Modified brucella strains and methods of use for treatment of disease

Abstract

A pharmaceutical composition contains an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole. The attenuated Brucella melitensis can contain a deletion of one or more of vjbR, asp 14, or mucR virulence genes, along with a first gene encoding for tryptophanase and a second gene encoding for toluene-4 monooxygenase. Methods of treatment of disease include administering a pharmaceutical composition containing an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole to the patient. Methods of treatment of disease include administering a pharmaceutical composition containing an attenuated bacterial strain of Brucella melitensis expressing an immunomodulatory factor to the patient. Methods of treatment of disease include further administering 5-hydroxyindole to the patient.

Description

Modified Brucella Strains and Methods of Use for Treatment of Disease Inventors: Jianxun Jim Song and Paul DeFigeiredo TECHNICAL FIELD

[0001] The present disclosure generally relates to treatment of disease by administration of indole- based compositions and modified brucella strains. More specifically, the disclosure relates to engineered bacterial strains of Brucella melitensis engineered to produce 5-hydroxyindole. treatment of disease by administration of hydroxyindole and modified brucella strains expressing cancer- associated antigens. BACKGROUND

[0002] Bacterial therapeutics have demonstrated efficacy in treating cancer. However, head-to-head comparisons of bacteria and state-of-the-art immunotherapeutic approaches (CAR-T, TCR-T, and immune checkpoint inhibitors [ICI]) have not been described. Moreover, a molecular analysis of the spectrum of responses induced by therapeutic bacteria, and hence, the full range of their therapeutic mechanisms of action, have not been determined.

[0003] Brucella melitensis is the etiological agent of brucellosis in livestock and wild animal populations. It is also the primary agent associated with human brucellosis, a disease marked by undulant fever and chronic symptoms. Over the past 30 years, live attenuated vaccine strains have been developed to protect animals against brucellosis. A novel class of attenuated mutants was created that comprise a deletion of the vjbR locus from Brucella melitensis 16M, referred to as BmΔvjbR. Importantly, this attenuated vaccine strain has displayed exceptional levels of safety following evaluation in tissue culture systems, as well as in immune-sufficient and immune-deficient mice, goats, sheep, and rhesus macaques. SUMMARY

[0004] Live attenuated vaccine strains of Brucella melitensis (Bm∆vjbR) possess attractive features for chassis development, including limited endotoxin activity, genetic tractability, demonstrated hallmarks of safety in immune-sufficient and -deficient murine, and pregnant sheep models, and relatively fast growth in culture. In addition, the bacterium homes to tumor tissue, supporting its deployment for development as an anticancer therapeutic. Finally, the chassis displayed synergistic activities when combined with adoptive cell transfer (ACT), indicating that it could enhance the performance of conventional immunotherapeutic approaches. Provided herein are pharmaceuticalcompositions containing an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole. Methods of treatment of disease include administering to the patient a pharmaceutical composition containing an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole. In certain embodiments, the attenuated bacterial strain of Brucella melitensis contains a mutation in one or more of vjbR, asp14, or mucR virulence genes. In certain embodiments, the attenuated bacterial strain of Brucella melitensis contains a deletion of one or more of vjbR, asp14, or mucR virulence genes. In certain embodiments, the attenuated bacterial strain of Brucella melitensis contains a first gene encoding for tryptophanase and a second gene encoding for toluene-4 monooxygenase.

[0005] Methods of treatment of disease include administering a pharmaceutical composition containing an attenuated bacterial strain of Brucella melitensis expressing an immunomodulatory factor to the patient. Methods of treatment of disease include further administering 5-hydroxyindole to the patient. Methods of treatment of disease include administration of indole-based compositions and modified brucella strains. Methods of treatment of disease include administration of hydroxyindole and modified brucella strains expressing cancer-associated antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0007] Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. To facilitate this description, like reference numerals designate like structural elements or procedures in a method. Embodiments are illustrated by way of example and not by way of limitation in the accompanying drawings.

[0008] The present disclosure can be better understood by referring to the following figures. These drawings illustrate the principles of the disclosure and no limitation of the scope of the disclosure is thereby intended.

[0009] FIGs. 1A – 1P demonstrates that hydroxyindole (HI) activates CD8+T cells and enhances their cytotoxic activity. FIG.1A is a set of representative flowcytometric analysis of proinflammatory cytokine TNF-α and cytotoxic markers perforin, granzyme B (GR-B) in mouse CD8+T cells. FIG. 1A is a quantitative analysis of flowcytometric dot-plot plots represented in FIG. 1A. FIG. 1C is a representative flowcytometric analysis demonstrating CD4+T cells differentiation into Th17 / Treg cell types upon treatment with HI, and FIG. 1D is a quantitative analysis of CD4+T cellsdifferentiation into T helper type 17 (Th17) cells or T regulatory (Treg) cells upon HI treatment. FIG. 1E is a representative flowcytometric assessment of M1 macrophage polarization by CD38 expression analysis. FIG.1F is a quantitative analysis of M1 macrophage polarization represented in FIG. 1E. FIG. 1G is an illustration of the genetic circuit used to construct SPIKE 1.0 (S1.0) bacterial strain producing hydroxyindole. FIG.1H is a representation of detection of 5-HI produced by the bacterial S1.0 determined by mass spectrometry analysis. Arrows: the standard (left) and the bacterial produced 5-HI (right). FIG.1I is a set of images presenting the immunofluorescence analysis of the intracellular S1.0 in mouse bone marrow-derived macrophages (BMDMs). FIG.1J is a representation of the dead- live flowcytometric assay (DLFA) of B16-Ova melanoma cells co-cultured with OT-1 CD8+T cells that had been pre-cocultured with S1.0, SPIKE-chassis (SC), or 1X PBS control (Ctrl) treated bone marrow-derived macrophages (BMDMs). FIG. 1K presents the quantitation of dead B16-Ova cells represented in FIG.1J. FIG.1L is a representative DLFA of mesothelin antigen specific CD8+T cells co-cultured with H1975 lung cancer cells and FIG.1M is a quantitation of dead H1975 cells in the indicated treatments. The representative DLFA data (FIGs.1J and 1M j-m) is shown from a CD8+T cells to cancer cells ratio of 1:1. FIGs.1N - 1P present the S1.0 distribution in tumor (FIG.1N), spleen (FIG. 1O), and liver (FIG. 1P) in Lewis lung carcinoma (LLC1) tumor-bearing mice administrated with various doses of S1.0. n = 10. Quantitative data represent mean ± standard error of measurement (SEM) from three independent experiments. *, **, ***, ****, p < 0.05, 0.01, 0.001, and 0.0001, respectively.

[0010] FIGS.1Q – 1X demonstrate that HI increases cytokine production and cytotoxicity of CD8+ T cells. FIG.1Q shows that HI regulates cytokines TNF-⍺ and IL-2, and immune checkpoint PD-1 expression levels in human CD8+ T cells. Human CD8+ T cells were isolated from human PBMCs and activated with anti-human CD3 / CD28 antibodies. FIG. 1R is a quantification of the indicated protein marker-positive in CD8+ T cell populations. n = 3. FIGs. 1S and 1T are flow-cytometry analysis of cells for dead live staining using Aqua zombie dead live staining dye and quantitation of dead cells (%) recovered from flowcytometric analysis, respectively. n = 3. The representative data is shown from a ratio of 1:1 (CD8+ T cells to cancer cells). FIGs.1U, 1V, and 1W are the analysis of the CFSE+ CD8+ T cells for intracellular cytokine production TNF-⍺ (1U), IFN-g (1V), and cytotoxic marker perforin (1W) as determined by flowcytometric analysis. FIG. 1X is a quantitation of the population of the CD8 cells that produce the indicated cytokines shown in (e-g), respectively. n = 3. Data represent mean ± standard error of measurement (SEM) from three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001.

[0011] FIGs.2A – 2T demonstrates that S1.0 inhibits tumor growth and increases survival of tumor- bearing mice. FIGs.2A – 2D are tumor growth and survival curves of C57BL / 6 mice bearing B16- Ova melanoma (FIGs. 2A – 2B) and Panc-02 (FIGs. 2C – 2D) tumors. FIGs. 2E – 2H are tumor growth curves of human H1975 lung tumor (FIG.2E) and mice survival curves (FIG.2F) and human pancreatic AsPC-1 tumor growth (FIG. 2G) and mice survival curve (FIG. 2H) in immunocompromised nonobese diabetic / severe combined immunodeficiency (NOD / SCID) γ mice. FIG.2I presents in vivo imaging of KPC-luciferase tumor-bearing mice treated with either S1.0, anti- PD-1 (^PD-1) antibody or a combination of S1.0 and ^PD-1 antibody. FIG. 2J is a quantitative representation of luciferase activity showing tumor mass growth in treated mice. FIG.2K is a survival curve of the treated KPC-luciferase tumor-bearing mice. FIG.2L is a set of images from a confocal microscopic analysis of OT-1 CD8+T cell infiltration into B16-Ova melanoma tumors in C57BL / 6 mice. FIG.2M is a representative flowcytometric analysis of OT-1 CD8+T cell infiltration into B16- Ova melanoma tumors in C57BL / 6 mice. FIG.2N is a quantitative presentation of the flowcytometric analysis shown in FIG.2M. FIG.2O is a percentage analysis of PD-1+cells to total CD4+and CD8+T cells. FIG. 2P is a multiphoton microscopic analysis of S1.0 localization in tumor microenvironments (TME) (green: S1.0 bacteria; red: Ly6g+cells) of explanted B16-Ova melanoma (white: SHG-second harmonic generation for collagen structure). FIGs. 2Q – 2R present hematoxylin and eosin (H&E) staining analysis of explanted tumor sections from tumor-bearing mice (FIG. 2Q) and heatmap showing the difference in the tumors with different treatments (FIG. 2R). Arrows indicate areas of lymphocyte infiltration. Tumor sections were scored in a blinded manner by a board-certified veterinarian. INV: local invasion, MAC: intraneoplasm macrophages, LYM: lymphocytes infiltration, TN: tumor necrosis. FIG. 2S is a representative multiparametric image analysis showing CD4+T cells, CD8+T cells infiltration, and Ki-67 proliferation into the TME of tumor-bearing mice. FIG. 2T is a quantification of immune cells in the TME obtained by multiparametric spatial image analysis shown in FIG. 2S. Quantitative data represent mean ± SEM from three independent experiments. *, **, ***, and ****: p ≤ 0.05, 0.01, 0.001, 0.0001, respectively.

