.t.) or bacteria (i.t.) plus monoclonal antibody (i.p.).

Extended Data Fig. 9 |. Tumor control by OmpA-mDR18 in B16F10 is mediated by CD8⁺ T cells and Natural Killer (NK) cells in the tumor microenvironment (TME).

a, C57BL/6 mice were subcutaneously (s.c.) engrafted with 0.5 × 10⁶ B16F10 cells. On day 6, mice were treated with monoclonal antibodies anti-CD8α, anti-NK1.1, anti-ly6G, and PBS intraperitoneally (i.p.). When tumor size reached 40 – 70 mm³ on day 7, mice were treated with OmpA-DR18 (0.5 × 10⁹ CFU) or PBS intratumorally (i.t.) and monoclonal antibody (mAb) intraperitoneally (i.p.) on day 7, day 10, day 14, day 17 and day 21. b, c, Mean tumor growth (b) and Kaplan–Meier survival curves (c) for mice bearing B16F10 treated with monoclonal antibodies or PBS, and bacteria or PBS. Two-way ANOVA test for tumor growth curve (b) and Mantel-Cox test for survival curve (c). Data are representative of one independent experiment, with n = 5 mice per group. Data represent means ± SEM (b).

Extended Data Fig. 10 |. Clustering hDR18 on the surface of bacteria mediates enhanced activation of the IL18 receptor.

a, The activity of purified hDR18 at different concentrations. b, Linear regression of purified hDR18 activity from 10pg/ml to 1000pg/ml. c, IL18 activity of bacteria control or bacteria displaying hDR18 by scaffold C-IgAP, YiaT232 or YiaT181 in MOI = 10 or MOI = 100. The activities of hDR18 were measured by HEK-Blue^™ IL18 reporter cells.

Extended Data Fig. 11 |. Gene expression changes in the MSLN-CAR NK cells induced by priming with E. coli-hDR18.

a, Log2 fold changes for differentially expressed genes (YiaT232-hDR18, E. coli vs PBS, 5% FDR, n=2923). Fold changes were calculated for YiaT232-E. coli, YiaT232-hDR18, E. coli and hDR18 compared with PBS (paired). Data is row normalized. b, GSEA enrichment FDR values for the comparisons YiaT232-hDR18, E. coli vs PBS, YiaT232-E. coli and hDR18. The left panel represents down-regulated gene sets, and the right panel represents up-regulated gene sets. The dashed line depicts 5% FDR. Highlighted in red are gene sets that passed 5% FDR in all comparisons.

Extended Data Fig. 12 |. Bacteria displaying human decoy-resistant IL18 (hDR18) mediate activation of NK cells (PBMCs gated on NK cells) as assessed by expression of CD25 and CD60 on NK cells.

a, The Median Fluorescence Intensity (MFI) of CD69⁺ cells in CD56⁺ cells. b, The percentage of CD25⁺ cells in CD56⁺ cells. Unpaired t-test. PBMCs were harvested from three donors. Data represent means ± SEM (a, b).

Extended Data Fig. 13 |. Bacteria displaying human decoy-resistant IL18 (hDR18) increase the expression of key activation markers in primary NK cells.

a, The fold change of the percentage of TNFα⁺, IFNγ⁺, CD107a⁺, DNAM-1⁺, NKp46⁺, NKp30⁺, NKG2D⁺, Tim-3⁺, NKG2A⁺, CD69⁺ and TIGIT⁺ cells of different treatment groups compared to PBS treated groups. Unpaired t-test. NK cells were harvested from PBMCs of two donors. Data represent means ± SEM (a).

Extended Data Fig. 14 |. Bacteria displaying human decoy-resistant IL18 (hDR18) increase the expression of activation markers in primary NK cells.

a, The fold change of the percentage of CD39⁺, CD94⁺, KLRG-1⁺, NKp44⁺ and Siglec-7⁺ cells of different treatment groups compared to PBS treated groups. Unpaired t-test. NK cells were harvested from PBMCs of two donors. Data represent means ± SEM (a).

Extended Data Fig. 15 |. Protein Structure

of a, YiaT232-hDR18. b, YiaT232-mDR18. c, OmpA-hDR18. d, OmpA-mDR18, predicted by Colabfold based on Alphafold 2.