[0012] FIGs.2U-2Z demonstrate the safety assessment of SPIKE 1.0 (S1.0) bacterial strain in mice and pregnant goat models. FIG.2U is a quantitative analysis of an organ weight analysis of mice (n = 5 / group) injected with different doses of S1.0 at 14 days post injection. FIG. 2V is a quantitative analysis of the CBC analysis of blood contents in mice (n = 5 / group) administered with different bacterial doses of S1.0 at 14 days post injection. MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration. FIG.2W is a quantitative analysis of the colony-forming unit(CFU) analysis of S1.0 distribution in tumor, spleen, lung, and kidney of B16-Ova melanoma tumor- bearing mice administrated with the dose of 5.0´107 bacteria of S1.0. FIGs. 2X and 2Y are the graphical representations of the CFU analysis of S1.0 distribution in lung (2X) and kidney (2Y) in Lewis lung 1 carcinoma (LLC1) tumor-bearing mice administrated with the indicated 5.0x107, 1.0x108, and 1.0x109doses of S1.0. n = 10. FIG.2Z is a graphical representations of the CFU analysis of S1.0 distribution in tumor and spleen of LLC1 tumor-bearing mice administrated with the doses of 5.0x107S1.0 with or without adoptive transfer of antigen specific CAR-T cells at X day post S1.0 injection.

[0013] FIGS.3A – 3M demonstrate that S1.0 promotes inflammatory immune activity and CD8+T cell infiltration in humanized mice. FIG.3A is a schematic experimental design of humanized-mouse MCC13 / H1975 tumor model. FIG.3B presents MCC13 tumor growth curves. FIG.3C is a graphical representation of the survival rate of MCC13 tumor-bearing mice. FIG.3D presents the H1975 tumor growth curves in tumor-bearing mice. FIGS. 3E and 3F are the survival curves and relative weight change, respectively, of H1975 tumor-bearing mice. FIGS. 3G – 3I present the flowcytometric analysis of the infiltration of CD8+T cells, CD19+B cells, and CD11b+monocytes infiltration into tumors. FIG. 3J presents the flowcytometric analysis of the PD-1+cells in the TME. FIG. 3K is a heatmap analysis of data showing in FIGs.3G – 3J. FIG.3L is a H&E image analysis of explanted tumors. Representative images are showing. Bars: 20 μm. FIG. 3M is a Heatmap analysis of H&E scoring in tumor sections showing in FIG. 3L. INV: local invasion, MAC: macrophage, NEU: neutrophils, LYM: lymphocytes, and TN: tumor necrosis. Quantitative data represents mean ± SEM from three independent experiments. *, **, and ***: p < 0.05, 0.01, and 0.001, respectively.

[0014] FIGs.3N – 3U demonstrates that SPIKE 1.0 inhibits tumor growth and increases survival of tumor bearing mice. FIGs.3N and 3O are schematic illustrations of mouse tumor model experiments in B6 wildtype mice (B16-Ova melanoma and Panc02 tumors) (3N) and in NSG mice (H1975 and AsPC-1 tumors) (3O). FIGs.3P and 3Q are quantitative analysis of the B16-Ova melanoma growth in tumor-bearing mice with the indicated treatments and the survival of B16-Ova melanoma bearing mice following SPIKE 1.0 administration, respectively. Ctrl: control, SC: SPIKE chassis, S1.0: SPIKE 1.0, αPD-1: anti-PD-1 antibody. n = 10 / treatment. FIGs. 3R and 3S are representative flowcytometric analysis of retroviral transduction of NY-ESO T cell receptor (TCR) and mesothelin specific chimeric antigen receptor (CAR), respectively. n = 3. FIGs.3T and 3U are survival curves of B16-Ova melanoma tumor-bearing CD8 knockout (KO) and Batf3 KO mice following administration of S1.0. n = 15 mice / group. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0015] FIGS.4A – 4Q demonstrate that S1.0 upregulates chemokine signaling and modulates amino acid (AA) receptors in immune cells. FIG. 4A is a representative UMAP (uniform manifold approximation and projection) clustering analysis of the immune cells (macrophages, neutrophils, T cells, and B cells) in the TME of Day 21 explanted tumor samples of Ctrl, S1.0, S1.0+CT and CT treated tumor-bearing mice. FIG.4B is a quantitative analysis of all cells and specific immune cells (macrophages, neutrophils, T cells, and B cells) in the TME from day 21 explanted B16-Ova melanoma tumors of different experimental groups. FIG. 4C is a set of representative violin plots showing the expression of antigen presentation genes and co-stimulatory markers on macrophages (M∅). FIG.4D is an AUCell score analysis of antigen presentation and co-stimulation of macrophages shown in FIG. 4C. FIGs. 4E and 4F are representative plots of chemokine ligands expression on macrophages and chemokine receptor expression on T cells, respectively. FIG.4G presents the AA transporter marker gene expression on the indicated immune cells. FIGs.4H, 4I, and 4J are heatmaps showing expression of marker genes indicating CD8+T cell activation, CD8+T cell memory, and CD8+T cell persistence, respectively. FIG.4K is a set of violin plots showing the expression of CD8+T cell exhaustion marker genes. FIGs.4L, 4M, and 4N are heatmap analyses of expression of markers showing CD4+T cell activation, CD4+T cell memory, and CD4+T cell persistence, respectively. FIG. 4O is a set of violin plots showing the expression of CD4+T cell exhaustion marker gene expression. FIG.4P is a heatmap showing expression of markers identifying M1 macrophages. FIG. 4Q is a AUCell score analysis of apoptosis-associated genes in melanocytes on day 21 post tumor- cell implantation n(PTCI). n=3. *: p < 0.05, *: p < 0.01, ***: p < 0.001.

[0016] FIGs. 4R – 4Z demonstrate that SPIKE 1.0 administration increases survival rate of tumor bearing mice and differentially increases immune cell activation in TME. FIG. 4R is a schematic illustration of tumor implantation, treatment schedule, treatment-efficiency evaluation, and the TME analysis in KPC- luciferase (KPC-Luc) orthotopic pancreatic ductal adenocarcinoma (PDAC) model. FIG.4S is a set of photographs capturing the tumor growth in KPC-Luc orthotopic PDAC model by the in vivo imaging system (IVIS). Representative IVIS images were obtained as the indicated time points after IV injection of tumor cells. n = 6. d: Days post-surgery. FIG.4T is a table presenting the data showing that S1.0 administration increases survival of the tumor bearing mice. FIGS.4U – 4Z are graphical representations demonstrating that SPIKE 1.0 administration differentially regulates CD4 T cells infiltration into TME (FIG.4U), frequency of activation status of CD8 T cells (FIG.4V), terminal differentiation of B cells and long-lived plasma cells (FIG.4W), frequency and activation status of NK cells (FIG. 4X), the infiltration and activation status of CD11c+ dendritic cells (DC)(FIG. 4Y), and the infiltration of tumor with myeloid derived suppressor cells (MDSC) (FIG. 4Z). MFI: mean fluorescence intensity. n = 3. *, **, ***: p < 0.05, 0.01, and 0.001, respectively.

[0017] FIGs. 5A – 5L present that S1.0 remodulates AA metabolic pathways impacting tumor metabolome. FIG.5A is a partial least squares-discriminant analysis (PLS-DA) of the metabolome of control, SC and S1.0 treated tumors. FIGs. 5B and 5C are heatmaps presenting the metabolomic profiles of the tumors at 21 days PTCI. FIG. 5B presents the comparison of metabolite levels of phospholipids-PC (phosphatidylcholine), PS (phosphatidylserine), PE (phosphatidylethanolamine), HI, and metabolite intermediates of glycolysis from the TME of the treated tumors. FIG. 5C is a representative heatmap of the comparative metabolomics analysis of amino acids. FIG. 5D is a representative pathway impact analysis derived from the metabolomics profile. FIGs.5E and 5F are network map analysis of glycolysis pathway and arginine biosynthesis pathway, respectively, in the TEM of tumor treated with Ctrl, SC or S1.0 at Day 21. The analyzed datasets are derived from the combined scRNA-seq data and metabolomics data (not included herein but available upon request). FIGs. 5G and 5H present the comparison of glycolysis levels and arginine biosynthesis levels, respectively, in CD 4+T cells across different experimental groups from tumors explanted on Day 21 and Day 28. The analyzed dataset is from scRNA-seq data. FIG.5I presents the qPCR validation of the upregulation of AA receptors and transporter markers in immune cells in TME of tumors treated with the indicated treatments. FIGs.5J and 5K are heatmaps showing different AA uptake (5J) and AA uptake profiling of T cells in TME of tumors treated with control or SC. FIG.5L is an illustration of the S1.0 immunomodulatory mechanisms in controlling tumors, according to an embodiment. Upon administration, S1.0 (1) releases hydroxyindole (HI) in the TME, promotes antitumor chemokine production, increases immune cells such as CD8+T cells infiltration into TME, and CD4+T cell polarization; (2) homes to the TME mediated by Ly6gHighcells and locates in these cells in the TEM. S1.0 then activates immune cell functions, including CTL directed tumor apoptosis, antigen uptake and presentation, functional M1 macrophage polarization, and cytotoxic T lymphocyte (CTL) activation and persistence; (3) S1.0 reprograms immune cell metabolomes and increases T cell amino acid uptake, resulting in increased CD8+T cell activation and CTL activity. Finally, these immunomodulatory activities suppress tumor growth. Data represents mean ± SEM, n=3. *: p < 0.05, *: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0018] FIGs. 5M – 5N are photographs of hematoxylin and eosin (H&E) staining analysis of explanted tumor sections from tumor bearing mice with the indicated treatments. FIGs.5M and 5N H&E image analysis of explanted tumors from B16-Ova (FIG. 5M) and pancreatic Panc-02 (FIG.5N) tumor sections from tumor-bearing mice treated with control, SPIKE chassis, SPIKE 1.0, and the combination of SPIKE 1.0 and CART cells (CT). The areas marked within yellow lines in each image display differences in macrophage, lymphocyte, and neutrophil infiltration in tumor sections induced by the indicated treatments shown in FIG.2Q and the enlarged sections shown in the right column. n = 5 mice / treatment.

[0019] FIGs.6A and 6B are chip-cytometry multiplex imaging analysis of biomarker expression in the TME. FIG. 6A is a set of single channel of chip-cytometry multiplex images showing different immune cells in the TME from day 28 post tumor implantation. Bars: 500 μm. FIG. 6B is a set of graphical representations of the expression biomarkers in the TME shown in FIG. 6A. *: p < 0.05, **: 0.01, ***: 0.001, ****: 0.0001. n = 3.

[0020] FIGs. 7A – 7I present the identification of different cell types by scRNA-seq analysis. FIG. 7A is a schematic representation of sample preparation and scRNA-seq experimental approach. FIG. 7A is a set of representative violin plots of different markers used to identify the indicated cells. MEL: melanocytes, IMM: immune cells, FB: fibroblasts, ENDO: endothelial cells, EPI: epithelial cells, and NEU: neutrophils. FIG.7C is a heatmap showing the expression of different markers used to identify MEL, IMM, FB, ENDO, EP, and NEU. FIG.7D is a set of UMAP clustering analysis of the different gene markers used for the identification of macrophages, neutrophils, T cells and B cells. FIG.7E is a set of representative violin plots of expression of the indicated gene markers used to identify macrophages, neutrophils, T cells and B cells. FIG. 7G is a Seurat based UMAP clustering of all different cell types derived from TME on Day 21 and Day 28 post tumor implantation. FIG.7H is a UMAP clustering and classification of the different cell types (MEL, IMM, FB, ENDO, EPI, and NEU) in scRNA-seq analysis. FIG.7H is a UMAP clustering and representation of the different cell types based on the treatment strategies. FIG.7I is a Seurat based UMAP clustering of immune cell types (B cells, T cells, and Neutrophils) in TME at day 28 post tumor implantation. n = 3.

[0021] FIGs. 8A – 8F are quantitative analysis of the comparative amino acid enrichment pathway scores on cells in the TME from day 21 and 28 post explanted B16-Ova melanoma tumor. FIGs.8A and 8B are the alanine and aspartate metabolism pathway at day 21 and day 28 post tumor implantation, respectively. FIGs. 8C and 8D are the valine, leucine and isoleucine biosynthesis pathway at day 21 and day 28 post tumor implantation, respectively. FIGs.8E and 8F are the arginine and proline metabolism pathway at day 21 and day 28 post tumor implantation, respectively. n = 3. *: p < 0.05, **: 0.01, ***: 0.001, ****: 0.0001. ns: not significant.

[0022] FIGs.9A – 9J present the scRNA-seq analysis of expression of marker genes used to identify different cell types and signaling pathways in TME. FIG. 9A presents the expression analysis of marker genes that identify B cells in TME with the indicated treatments at day 21 and 28 PTCI. FIG. 9B presents the AUCell score analysis of cytokine-cytokine receptor signaling pathway in B cells. FIG.9C presents the violin plots showing the differential expressed genes of the cytokine-cytokine receptor signaling pathway in B cells among different groups. FIG.9D presents the AUCell analysis of gene sets of the aryl hydrocarbon receptor (AhR) signaling pathway in macrophages and in all cells. FIG.9E presents the expression analysis of the marker genes in the AhR signaling pathways in the indicated cells. FIGs. 9F- 9H present the expression analysis of marker genes that identify neutrophils (FIG.9F), M2 macrophages (FIG.9G), and myeloid-derived suppressor cells (MDSCs) (FIG.9H) in TME at day 21 and 28 PTCI. FIGs.9I and 9J are UMAP clustering analysis and violin plots, respectively, showing the expression of marker genes that identify N2 neutrophil in TME at day 21 post tumor implantation. n = 3.

[0023] FIG.10 is a set of graphical representations of the amino acid uptake profiling of tumors from B16-Ova melanoma tumor-bearing mice treated with SPIKE and control. Tumors from control and SPIKE chassis treated were homogenized, metabolites were extracted and analyzed as the methods described in the Methods section. n = 4.

[0024] FIG.11 presents the constructs used to generate certain embodiments of the BmΔvjbR strains. Detailed plasmid maps of the cassettes used to generate the bacterial strains are included in FIGs.12 and 13. DETAILED DESCRIPTION

[0025] Solid tumors, which account for about 90% of adult cancers, are responsible for millions of deaths annually. Synthetic Programmable bacteria for Immune-directed Killing in tumor Environments (SPIKEs) represent a transformative approach to cancer immunotherapy for solid tumors. A single systemic administration of SPIKEs demonstrated robust efficacy while maintaining a favorable safety profile, even at high doses. SPIKEs reshaped the tumor microenvironment, enhanced both innate and adaptive antitumor immunity, and significantly improved key hallmarks of T cell function, including activation, persistence, survival, and memory, while reducing markers of exhaustion. These metabolically engineered bacteria outperformed immune checkpoint inhibitors (ICIs) and adoptive cell transfer (ACT) therapies in multiple solid tumor models, including pancreatic, lung, and skin cancers, and exhibited synergistic effects when combined with these treatments. Integrative single-cell transcriptomics, immunological profiling, and metabolomicsanalyses identified SPIKE-mediated remodeling of amino acid availability in immune cells within the TME as a central mechanism of action. SPIKEs are a scalable and cost-effective immunotherapeutic platform for overcoming therapeutic resistance and enhancing immune responses in solid tumors.

[0026] Bacteria-based cancer immunotherapy (BCIT) has emerged as a transformative strategy for cancer treatment, offering unique advantages over conventional therapies. Therapeutic bacteria preferentially accumulate and proliferate within the TME, disrupting tumor cell metabolism, enhancing antitumor immune responses, and reversing immune suppression. Furthermore, advances in genetic engineering have enabled bacteria to deliver therapeutic agents, including cytokines, antibodies, and other anticancer molecules, directly to tumors. BCIT has shown promise as both a monotherapy and in combination with other modalities, improving clinical outcomes. Despite these advances, direct comparisons between bacterial therapies and state-of-the-art immunotherapies, such as adoptive cell transfer (ACT) therapies (e.g., CAR-T and TCR-T) and immune checkpoint inhibitors (ICIs), remain limited. Additionally, the molecular mechanisms underpinning bacterial therapeutic efficacy are not fully elucidated.

[0027] Live attenuated strain of Brucella melitensis (Bm∆vjbR) is a promising chassis for therapeutic development. This strain exhibited minimal endotoxin activity, genetic tractability, and a strong safety profile across various immune-sufficient and -deficient animal models. Bm∆vjbR specifically homed to tumor tissues, remodeled the TME, promoted proinflammatory M1 macrophage polarization, and enhanced CD8+T cell infiltration and activity. Moreover, this strain synergized with ACT therapies, stressing its potential as a robust adjunct to conventional immunotherapy. In an embodiment, the live attenuated bacterial strain of Brucella melitensis is Brucella melitensis 16M ΔvjbR (BmΔvjbR). Brucella melitensis 16M is available, for instance, as ATCC #23456.

[0028] Tryptophan-derived metabolites, such as indole and its derivatives, display antitumor activities by modulating T cell fate and function. Indole, known for its role in influencing regulatory T cells (Tregs), has been previously shown to attenuate inflammation in a murine rheumatoid arthritis model when expressed by engineered Bm∆vjbR. Notably, tryptophan-derived metabolites like indole- 3-propionic acid (IPA) enhance CD8+T cell-mediated responses in pancreatic cancer immunotherapy. Despite these findings, the role of specific indole derivatives in modulating immune responses within the TME remains underexplored.

[0029] Provided here are compositions of attenuated strains of Brucella with engineered immunomodulatory activities. Provided herein are live attenuated bacterial strain (BmΔvjbR) that can be utilized for treatment of various diseases such as cancer, autoimmunity, and inflammation. Asdescribed herein, pharmaceutical compositions containing the strain can be used as therapeutic tools to modulate immune response in various disease states and to provide advantageous strategies to improve or supplement existing immunotherapies.

[0030] In an embodiment, the patient needs treatment for cancer. In an embodiment, the cancer is breast cancer, prostate cancer, lung cancer, pancreatic cancer, and colorectal cancer. In an embodiment, the cancer is partially or completely resistant to a chimeric antigen receptor (CAR)-T cell therapy.

[0031] Provided herein are pharmaceutical compositions containing an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole (BmΔvjbR-HI). In certain embodiments, the attenuated bacterial strain of Brucella melitensis contains a mutation in one or more of vjbR, asp14, or mucR virulence genes. In certain embodiments, the attenuated bacterial strain of Brucella melitensis contains a deletion of one or more of vjbR, asp14, or mucR virulence genes. In certain embodiments, the attenuated bacterial strain of Brucella melitensis contains a first gene encoding for tryptophanase and a second gene encoding for toluene-4 monooxygenase. The pharmaceutical composition can further include a second therapeutic agent, such as an anti-cancer therapeutic agent, an auto-immune therapeutic agent, or an anti-inflammatory therapeutic agent, or any combination thereof.

[0032] Methods of treating disease in a patient include administering a pharmaceutical composition comprising an attenuated bacterial strain of Brucella melitensis engineered to produce 5- hydroxyindole to the patient. Methods of treating disease in a patient include administering a pharmaceutical composition comprising an attenuated bacterial strain of Brucella melitensis expressing an immunomodulatory factor to the patient. The immunomodulatory factor can be one or more of a nanobody that interacts with and stimulates CCR7, a nanobody that interacts with and antagonizes an immunosuppressive protein, a cancer-associated antigen, and a neoantigen. Certain embodiments of the immunomodulatory factor include Hormad1, Mesothelin, NY-SEO-1, VGLL1, MAGE-A4, MUC1, WT1, or PRAME. Methods of treating disease in a patient include co- administration of an indole compound. An indole compound can be one or more of indole, hydroxyindole (e.g., 2-hydroxyindole, 3-hydroxyindole, 7 hydroxyindole), 1-(2- carboxyphenylamino)-1-deoxy-D-ribulose-5-phosphate, 5-Hydroxy-L-tryptophan, Indoleglycerol phosphate, indolepyruvate, N-(5-Phospho-D-ribosyl)anthranilate, tryptamine, indole-3-acetate, L- formylkynurenine, L-Tryptophanyl-tRNA(Trp), indole-3-acetamide, indole-3-pyruvate, indole-3- lactic acid, tryptophol, indole-3-acetaldehyde, indole-3-aldehyde, isatin (indole-2,3-dione), isoindigo,indirubin, indoxyl-sulfate, and 2-oxyindole. Methods of treating disease in a patient include co- administration of 5-hydroxyindole.

[0033] As used herein, administering means oral administration, administration as a intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration to a subject. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0034] The terms “treat,” “treating” or “treatment,” and other equivalents as used herein, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.