Extended Data Fig. 16 |. Flow cytometry gating strategy.

a, b, The gating strategy for analyzing granulocytes, M-MDSCs, monocytes, macrophages (a), CD4 T cells, CD8 T cells, NK cells, and NKT cells (b). The cells were harvested from tumors of mice bearing MC38 cells. c, The gating strategy for analyzing NK cells in human PBMCs. d, The gating strategy for analyzing MSLN-CAR NK cells in NSG mice.

Extended Data Fig. 17 |

Transduction rates of MSLN-CAR NK cells

Fig. 1 |. Murine decoy-resistant IL18 displayed by bacterial scaffold Lpp-OmpA induces potent anti-tumor responses in an immune-competent syngeneic mouse model (MC38).

a, Schematic figure showing the surface display of cytokines in E. coli by their transformation with the cytokine-scaffold coding plasmids and the surface expression assessed by flow cytometry. b, Heatmap showing the expression levels of 8 cytokines (murine interleukin-15, mIL15; decoy resistant murine interleukin-18, mIL18-CS2; murine interleukin-21, mIL21; human IL15, hIL15; human IL18, hIL18; two types of human decoy resistant IL18, human hIL18–6-12 and, hIL18–6-29; human IL21, hIL21) displayed by 5 different bacterial scaffolds: C-IgAP, Neae, Lpp-OmpA (OmpA), YiaT181 and, YiaT232. The display levels represent median fluorescence intensity (MFI) measured by flow cytometry. c, Mean tumor volumes as assessed on day 15 post-tumor inoculation in different groups of mice treated with PBS, mIL18-CS2 (mDR18, 0.4mg/kg), mIL15 (0.4mg/kg), bacteria displaying mDR18, mIL15 or scaffold only by C-IgAP, Lpp-OmpA, YiaT181, YiaT232 or Neae (10⁹ CFU) intratumorally (i.t.) three times on days 7, 10 and 14 (n = 6 each group)]. d, C57BL/6 mice were subcutaneously (s.c.) engrafted with 0.5 × 10⁶ MC38 cells on one side of the flank. Starting on day 7 after tumor cell injection (tumor size 50–100 mm³), mice were treated with PBS, mDR18 (mIL18-CS2, 4mg/kg), OmpA (0.25 × 10⁹ CFU), OmpA-mDR18 (0.25 × 10⁹ CFU). e, f, Mean tumor growth (e) and, Kaplan–Meier survival curves (f) for mice (n = 8–10 each group) bearing MC38 tumors after treatment with OmpA-mDR18 (0.25 × 10⁹ CFU), OmpA (0.25 × 10⁹ CFU), mDR18 (4mg/kg) and PBS. Two-way ANOVA test for tumor growth curve (e) and Mantel-Cox test for survival curve (f). Data represent means ± SEM (c, e).

Fig. 2 |. Systemically delivered tumor-homing OmpA-mDR18-E. coli induce an abscopal effect and are safe.

a, Mice (n = 5 each group) were subcutaneously (s.c.) engrafted with 0.5 × 10⁶ and 0.3 × 10⁶ MC38 cells into the left and right flanks. Starting from day 7, tumors on the left side were treated with PBS, OmpA-mDR18 (0.5 × 10⁹ CFU), or OmpA (0.5 × 10⁹ CFU) (i.t.) five times on days 7, 11, 14, 17, and 21. b, c, Mean tumor growth on the treated side (b), and untreated side (c) of mice. d, Mice (n = 8–10 each group) engrafted with 10⁶ MC38 cells were treated on days 8, 11, 15 and 18 intravenously (i.v.). e, f, Mean tumor growth (e) and Kaplan–Meier survival curves (f) of mice. g, On day 8 after engraftment of 10⁶ MC38 cells, mice (n = 3–5 each group) were treated once (i.v.). Plasma was isolated from blood collected from the submandibular vein on days 11 and 15 for cytokine measurement. Tissues were collected on day 15 for bio-distribution. h, Bacterial distribution in tumor, liver, and spleen on 7 days post-injection. CFU/g is bacterial concentration. i, The concentration of cytokines associated with CRS: interleukin-1β (IL1β), interleukin-6 (IL6), monocyte chemoattractant protein-1 (CCL2), interferon-γ (IFNγ), interleukin-2 (IL2) and granulocyte macrophage colony-stimulating factor (GM-CSF) in the plasma (on days 3 and 7 post-treatment unless other mentioned) from mice treated with LPS (5-hour post-treatment), PBS, mDR18, OmpA and OmpA-mDR18. Mice were treated with PBS, 4mg/kg mDR18, 10⁹ CFU OmpA or OmpA-mDR18 (d, e, f, g, h). Two-way ANOVA test for growth curve (b, c, e), Mantel-Cox test for survival curve (f), one-way ANOVA test and unpaired t-test for comparison (i). Data represent means ± SEM (b, c, e, i).