[0035] Immunotherapies have emerged as a powerful avenue for combatting cancer. However, their efficacies against solid cancers are limited. Embodiments provided herein include systemically administered SPIKEs (Synthetic Programmable Bacteria for Immune-directed Killing in tumor Environments) that promoted innate and adaptive antitumor immunity, dramatically improved the tumor microenvironment, substantially enhanced markers of T cell activation, function, survival, persistence, and memory, greatly reduced markers of T cell exhaustion, and outperformed immune checkpoint inhibitors (ICI) and adoptive cell transfer (ACT) -based immunotherapies against diverse solid cancer types in murine and humanized models of cancer, including cancers of the pancreas, lung, and skin. In addition, SPIKEs acted synergistically with ACT and ICI approaches. Single-cell transcriptomics, immunological profiling, and metabolomics analyses demonstrated that SPIKE- directed remodeling of amino acid availability in the TME was a key driver of these activities. Taken together, SPIKEs provide compelling interventions for modulating the tumor metabolome of solid cancers, and are suitable compositions for developing scalable, affordable, and efficacious interventions for combatting intractable cancers.

[0036] The highly immunosuppressive tumor microenvironment stimulates cancer cells resistance to immunotherapy. A live-attenuated Brucella melitensis ΔvjbR (BmΔvjbR) bacterial strain, breaks cancer cell resistance to immunotherapy by remodeling the TME and facilitating infiltration of cytotoxic CD8+T cells (CTLs) into the tumor. As a result, there was shrinkage of tumor size and significant improvement in survival of these animals. However, BmΔvjbR was efficient in controlling colorectal tumors, but was rendered ineffective in B16 melanoma. Here, the efficacy of BmΔvjbRin in controlling different cancers was improved by engineering it to augment the production of 5HI, i.e., BmΔvjbR-HI. The effect of BmΔvjbR-HI in modulating the function and activity of CTLs was assessed by flowcytometry in in vitro assays. The tumor modulating activity of the BmΔvjbR-HI was also assessed by intravenous injection of this bacteria followed by adoptive CTL therapy in colon carcinoma, melanoma and pancreatic orthotropic tumors. BmΔvjbR-HI increased inflammatory cytokines in CD8+T cells. The granzyme-B production and cytotoxicity of CD8+T cells was also enhanced on treatment with the bacteria. BmΔvjbR-HI remodeled the TME and ameliorated MC32- CEA colorectal cancer, B16-OVA melanoma and Panc-02 orthotropic pancreatic tumors. Moreover, the BmΔvjbR-HI homed into the TME by uptake in myeloid derived suppressor cells. Overall, BmΔvjbR-HI remodels heterogeneous TMEs of different cancers to promote CTL-mediated antitumor immunity.

[0037] In an embodiment, SPIKE 1.0 (S1.0), a metabolically engineered bacterial strain, is designed to reshape the TME through persistent production of HI. By dynamically modulating metabolic and immune landscapes within solid tumors, S1.0 bypassed conventional immunosuppressive barriers, enhanced tumor immunogenicity, and reinvigorated exhausted T cells. In preclinical models, S1.0 significantly outperformed ICIs, CAR-T, and TCR-T therapies across multiple refractory cancers, including pancreatic, lung, and skin malignancies. Mechanistically, S1.0 remodeled amino acid bioavailability within the TME, rewiring CD8⁺ T cell metabolism to sustain robust antitumor immunity, prolong effector function, and prevent exhaustion. Unlike traditional immunotherapies that rely on bolus cytokine delivery or systemic immune activation, S1.0 operated as a continuous metabolic factory, locally enhancing immune fitness while minimizing systemic toxicity. S1.0 is a therapeutic platform that integrates tumor metabolism and immunotherapy, offering a scalable, cost- effective, and broadly applicable therapy for overcoming resistance in solid tumors. By leveraging synthetic biology to reprogram the TME from an immunosuppressive niche into an immunostimulatory environment, S1.0 establishes a paradigm in bacterial-based cancer immunotherapy, paving the way for next-generation precision immuno-metabolic interventions.

[0038] These engineered bacteria displayed several desirable anticancer features, including the activation of both murine and human CD8+T cells and CD4+T cells, the polarization of naive CD4+T cells into Th1 and Th17 cells, the polarization of macrophages toward an M1 phenotype, and the suppression of Treg polarization when T cells were placed under Treg skewing conditions. Sequence analysis indicated that the SPIKE chassis did not contain genes to encode enzymes that catalyze the production of HI, and biochemical analyses confirmed this lack of HI, as no HI production was detected by the chassis strain. Embodiments include improvement of the anti-cancer activity of the chassis by engineering HI production into it. This resulting strain (SPIKE 1.0) efficiently produced HI. Survival and tumor size in tumor-bearing mice treated with a single intravenous dose of the intervention (or mock treated with saline) were measured. Two syngeneic heterotopic murine cancer models were used: melanoma [B16], a warm tumor that displays responsiveness to treatment with immunomodulatory agents, and pancreatic cancer [PANC02], a cold tumor that limits immune cell infiltration into the tumor microenvironment and is recalcitrant to treatment with antibody or cellular therapeutic agents. SPIKE treatment significantly increased survival and reduced tumor sizes in each of the tested systems. The activity of SPIKEs was also evaluated in an orthotopic pancreatic cancer model. Administration of SPIKE dramatically inhibited tumor growth and extended survival in these systems. In addition, SPIKE acted synergistically with ICI to inhibit tumor progression. SPIKEs have broad spectrum anti-cancer activity.

[0039] Having demonstrated SPIKE efficacy in cancer models, the studies were conducted in two heterotopic xenograft models (i.e., lung [H1975] and pancreas [ASPC-1]). Certain experimental groups were treated with chimeric antigen receptor (CAR-T) (mesothelin; AsPC-1; CEA; PANC-02) or T cell receptor T (TCR-T) (NY-ESO1; H1975) cells that target antigens expressed on the corresponding tumor surfaces. The CAR-T, TCR-T, and ICI interventions worked synergistically with SPIKE treatment. Finally, in several models, SPIKE treatment performed as well or better than corresponding CAR-T, TCR-T, or ICI benchmark treatments, thereby demonstrating that the bacterial intervention with HI induced potent anticancer effects.

[0040] These compositions were evaluated in humanized mouse models of lung (H1975) and pancreatic (ASCB-1) cancer. In this model NSG (NOD.Cg-Prkdcˢ^^^ Il2rg^^¹^ʲˡ / SzJ) mice were first engrafted with human CD34+hematopoietic stem cells, resulting in animals that carry human B cell, T cell, and monocyte populations. Next, lung (H1975) or pancreatic (ASCB-1) cancer cells were heterotopically transplanted into the humanized mice, and either treated with the bacterial therapeutic,TCR-T cells that target the tumor, or with both interventions. Bacterial intervention substantially reduced tumor growth and increased the survival of the treated animals.

[0041] SPIKEs remodeled the cellular immune profile of the TME. SPIKE-treated tumors were enriched in immune cell types, including CD4+T cells, CD8+T cells, B cells, and macrophages. In addition, immunohistopathological analysis showed that immune infiltrates of these cell types were increased in SPIKE-treated animals, consistent with the observation that SPIKEs reversed the immune-suppressive TME.

[0042] Second, SPIKE treatment influenced T cell activation, function, survival, and persistence / memory in the TME. Results from the in vitro immune profiling experiments showed that live SPIKE- treated BMDMs induced significantly higher production of perforin, granzyme B (GrzB), TNFα, and IFNγ, from CD8+T cells. Activated CD8+T cells retained functional recall ability, a feature critical for antitumor efficacy. This was demonstrated by use of anti-CD3 / anti-CD28 Abs to restimulate CD8+T cells at 3 days post-activation. The CD8+T cell recall responses were enhanced post-restimulation, exhibiting lower PD-1 expression and higher expression of proinflammatory cytokines. The surface protein expression of T cell markers and activation proteins (e.g., CD25, CD44, CD69, PD-1, Tim3) were also examined on control and SPIKE-treated T cells. Expression of these markers was significantly upregulated in SPIKE-treated tumor-bearing animals. Naive CD8+T cells (effector) from OT-I T cell receptor (TCR) transgenic (Tg) mice were co-cultured with B16-OVA tumor cells (targets). These experiments revealed that SPIKE treatment significantly enhanced CD8+T cell- mediated tumor-killing activities.

[0043] Finally, macrophages and monocytes were analyzed. ScRNA-seq analysis revealed that M1 polarized macrophages (i.e., TNF^+, MHC class II+, CD68+, B7-1 / CD80+, B7-2 / CD86+, and iNOS) were enriched whereas their M2-polarized counterparts (i.e., CD206 (mannose receptor C type 1), CD163 (scavenger receptor), CD68, arginase-1 (ARG1), DECTIN-1, FIZZ1, Ym1, and Ly6C) were depleted in SPIKE-treated mice (compared to control). The presence of immune suppressive MDSCs, as indicated by the expressions of CD11b, Gr-1, Ly-6G, Ly-6C, F4 / 80, M-CSF R / CD115, IL-4 R alpha / CD124, and CCR2, was also reduced. Taken together, this SPIKE-directed remodeling of immune cell populations toward a less immune-suppressive profile substantially contributed to cancer suppression.

[0044] The molecular mechanisms driving the remodeling of the cellular immune profile in the TME were interrogated. SPIKEs induce alterations in molecular genetic networks in immune cells that favor tumor killing. Single-cell RNA-seq (scRNA-seq) of tumor tissue was performed, these tumortissue being harvested from mice that were mock-treated or treated with a single dose of intravenously delivered SPIKEs . First, the scRNA-seq analysis confirmed that TME was enriched in immune cells, including M1 polarized macrophages, neutrophils, B cells (CD19, CD80, CD73, and PD-L2 / CD273), NK cells (CD27, NK1.1, NKp46, T-bet, and CD127 / IL-7 R alpha), CD8+T cells, Th17 CD4+cells, and Th1 CD4+T cells. Importantly, reductions in suppressive cell types were noted, including M2- polarized macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Treg cells, FoxP3, CD25, GITR, OX40, TNFR2).

[0045] Second, T cells from SPIKE-treated mice displayed an increase in the expression of genes associated with tumor cell killing. For example, these cells displayed increased expression genes associated with T cell activation (Granzyme B (GrB), Perforin (PRF1), CCL5, GBP5, GZMA, GZMH, IRF1, LAG3, NKG7, and PSMB10), proliferation (CD25), and central and effector memory (e.g., CD69, CD103, CD44, CD62L, CD127 / IL-7Rα). In addition, these cells showed a reduction in the expression of genes associated with suppressive activities or exhaustion (e.g., PD-1, LAG-3, TIM- 3, FASL, GZMB, CXCL13, CTLA-4, and TIGIT). Third, neutrophils in the TME of SPIKE-treated mice displayed increased expression of genes associated with activation, including NE (neutrophil elastase) and MPO (myeloperoxidase). Fourth, the monocyte cell populations in the TME of SPIKE- treated mice displayed increases in the expression of tumor-killing genes, including HLA-DR, CD11c, CD86, iNOS, and pSTAT. Finally, the cells in the immune cells in the TME displayed dramatic changes in the expression of genes controlling cellular metabolism, including glucose synthesis and utilization, nucleotide, metabolism, lipid metabolism, and amino acid metabolism. In addition, enhanced expression of amino acid transporters was observed on the surfaces of immune cells (compared to untreated controls), indicating that SPIKE treatment facilitated uptake by immune cells.

[0046] SPIKE treatment altered the expression of metabolic pathways, including pathways encoding the uptake, metabolism, and biosynthesis of amino acids, indicating that this amino acid remodeling may contribute to driving the observed immune alternations in the TME. The metabolomes of tumors from mock-treated and SPIKE-treated animals were analyzed. The metabolites of cells treated with CAR-T cells that target the corresponding tumors were also analyzed. First, the abundance of all the examined amino acids was substantially depleted in tumors harvested from SPIKE-treated mice (compared to mock-treated controls). Second, an integrative analysis was performed where concordance between the scRNA-seq and metabolomics findings were considered in the same cancer model. This analysis amplified the role for amino acid metabolism as a central component of immuneremodeling in the TME. SPIKE-directed remodeling of the TME metabolome controls tumor growth, and the tumor-suppressive outcomes of SPIKE treatment.

[0047] Metabolic remodeling of the TME contributed to the observed anti-cancer immunological activities induced by SPIKEs. Importantly, amino acid availability to diverse immune cell types, including T cells and macrophages, was altered in SPIKE-treated animals. Because amino acids uptake and utilization have recently emerged as key regulators of anti-cancer immune cells functions, these findings indicate that the enhanced utilization of amino acids in the TME by bacterial-activated immune cells constitutes an important mechanism that contributes to cancer cell killing. Additionally, several additional anti-cancer features were observed, including the enhancement in the expression of markers of T cell exhaustion, and suppression of pro-cancer checkpoint proteins, including PD1, CTLA-4, and TIM3. Furthermore, a combination of effects further contributes to the ultimate favorable outcomes, including bacterial scavenging of amino acids and activation of immune cells (and their amino acid scavenging programs) that contribute to depletion. Finally, current state-of-the- art cellular therapies, including CAR-T and TCR-T interventions, are expensive. Bacterial therapeutics can be produced at a fraction of the cost, and therefore, can make cancer treatment more affordable for cancer patients and their survivors.

[0048] Provided herein are engineered live attenuated strains of Brucella to address cancer, especially in the context of cellular immunotherapy. In an embodiment, the Brucella melitensis strain has been engineered to contain a mutation in one or more of its virulence genes, such as vjbR, asp14, and mucR that inactivates the virulence gene, and includes an exogenously introduced gene (e.g., tnaA) that encodes for tryptophanase activity and another that encodes for toluene-4 monooxygenase (e.g., tmo). Provided herein are live attenuated strains of Brucella melitensis in which a gene encoding a factor in a two-component master regulator of bacterial virulence (vjbR) is deleted (BmΔvjbR) and displays anticancer activity when delivered systemically (intravenous installation). Moreover, this engineered bacterium is engineered to produce tryptophan metabolites with immunomodulatory activities. Embodiments include engineered strains of Brucella (BmΔvjbR) that produce a variety of immunomodulatory factors, individually or in combination, including: (1) Nanobodies that interact with and stimulate CCR7, the CXCL19 immune receptor; (2) Nanobodies that interact with and antagonize the immunosuppressive proteins PD1 and / or PDL1; and (3) Peptides that encode cancer- associated antigens and / or neoantigens, including, but not limited to antigens encompassing or within the following:a. Hormad1 with the amino acid sequence: MVVLATISEC YQFKFKYTNN GPLMDFISKN QSNESSMLST DTKKASILLI RKIYILMQNL GPLPNDVCLT MKLFYYDEVT PPDYQPPGFK DGDCEGVIFE GEPMYLNVGE VSTPFHIFKV EVTTERERME NIDSTILSPK QIKTPFQKIL RDKDVEDEQE HYISDDLDIE TKMEEQEKNP ASSELEEPSL VCEEDEIMRS KESPDLSISH SQVEQLVNKT SELDMSESKT RSGKVFQNKM ANGNQPVKSS KENRKRSQHE SGRIVLHHFD SSSQESVPKR RKFSEPKEHI b. Mesothelin with the amino acid sequence: SMYGFYAVALFYAAS c. NY-SEO-1 with the amino acid sequence: SLLMWITQC d. VGLL1 with the amino acid sequence: LSELETPGKY e. MAGE-A4 with the amino acid sequence: GVYDGREHTV f. MUC1 with the amino acid sequence: AHGVTSAPDTRPAPGSTAPPAHGVTS g. WT1 with the amino acid sequence: MGQQGSLG h. PRAME with the amino acid sequence: RLVELAGQSLLKDEA

[0049] Embodiments include nucleic acid constructs containing genes that encode the expression of these factors under the control of constitutive or inducible promoters. Constitutive promoters include the lac, kan, gro-L or similar promoters. Inducible promoters include hypoxia- or lactic acid-inducible promoters. Because these acid-inducible genes will only be expressed under conditions expected to present in the tumor microenvironment, including hypoxia, and lactic acid presence.

[0050] Embodiments includes systems used for secretion of proteins, such as engineered live attenuated strains of Brucella expressing a hemolysin export system. The hemolysin export system is a type I secretion system that is used by gram-negative bacteria to transport a wide range of proteins across the outer membrane. This system can also be used to export recombinant proteins from bacterial cells efficiently.

[0051] To utilize the hemolysin export system for recombinant protein expression, genes of interest are first cloned into plasmid vectors (for example see FIGs.11-13) that contain the necessary elements for expression and secretion. These plasmids will include a promoter to drive gene expression, a signal peptide sequence to direct the protein to the hemolysin secretion machinery, and a coding sequence for the recombinant protein of interest (e.g., nanobody or protein or peptide cancer antigen). Once the plasmid has been transformed into the BmΔvjbR, the recombinant protein will be produced and targeted to the hemolysin export system. The protein will then be transported across the inner membrane and into the periplasmic space. It can be properly folded and modified before being transported across the outer membrane by the hemolysin export system. An advantage of using thehemolysin export system for recombinant protein expression is that it can accommodate proteins of varying sizes and structures. Additionally, this system allows for the efficient secretion of proteins into the extracellular environment, enabling the target protein to interact with cellular targets of interest.

[0052] Embodiments will also include methods that target several loci in BmΔvjbR to integrate the recombinant cassettes, including vjbR, Asp24 and hdeA. These loci are known to attenuate the bacterium (and thereby, disruption of the target genes will generate recombinant strains with enhanced safety profiles).

[0053] Provided herein are a pharmaceutical composition containing an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole. Methods of treatment of disease include administering to the patient a pharmaceutical composition containing an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole. Methods of treatment of disease include administering a pharmaceutical composition containing an attenuated bacterial strain of Brucella melitensis expressing an immunomodulatory factor to the patient. Methods of treatment of disease include further administering 5-hydroxyindole to the patient. Methods of treatment of disease include administration of indole-based compositions and modified brucella strains. Methods of treatment of disease include administration of hydroxyindole and modified brucella strains expressing cancer- associated antigens. EXAMPLES

[0054] Various examples are describe to illustrate selected aspects of certain embodiments of the pharmaceutical compositions and their methods of use.

[0055] HI activates CD8+T cells and enhances cytotoxic activity.

[0056] HI was found to exhibit potent immunomodulatory effects in vitro. As shown herein, HI possesses several anticancer properties, including the activation of both murine and human CD8+T cells and the promotion of murine CD4+T cell differentiation into a Th17 inflammatory subtype (FIGs. 1A - 1D, FIGs. 1Q – 1R). Specifically, HI increases the production of TNF-α, perforin, granzyme B (GR-B), and IL-2 in murine CD8+T cells as compared to controls (FIGs.1A-1B; FIGs. 1Q – 1R). In human CD8+T cells isolated from peripheral blood mononuclear cells, 5-HI similarly enhances TNF-α production (FIGs.1Q – 1R).

[0057] To investigate whether HI promotes the polarization of naive CD4+T cells into Th17 cells, CD4+T cells were isolated, activated, and cultured under Th17 differentiation conditions in the presence of HI. This treatment resulted in the enhanced differentiation of naïve CD4+T cells intoTh17 cells (FIGs.1C-1D). Additionally, HI promoted the polarization of macrophages towards the M1 phenotype (FIGs.1E-1F), as indicated by increased CD38 expression on murine bone marrow- derived macrophages (BMDM) determined by flow cytometric analysis.

[0058] The BmΔvjbR (SPIKE chassis, hereinafter SC) was engineered to express genes that direct the synthesis of this metabolite to enhance its anticancer activity. First, a modified strain was engineered that carried tryptophanase (Tna) and toluene-4 monooxygenase (Tmo) genes, referred to as SPIKE 1.0 (S1.0), that constitutively produced HI (FIGs.1G - 1H). Importantly, safety assessments demonstrated that S1.0 was non-pathogenic in tested animals. Third, the production of HI by S1.0 did not alter its ability to be internalized by host cells. Fluorescence microscopy analysis showed that S1.0 entered BMDMs and replicated intracellularly (FIG. 1I). Fourth, S1.0 displayed enhanced antitumor activities. BMDMs were co-cultured and pre-treated with SC or S1.0, with OT-I CD8+T cells and co-cultured these chicken ovalbumin (Ova)-specific CD8+T cells with B16-Ova melanoma cells, and then the survival of tumor cells was determined at various time post-co-incubation. CD8+T cells co-cultured with S1.0 induced significantly higher B16-Ova melanoma cell killing than that of the SC parental strain. Further, S1.0 had significantly higher cytotoxic activity compared to SC as demonstrated by flow cytometric analysis (FIGs.1J-1K). Finally, mesothelin (Mes)-specific human CD8+T cells demonstrated significantly higher cytotoxic activity against human H1975 lung cancer and Panc02 murine pancreatic cancer cells after treatment with 5-HI (FIGs.1L-1M, FIGs.1S – 1X). Taken together, the pro-inflammatory activity of HI induced cytotoxic activity against diverse types of cancer cells.

[0059] S1.0 is non-pathogenic in animal models.