Fig. 3 |. Tumor control by OmpA-mDR18 is mediated by CD8⁺ T cells and Natural Killer (NK) cells in the tumor microenvironment (TME).

a, C57BL/6 mice were subcutaneously (s.c.) engrafted with 0.5 × 10⁶ MC38 cells in the flanks. When tumor size reached 150–200 mm³ on day 10, mice (n = 3–5 each group) were treated with PBS, OmpA-mDR18 (0.5 × 10⁹ CFU), OmpA (0.5 × 10⁹ CFU) or mDR18 (4mg/kg) intratumorally (i.t.). The mice were sacrificed, and the tumors harvested for flow cytometric analysis of tumor-infiltrating immune cells (CD8⁺ T cells, CD4⁺ T cells, NK cells, NKT cells, macrophages, monocytes, granulocytes, and mononuclear myeloid-derived suppressor cells, M-MDSCs) 3 days after the bacterial injection. b, The percentages of different cell types (as a proportion of live cells): CD8⁺ T cells (CD45⁺CD3⁺CD8⁺), CD4⁺ T cells (CD45⁺CD3⁺CD4⁺), NK cells (CD45⁺CD3⁻NK1.1⁺), T like NK cells (NKT, CD45⁺CD3⁺NK1.1⁺), granulocytes (CD45⁺CD11b⁺Ly6C⁺Ly6G⁺), monocytes (CD45⁺CD11b⁺Ly6C^lowLy6G⁻), macrophages (CD45⁺CD11b⁺F4/80⁺) and M-MDSCs (CD45⁺CD11b⁺Ly6C^highLy6G⁻) in the tumor microenvironment. c, C57BL/6 mice (n = 5 each group) were subcutaneously (s.c.) engrafted with 0.5 × 10⁶ MC38 cells and on day 6, treated with monoclonal antibodies anti-CD8α, anti-NK1.1, anti-ly6G, or PBS intraperitoneally (i.p.) and then treated with OmpA-DR18 (0.5 × 10⁹ CFU) or PBS intratumorally (i.t.) on days 7, 10, and 13. d, e, Mean tumor growth (d) and Kaplan–Meier survival curves (e) for mice bearing MC38 treated with monoclonal antibodies or PBS, and bacteria or PBS. Unpaired t-test for percentage data (b), two-way ANOVA test for tumor growth curve (d) and Mantel-Cox test (e) for survival curve. Data represent means ± SEM (b, d).

Fig. 4 |. Bacteria displaying human decoy-resistant IL18 (hDR18) enhance anti-tumor responses of mesothelin (MSLN)-CAR NK cells.

a, Schematic figure showing the use of HEK-Blue^™ IL18 reporter cells to screen multiple bacterial scaffolds expressing human decoy-resistant IL-18 (hDR18) for activation. b, The activity (assessed using HEK-Blue^™ in (a) of wildtype human IL18 (WT) and two variants of hDR18 (6–12 and 6–29) displayed by five bacterial scaffolds: Neae, C-IgAP, OmpA, YiaT232, YiaT181 with the multiplicity of infection (MOI) of E. coli displaying cytokines and HEK-Blue^™ IL-18 reporter at 10. Human IL18 activity is represented by OD620. c, The schematic figure for in vitro coculture killing assay with MSLN-CAR NK and tumor cells. Briefly, MSLN-CAR NK cells were primed by bacteria or other control groups overnight and then cocultured with the tumor cells for 4 hours, viability of the tumor cells was assessed by Zombie NIR^™ viability dye. d, e, Viability of H226 (d) and H2591 (e) cell lines after co-culture with MSLN-CAR NK primed by hDR18 displaying bacteria (YiaT232-IL18–6-12, YiaT232-hDR18; YiaT181-IL18–6-12, YiaT181-hDR18; C-IgAP-IL18–6-12, C-IgAP-hDR18) or control groups for overnight. The MOI of E. coli displaying cytokines and MSLN-CAR NK was 1000. E: T = 1:1. Unpaired t-test. Data are representative of three independent experiments with MSLN-CAR NK cells from three independent donors. Data represent means ± SEM (d, e).