[0060] The safety profile of S1.0 was examined in mouse and ruminant animal models. First, complete blood count (CBC) and organ weights of treated (5 ^ 107S1.0 cells) and control mice did not display significant differences (FIGs.2U – 2V). Second, S1.0 persisted in tumor and spleen at 12 days post-injection (DPI), and only in tumor, but none of the organs of tumor-bearing mice, at 16 DPI (FIGs. 1N-1P; FIGs. 2W – 2Z). However, when tumor-bearing mice were treated with the combination of S1.0 and adoptively transferred CD8+T-cells, viable S1.0 was not detected in the tested organs of treated mice at 12 DPI (FIG. 2UX). Finally, S1.0 was tested in pregnant goats, a highly sensitive testbed for evaluating the safety of a Brucella-based bacterial therapeutic. No abortions in mid-gestational pregnant goats were detected following S1.0 treatment. Serological renal, hepatic, and hematologic toxicology screens showed no statistically significant changes in blood urea nitrogen, red blood cell count, hemoglobin, and hematocrit between animals treated with S1.0 andvehicle alone control. These findings were further corroborated by histologic organ analyses that did not identify signs of necrosis, cellular infiltration, inflammation, or hemorrhage in lung, kidney, liver, and spleen tissue excised from treated animals (TABLE 1).

[0061] TABLE 1

[0062] Finally, no S1.0 wasspecimens ofor kids throughout the experiment (TABLE 1), consistent with a favorable safety profile. Collectively, these data demonstrated the favorable safety features and an absence of induction of acute toxic effects by S1.0 in these tested animal systems.

[0063] S1.0 inhibits tumor growth and improves survival in multiple animal tumor models. To test whether S1.0 displays anticancer activity in animal models of solid tumors, tumor size and survival were measured in tumor-bearing animals treated with a single intravenous dose of the intervention (or mock treated with 1^ PBS). Two syngeneic heterotopic murine cancer models were used: B16-Ova melanoma—a warm tumor that displays responsiveness to treatment with immunomodulatory agents, and pancreatic cancer Panc02—a cold tumor that limits immune cell infiltration into the TME and is recalcitrant to treatment with antibody or cellular therapeutic agents. To extend the findings to human solid tumor models, lung cancer (H1975) and pancreatic cancer (AsPC-1) xenograft models were also used in which immunocompromised non-obese diabetic (NOD)-scid IL2rγnull(NSG) mice were engrafted with these tumor cells. The NSG mouse model is well-suited for evaluating CAR-T cell efficacy, as its lack of endogenous T cells minimizes confounding adaptive immune responses. Results from these experiments showed that in both syngeneic (FIG. 3N) and xenograft (FIG. 3O) mouse-tumor models, S1.0 treatment significantly reduced tumor sizes and increased survival of tumor (i.e., B16-Ova melanoma, Panc02, H1975, and AsPC-1)-bearing mice (FIGs. 2A-2H; FIGs.3P – 3Q).

[0064] S1.0 significantly out-performed the parental SC in anti-cancer treatment efficacy in both mouse tumor models (B16-Ova melanoma and Panc02) and human solid tumor models of H1975 and AsPC-1 tumor (FIGs.2A-2H; FIGs.3P – 3Q). Next, these compositions were evaluated in heterotopic xenograft models (i.e., lung [H1975] and pancreas [AsPC-1]) (FIGs.2E -2H; FIG.3O) as well as a Kras(G12D) / Trp53 null / Pdx1-cre (KPC)-luciferase orthotopic pancreatic ductal adenocarcinoma (PDAC) model (FIGs. 2I-2K, FIGs. 4R – 4Z). In the KPC-luciferase PDAC model, a single dose treatment with S1.0 inhibited tumor growth, and extended survival, to a similar level as treatment with 5 doses of ICI ^PD-1 (anti-PD-1) antibody (FIGs. 2I-K; FIGs. 4R – 4T). When S1.0 was combined with ^PD-1 therapy, the survival benefit was enhanced (FIGs. 2I-2K; FIGs. 4R – 4T). Moreover, several mice in the S1.0-treated or combination-treated experimental groups displayed complete resolution of the tumor (FIG. 4S), indicating that S1.0 contributed potent anticancer activities. Finally, S1.0 also acted synergistically with ^PD-1 or CAR-T (CEA) therapy in treating these tumors (FIGs. 2A-2K; FIGs. 3P – 3Q; FIGs. 4S – 4T). Taken together, S1.0 promotes broad spectrum anti-cancer activities.

[0065] To benchmark these studies, some experimental groups were also treated with CAR-T (mesothelin; AsPC-1), T cell receptor T (TCR-T) (NY-ESO1; H1975) cells, or with ICI ^PD-1 (KPC- luciferase orthotopic PDAC) that target antigens expressed on the corresponding tumor surfaces (FIGs.3P-3Q; FIGs.4S – 4T). In several tested models, S1.0 treatment performed as well or better than corresponding CAR-T, TCR-T, or ICI benchmark treatments (FIGs.2A-2K, FIGs.3P-3Q; FIGs. 4S – 4T). In addition, CAR-T, TCR-T, and ICI interventions worked additively or synergistically with S1.0 treatment. These findings indicated that the bacterial intervention induced potent anticancer effects.

[0066] To dissect the molecular and cellular mechanisms by which S1.0 inhibited tumor growth and increased survival rates of tumor-bearing mice in divergent tumor models, the responses of immune cells in the TME were measured. In the mouse B16-Ova melanoma model, the infiltration of OT-1 CD8+T cells into the TME increased significantly compared to controls (FIGs.2L-2N), implying that CD8 T cells may play important roles in immunomodulation and antitumor activity in the presence of S1.0. The antitumor roles of CD8 T cells were tested using CD8 and Batf3 knockout (KO) mice as Batf3 controls the development of CD8α+dendritic cells (DCs), which are crucial for priming CD8+T cells and initiating effective immune responses against tumors. S1.0 treatment of tumor- bearing mice with deficiencies in CD8 or Batf3 significantly prolonged survival of these tumor- bearing CD8 and Batf3 KO mice; however, these cell functions are not essential for the anticanceractivities in the presence of S1.0 (FIGs. 3T-3U). Moreover, S1.0 promoted reduction in PD-1 expression both independently and in combination with ^PD-1 antibody in CD4+T and CD8+T cells derived from the KPC-luciferase orthotopic PDAC mouse model (FIG. 2O) and increased immune cells infiltration into the TME (FIGs.4U – 4Z). Multi-photon microscopy analysis showed that S1.0 localized in a high-expression level of Ly6g+cells (FIG. 2P). The hematoxylin and eosin (H&E) staining analysis showed increased intraneoplasm macrophages (INM), lymphocytes (LYM) infiltration, and tumor necrosis (TN) in S1.0 or the combination of S1.0 and antigen specific CAR-T cell treated groups (FIGs.2Q-2R, FIGs.5M-5N). A multiparametricspatial imaging analysis showed increased infiltration of F4 / 80+macrophages, CD8+T cells, CD4+T cells, CD11b+monocytes, NK 1.1 cells and Ly6g+cells into the TME of B16-Ova melanoma (FIGs.2S-2T, FIGs. 6A – 6B). The S1.0 treatment regressed tumor size and enhanced mouse survival by increasing immune cell infiltration into the TME.

[0067] S1.0 promotes inflammatory activity and CD8+T cell infiltration in humanized mice.

[0068] To more accurately assess bacterial modulation of innate immune responses, studies were performed using humanized mouse models of lung cancer (H1975) and Merkel cell carcinoma (MCC13) (FIGs.3A – 3M) as NSG mice are deficient in innate immune functions. In these models, NSG (NOD.Cg-Prkdcˢ^^^ Il2rg^^¹^ʲˡ / SzJ) mice were first engrafted with human CD34+hematopoietic progenitor cells, resulting in animals that carry human B cell, T cell and monocyte populations. H1975 or MCC13 cancer cells were heterotopically transplanted into the humanized mice, and either treated with S1.0, TCR-T cells that target the tumor, or with both interventions (FIGs. 3A). These experiments revealed that the bacterial intervention substantially reduced tumor growth and increased the survival of the treated animals (FIGs.3B-3E). The improvement in mice weight with the treatment also indicated that the S1.0 intervention alone or in combination with CAR-T cells significantly improved the outcome of treatment (FIGs.3F). These data therefore supported the human-relevance of the observations. There was significantly increased CD8+T cells infiltration into the TME (FIG. 3G), increased CD19 expression (FIG. 3H), CD11b expression (FIG. 3I) and reduced PD-1 expression (FIG. 3J) of humanized mice treated with S1.0 and CT (FIGs. 3G-3K). Consistent with B16-Ova tumors, H&E staining analysis also showed that S1.0 treatment increased infiltration of immune cells, including macrophages and lymphocytes, and tumor necrosis in S1.0 or combination treatments (FIGs. 3L-3M). These results demonstrated that in the humanized mouse model, S1.0 promotes infiltration of immune cells, regresses tumor size, and improves survival of tumor-bearing mice, indicating the versatility of our engineered bacteria.

[0069] S1.0 upregulates chemokine signaling and modulates the expression of amino acid receptors on T cells.

[0070] S1.0 remodeled the cellular immune profile of the TME. Single-cell RNA sequencing (scRNA-seq) analyses of treated or mock-treated explanted murine B16-Ova melanoma tumors were performed on day 21 or day 28 post tumor cell implantation (PTCI) (FIG. 7A). Hundreds of differentially expressed genes (DEGs) were identified in tumors treated with S1.0 compared to controls. Using cell type-specific molecular markers (FIGs.7B-7E) and the Seurat pipeline, clustering analysis was performed to characterize the abundance of assorted cell types in the TME (FIG. 4A, FIGs. 7F-7I). S1.0 promoted increased immune cell infiltration (B cells, T cells, neutrophils, and macrophages) compared to the untreated control or CAR-T groups on day 21 PTCI. This effect was further amplified treatments that combined S1.0 with CAR-T (FIG.4B). As expected, the expression of antigen presentation proteins such as H2Aa1 and H2Ab1 in macrophages of S1.0 treated tumors, was significantly increased on day 21 PTCI compared to the controls (FIGs.4C-4D). Similarly, the expression of chemokine ligands like CCL-3 and CCL-4 on macrophages was increased in S1.0- treated tumors (FIG. 4E). These results indicated that S1.0 not only promoted inflammatory and activated macrophages but also provided the conditions for increased chemotaxis of adaptive immune cells into the TME.