Fig. 5 |. Bacteria displaying hDR18 enhance the proliferation, tumor trafficking, and efficacy of MSLN-CAR NK cells in vivo leading to improved tumor control.

a, NOD scid gamma (NSG) mice were subcutaneously (s.c.) engrafted with 5×10⁶ H226 cells. Starting on day 30, mice (n = 10 each group) were treated with PBS, YiaT232 (10⁹ CFU), YiaT232-hDR18 (10⁹ CFU) and purified hDR18 (4mg/kg) (i.t.) three times (on days 30, 37 and, 44). 3–5 million MSLN-CAR NK cells were administrated intravenously (i.v.) except for mice in the tumor-only groups. All the mice were injected with 75 kU human recombinant IL2 every other day intraperitoneally (i.p.) to support the survival of human NK cells in vivo. b, c, Mean tumor growth (b) and Kaplan–Meier survival curves (c) for the tumor-bearing after treatment. Mean tumor growth and survival curves are the combination of two independent experiments, with n = 10 mice per group. Data represent means ± SEM. d, NSG mice were subcutaneously (s.c.) engrafted with 5×10⁶ H226 cells, on day 40, mice (n = 5 each group) were treated with PBS, YiaT232 (10⁹ CFU), YiaT232-hDR18 (10⁹ CFU) and purified hDR18 (4mg/kg) (i.t.). 5 million MSLN-CAR NK were administrated intravenously (i.v.) except in the mice from tumor only groups. All the mice were injected with 75kU human recombinant IL2 every other day intraperitoneally (i.p.). On Day 47, mice were sacrificed and organs (tumor, liver, lung, spleen, and bone marrow) of all mice were collected for analysis by flow cytometry. e, The percentage of human CD45⁺ cells in livers, tumors, spleens, lungs, and bone marrow post-treatment. Two-way ANOVA test for tumor growth curve (b) and Mantel-Cox test for survival curve (c), unpaired t-test for NK percentage (e).

Fig. 6 |. Gene expression changes in the MSLN-CAR NK cells induced by priming with E. coli-hDR18.

mRNA transcript levels were measured in MSLN-CAR NK cells 73 hours after treatment with YiaT232-hDR18, E. coli (MOI = 1000), YiaT232, E. coli (MOI = 1000), hDR18 (no bacteria, 100ng/ml) and PBS (a, b, c, d, e). a, Principal component analysis of genes that were differentially expressed between YiaT232-hDR18, E. coli, and PBS (5% FDR, n=2923). Samples were labeled with the different treatment groups (Top) and different donors (Bottom). b, Volcano plot showing gene expression of YiaT232-hDR18, E. coli, and YiaT232-E. coli treated MSL-CAR NK cells. Highlighted in green are genes that passed 5% FDR comparing YiaT232-hDR18, E. coli, and PBS with log2FC>0.5 (or log2FC<−0.5) and had p-value<0.01 comparing YiaT232-hDR18, E. coli and YiaT232, E. coli with log2FC>0.5 (or log2FC<−0.5). n=43 up-regulated, n=6 down-regulated genes. The dashed line depicts a p-value of 0.05. c, Normalized expression levels (z-scores) of the genes highlighted in B. d, String network analysis for the upregulated genes highlighted in B (n=43). Genes were labeled according to the significantly enriched msigDB gene sets; TNFα signaling via NFκB, cytokine signaling, and GPCR signaling. e, GSEA enrichment plot for the KEGG TNFα signaling via NFκB in YiaT232-hDR18, E. coli, and YiaT232-E. coli groups.

Summary Fig. |

Schematic representation of our approach to surface display murine and human cytokines in non-pathogenic E. coli as a promising platform for immunotherapy, with E. coli displaying decoy-resistant IL18 mutein (DR18) being most effective in vitro and in immune-competent MC38 and B16 models and NSG mice bearing mesothelioma tumor cells treated with CAR NK cells.