[0071] To test the hypothesis that S1.0 increased adaptive immune surveillance via enhanced macrophage activity, functional T cell activity was analyzed there. First, the chemokine receptor and ligand activation on tumor-associated T cells were analyzed. The chemokine receptor activation increased in the S1.0 treated group, which was further enhanced by combined treatment of S1.0 with CT (FIG.4F). Interestingly, S1.0 alone promoted Ccr-7 expression, a marker for central memory T cells (Tcm), whereas S1.0 in combination with CAR-T promoted effector memory T cells (Tem) by increasing the expression of the CCR-5 chemokine receptor marker (FIG. 4F). These observations supported the hypothesis that the increased chemokine expression on macrophages and other immune cells promoted by S1.0 may increase infiltration and alter the polarization of immune cells penetrating the TME.

[0072] Amino acids are essential nutrients for the survival and growth of immune cells and cancer cells. Interestingly, amino acid transporter expression was significantly increased in the tumor- associated T cells of S1.0-treated mice (FIG. 4G; FIGS. 8A – 8F). Importantly, the expression of SLC38a1 and SLC38a2, two members of the solute carrier family 38 (SLC38) involved in the transport of glutamine and other amino acids such as alanine, serine, proline, and glutamine, intocells, was increased in S1.0-treated tumor-bearing mice. Moreover, the expression of GLS, GLUD-1 and GoT 1 were also increased by S1.0 treatment. The increased expression of these markers was sustained in tumor-bearing mice treated with S1.0 and CAR-T (FIG. 4G). These findings indicated that S1.0 treatment metabolically reprograms T cells by modulating amino acid receptor signaling pathways, potentially mitigating early exhaustion and enhancing effector functions.

[0073] The observed metabolic rewiring might facilitate a transition of some CD4+T and CD8+T cells toward an effector / central memory phenotype, further protecting against exhaustion. Importantly, this metabolic modulation may confer a competitive advantage to T cells by depriving cancer cells of essential nutrients critical for tumor growth. Comparative analysis of amino acid metabolism enrichment in T cells and melanocytes was performed. This analysis revealed an increase in alanine and aspartate metabolism in T cells, while these pathways were significantly suppressed in melanocytes in day-21 explanted tumors (FIGS. 8A – 8F). Furthermore, global amino acid metabolism was selectively enhanced in tumor-associated T cells treated with S1.0 compared to controls, underscoring the cell-type specificity of S1.0-induced metabolic reprogramming.

[0074] To investigate whether enhanced amino acid utilization and metabolic rewiring in CD4+and CD8+T cells translated into increased functional capacity within the TME, their activation, persistence, memory formation, and suppressive capabilities were analyzed. There was a marked increase in CD8+T cell activation in tumors at day 21 PTCI from the S1.0-treated group compared to the untreated control (FIG. 4H). Notably, S1.0-treated CD8+T cells demonstrated an enhanced effector / central memory phenotype (FIG.4I), which was attributed to reduced activation-induced cell death and a prolonged persistence of functional CD8+T cells (FIG. 4J). In contrast, the initial heightened activation observed in the antigen-specific CAR-T-treated group appeared to contribute to early exhaustion of CD8+T cells (FIGs. 4J-4K). Importantly, S1.0 and combination of S1.0 and CAT-T treatments induced a distinct population of tumor-associated CD8+T cells characterized by a CCR5highIL7highGzmBhighCD27highCD28highphenotype on day 21 PTCI (FIGs. 4F-4J), suggesting robust and sustained anti-tumor activity. Similar phenotypes were observed in tumor-associated CD4+T cells with the expression of genes indicating lower initial activation (FIG. 4L), higher memory phenotype (FIG.4M), higher persistence (FIG.4N) and lower suppressive capacity on day 28 PTCI (FIGs. 4L-4O), compared to controls. Consistent with the hypothesis, reduced expression of exhaustion markers PD-1, Tim-3 (Havcr2) and Lag3 and increased IL-7 signaling in CD4+T cells and CD8+T cells was observed (FIGs.4I, 4K, 4M, 4O), in the combination treatment group of S1.0 and CAR-T, corroborating their enhanced functional state, compared to the S1.0 or CAR-T groups,particularly on Day 28 PTCI, with significant differences observed in Tim-3 and Lag-3 expression (FIGs.4K, 4O). PD-1 expression analysis revealed similar mean values between the S1.0 and CAR- T groups, likely due to an increase in tumor-resident CD4 / CD8 T cells, indicating enhanced activation, differentiation, and function. However, the combined treatment group (S1.0+CAR-T) showed significantly lower expression of PD-1, Lag-3, and Tim-3 on Day 28 in both tumor-associated CD4+and CD8+T cells (FIGs.4K, 4O). S1.0 modulates the TME to enhance tumor-infiltrating CAR- T cell activity, reducing exhaustion while promoting cytotoxic function with limited differentiation. Furthermore, increased expression level of proinflammatory M1 macrophages was observed (FIG. 4P), B cell activity (FIGs. 9A-9C), aryl hydrocarbon receptor (AhR) signaling in macrophages and other immune cells (FIGs.9D-9E) in tumors treated with S1.0 or a combination of S1.0 and CAR-T. Decreases in the expression of immune cell markers associated with tumor-promotion, including M2 macrophages, and MDSCs, was also observed in the tumors of S1.0-treated mice (FIGs.9F-9H). The upregulated expression patterns of apoptosis-associated genes implied more apoptosis in melanocytes in the TME of tumors on day 21 post-treatment with S1.0 (FIG.4Q). Similarly, down-regulation of markers associated with N2 neutrophils was observed (FIGs.9I-9J) that are pro-tumorigenic and have strong immunosuppressive properties, promote tumor angiogenesis, and metastasis.

[0075] Overall, S1.0 promotes the infiltration of immune cells into the TME and increases the antigen presentation and chemokine expression activity in innate immune cells, thereby resulting in the increased functional capacity of CD8+and CD4+T cells. Furthermore, S1.0 treatment selective promotes in CD8+and CD4+T cells increases in the expression of genes that control the metabolism of amino acids.

[0076] S1.0 remodels amino acid metabolic pathways.

[0077] The regulation of amino acid transporters in CD8+and CD4+T cells by S1.0 encouraged the performance of a metabolomics analysis of the TME in mock- or S1.0-treated tumor-bearing mice. The PLSDA (partial least squares-discriminant analysis) score plots based on the TME metabolome database of B16-Ova melanoma excised tumors at 21 days PTCI revealed significant separation of the control, SC, and S1.0 treatments (FIG. 5A). Metabolite analysis demonstrated increases in HI, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and methylthioadenosine, a naturally occurring sulfur-containing nucleoside that suppresses tumors by inhibiting tumor cell proliferation, invasion, and the induction of apoptosis (FIG. 5B, left). Metabolites associated with tumor proliferation and growth, including lactate, glutamine and glutamate decreased in the TME of tumors from mice treated with S1.0 (FIG. 5B, right; FIG. 5C). Metabolic Set Enrichment Analysis(MSEA) revealed that in the S1.0-treated TME, metabolic pathways associated with tumor cell development, proliferation and metastasis, including arginine biosynthesis, glycolysis or gluconeogenesis, alanine, aspartate and glutamate metabolism, were modulated (FIGs. 5D-5F). Moreover, scRNA-seq gene set enrichment analysis demonstrated that in CD4+T cells, the upregulation of genes involved in glycolysis and down-regulation of genes in arginine metabolism pathway (FIGs.5G-5H) on day 21 and day 28 PTCI, respectively.

[0078] To validate these findings in the scRNA-seq and metabolomics analyses, quantitative real- time PCR (qPCR) and amino acid uptake profiling assays were performed to analyze the expression of some key identified amino acid transporters and the uptake levels of amino acid in the TME. The validation analyses demonstrated that the expression of these tested amino acid transporters were upregulated (FIG.5I) and the uptakes of the tested amino acids increased (FIGs.5J-5K; FIG.10) in the TME of B16-Ova melanoma tumor-bearing mice treated with SPIKE bacteria S1.0 or SC, indicating the reprograming of amino acid availability and metabolomics in immune cells within the TME.

[0079] Collectively, the metabolomics and scRNA-seq analyses demonstrated that metabolites and / or metabolic synthesis pathways related to immunomodulation increased and those associated with tumor cell growth and metastasis decreased in the S1.0-treated TME, thereby revealing metabolic rewiring of CD8+T cells, thereby promoting their differentiation into effector / central Tcmalong with optimal sustained cytotoxicity preventing early induction of cell death (FIG.5O). This metabolic shift can be characterized by a gradual transition from glycolysis to oxidative phosphorylation, which supports the energetic and biosynthetic demands of long-lived, functionally active T cells.

[0080] A single systemic administration of SPIKEs displayed efficacy across diverse murine and humanized cancer models while maintaining a favorable safety profile, even at high doses, in animal models. S1.0 serves as a platform in BCIT, demonstrating that metabolic engineering of live bacteria can dynamically reprogram the TME to enhance antitumor immunity. By producing HI, S1.0 not only amplifies CD8+T cell-mediated cytotoxicity but also diversifies immune engagement across multiple cell types, including CD4+T cells and macrophages, fostering a highly pro-inflammatory and tumoricidal environment. The dual activation of both innate and adaptive immune pathways, marked by increased TNF-α, perforin, and GR-B secretion, positions S1.0 as a potent immunomodulator capable of overcoming immune exclusion in cold tumors and reinvigorating exhausted T cells in hot tumors. The ability of HI to upregulate CD38 expression in macrophages further underscores its role in reshaping tumor metabolism, a novel strategy that directly links bacterial metabolic engineeringwith immune potentiation. Notably, S1.0 outperformed its parental strain in tumor cell cytotoxicity without compromising bacterial invasion efficiency, highlighting the precision of its engineered immunostimulatory functions. Moreover, the observed enhancement of antigen-specific human CD8+T cell cytotoxicity against lung cancer cells underscores the translational effectiveness of this platform. S1.0 is a bacterial immunotherapy with the capacity to remodel immune landscapes across diverse tumor types, setting the stage for innovative metabolic-immune interventions in oncology.

[0081] S1.0 reshapes the TME profoundly, enhancing immune cell infiltration, metabolic reprogramming, and functional immune activity. scRNA-seq analysis revealed increased infiltration of innate and adaptive immune cells, including CD8+and CD4+T cells, macrophages, and neutrophils, particularly in tumors treated with S1.0. Given the role of the AhR as a critical modulator of immune responses, indole derivatives like 5-HI drive AhR activation was supported by the data. AhR activation in T cells, macrophages, and other immune cells was linked to enhanced cytotoxic activity, memory formation, and recruitment of effector CXCR3⁺ CD8+T cells into the TME. These effects were further complemented by the upregulation of antigen presentation machinery and chemokine expression in macrophages, facilitating robust T cell activation and chemotaxis. The reduction of pro-tumorigenic N2 neutrophils further supports the ability of S1.0 to mitigate immune suppression within the TME.

[0082] The metabolic reprogramming induced by S1.0 represents another significant mechanism by which this engineered strain enhances anti-cancer immunity. Amino acid availability, critical for immune cell function, was markedly altered in the TME of S1.0-treated animals. Key transporters such as SLC38A1 and SLC38A2, involved in glutamine uptake, were upregulated in CD4+and CD8+T cells, supporting their activation, proliferation, and effector functions under nutrient-deprived conditions. These observations align with previous reports linking amino acid metabolism to enhanced T cell functionality. Elevated expression of glucose transporter 1 (GLUD1), which facilitates glutaminolysis, and glutamate oxaloacetate transaminase 1 (GOT1), which promotes aspartate metabolism, underscores the metabolic adaptations of immune cells to the hostile TME. These pathways not only sustain cytokine production and cytotoxic activity but also promote the formation of memory T cells, which rely on mitochondrial health and oxidative phosphorylation (OXPHOS) for persistence.

[0083] Additionally, the upregulation of CCR7 and IL-7R in T cells within the TME highlights their role in enhancing T cell trafficking, survival, and memory differentiation. CCR7 directs T cells to tumor-draining lymph nodes (TDLNs) for priming and expansion, while IL-7R signaling supportseffector and memory T cell survival in the immunosuppressive TME. These mechanisms collectively enhance the endurance and readiness of T cells to mount effective antitumor responses. Furthermore, the observed increase in Ccr5 and arginine amino-acid expression indicates a higher potential for differentiation into effector memory phenotypes, critical for sustained immune surveillance.

[0084] Collectively, the data support the proposed immunoregulatory mechanisms triggered by S1.0 (FIG.5L). Upon administration, S1.0 homes to tumor, which may be mediated by Ly6gHighmyeloid derived suppressor cells (MDSCs), and locates in these Ly6gHighcells in the TME. Next, S1.0 releases HIs, promotes antitumor chemokine production, and increases immune cells such as CD8+T cells infiltration into TME, and CD4+T cell polarization. S1.0 activates immune cell functions, including cytotoxic T lymphocyte (CTL) directed tumor apoptosis, antigen uptake and presentation, functional M1 macrophage polarization, and CTL activation and persistence. S1.0 reprograms immune cell metabolomes and increases T cell amino acid uptake, resulting in increased CD8+T cell activation, the numbers of memory CD8+T cells and their persistence and CTL activity. Finally, these immunomodulatory activities stimulated by S1.0 suppresses tumor growth. In comparison to current cellular therapies like CAR-T or TCR-T, which are often prohibitively expensive and complex, bacterial therapeutics like S1.0 offer an affordable, scalable, and efficacious alternative. Materials and Methods

[0085] Bacterial culture and metabolic engineering. Bacterial strains SC and S1.0 were cultured. For SC or S1.0 administration, tryptone Soy Broth (SOB) (Millipore Sigma, USA) was used to culture the bacterial strains. The bacteria were collected by centrifugation followed by washing and resuspending in 1^^ PBS. For co-culture bacteria with cells, bacteria were added to each well of a 24- well plate at an MOI (Multiplicity of infection) of 20 of macrophage monolayer in DMEM medium (Invitrogen, CA, USA).

[0086] Cell culture. H1975 human non-small cell lung cancer cells, Panc02 mouse pancreatic ductal adenocarcinoma cells, and AsPC-1 (human pancreatic cancer cells derived from nude mouse xenografts) cells were cultured in 10% FBS, 1% penicillin-streptomycin supplemented RPMI 1640 medium (Invitrogen, CA, USA). B16-Ova melanoma cells were grown in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin antibiotics.

[0087] Flowcytometric assessment of 5-HI activated CD8+T cells and CD4+T cells. Mouse CD8+T cells were isolated from C57BL / 6 mice (n=15) and activated with anti-CD3 / CD28 antibodies. At 3 days post-activation (dpa), the cells were treated with 50 µM of HI or indole or vehicle control (Control). The levels of TNF-⍺, Perforin, and GR-B were tested at 48 h post treatment byflowcytometric analysis. The same protocol was followed for assessing CD4+T cells differentiation in Th17 cells.

[0088] In-vitro cytotoxic assessment of CD8+T cells activity against various cancer cells. BMDMs were isolated from C57BL / 6 mouse (n=15) and expanded with GM-CSF for 24 hours. Subsequently, these BMDMs were co-cultured with S1.0, SC at an MOI of 20 or remained untreated in the presence of antigen specific CD8+T cells. The CD8+T cells were then retrieved from the culture and added to the target B16-Ova or H1975 cancer cells at various target to effector ratios of 1:1, 1:2 and 1:5. Dead / live staining was performed using the Aqua Zombie Fixable Dead / Live Staining Kit (BioLegend, #423101) on CFSE-pre-stained CD8+T cells to distinguish them from cancer cells and assess cancer cell death.

[0089] Identification of HIs from S1.0 bacterial culture filtrate. S1.0 bacterial culture filtrate was prepared. Briefly, S.10 bacteria were cultured in SOB for 48 hours, culture media were collected and centrifuge at ~20,160 ^g for 15 min to remove cell debris, media were then separated by methanol- chloroform-mQ water extraction, the upper (aqueous) phase was transfer into an Eppendorf tube and dried using a SpeedVac vacuum concentrator (Thermo Fisher Scientific Inc., USA). Detection and identification of HIs were performed by using Liquid Chromatography-Mass Spectrometry (LC-MS) analysis.

[0090] Mouse lines. The wild-type C57BL / 6 (B6) Thy 1.1 mice were purchased from the Jackson Laboratories. Non-obese diabetic-Severe combined immunocompromised γ (NOD.Cg-Prkdcˢ^^^ Il2rg^^¹^ʲˡ / SzJ; NSG) mouse model mice are severely immunocompromised mice as they lack the IL- 2 receptor γ chain. The mice were procured from Jackson laboratories and maintained in 12 h light and dark cycles with their littermates. The humanized mice (huCD34 mice) are created by transferring CD34+hematopoietic stem cells into NSG mice to mimic a humanized immune system. The humanized mice were procured from Charles River laboratories and maintained under similar conditions. The OT-1 transgenic mice produce a transgenic T- cell receptor on CD8+T cells recognizing ovalbumin peptide residues 257-264 (OVA257-264). OT-1 T cell receptor transgenic mice were procured from Jackson Laboratories. Six to eight weeks old mice, unless otherwise indicated, were used in each specific mouse tumor model described details as the below.

[0091] S1.0 safety assays

[0092] Mouse complete blood count (CBC) assays. C57BL / 6 mouse organ weight analysis was conducted on mice injected with varying doses of S1.0 (n=5 per group), receiving either 100 µl of the S1.0 bacterial dose or 100 µl of 1× phosphate-buffered saline (PBS). Organ (spleen, liver, lung,kidney, brain) weights were collected on 14 dpi. Additionally, a CBC analysis, including red cell distribution width, neutrophil count, reticulocyte absolute count (Retic Count), lymphocyte count, hematocrit, hemoglobin, red blood cell count, white blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (MCHC), and plasma protein, was performed on mice administered with different S1.0 bacterial doses or 1× PBS. Results were analyzed on 14 dpi.

[0093] S1.0 biodistribution in tumor-bearing mice. Male C57BL / 6 wild type mice were subcutaneously injected with 1×106LLC1-Ova cells (Vitro Biotech, CA, USA) in the right lateral flank on day 0. The mice were divided into three groups (n=15 per group). On day 8, once the tumor volume reached approximately 150 mm3, mice were intravenously injected with 5.0 × 107S1.0 (group 1), 1.0 × 108S1.0 (group 2) and 1.0 × 109S1.0 (group 3) respectively. The colony forming unit (CFU) assay was performed to determined S1.0 bacterial distribution in tumor and other organs of mice pre- implanted with or without tumor cells and injected with S1.0 as a tumor therapy treatment. In brief, mice were sacrificed at 4-, 8-, 12-, 16-, and 20-days post-injection (dpi), and tumor, spleen, lungs, kidney, liver, brain, bone marrow, fecal and urine samples were collected. The harvested organs were homogenized and plated on Tryptic Soy Agar (TSA) (Millipore Sigma, USA) plates supplemented with chloramphenicol (30 μg / mL) (Research product international, IL, USA). The CFU was recorded after 3-4 days of post-cultivation of the bacteria. The CFU was calculated using the following formula: CFU / g = (Number of colonies*dilution factor) / weight of tissue sample in gm.

[0094] Goat S1.0 safety assessment. Two groups of pregnant goats (n=7 / group) at 65 days of gestation were inoculated bilaterally in the conjunctival sac with a total dosage of 1^108and 1^109S1.0 bacteria, respectively. Clinical evaluations were conducted daily, and blood samples were collected bi-weekly for card test and ELISA analysis through 84 days post S1.0 inoculation and at euthanasia. The dams had normal parturitions and healthy kids. At parturition, colostrum, vaginal swab, and urine were collected, and following humane euthanasia at approximately 10 days post- parturition, multiple tissues were collected from the dam and kids for histology, bacteriology, and quantitative PCR to detect DNA from S1.0. The samples of vaginal swabs, lung, abomasum and / or placenta, at various dpi, were collected for quantitative PCR using a S1.0-specific probe (IS711).

[0095] CAR-T and TCR-T cell preparation. The MSGV1 γ retroviral vector backbone was modified to express mesothelin antigen specific scFv as described in our previous studies. CAR-T cells were prepared by transduction of viral supernatants containing the respective antigen with 5 µg / mL polybrene. The lentivirus to modify NY-ESO-TCR specific CD8+T cells derived from human bloodperipheral blood mononuclear cell sample - MSGV1 γ retroviral vector backbone was kindly provided by Dr. Wendell Yang (Imgen Lifesciences Inc.).

[0096] Murine tumor models. Tumor cell inoculation and cancer treatments using divergent murine tumor models are illustrated in FIGS.3N – 3O. Six to eight weeks old mice were used in each specific mouse tumor model described details as the below.

[0097] Lung cancer model in NSG mice and hCD34+humanized mice. Wild-type NSG mice or hCD34+NSG humanized mice were subcutaneously injected with 1×106H1975 human lung cancer cells in the right lateral flank on day 0. The mice were then divided into five different groups (n = 5 mice / per group). The first 3 groups of mice were injected intravenously with either 1× PBS control (untreated control), live attenuated 1 × 107SC bacteria or engineered live attenuated S1.0 bacteria on day 7 post tumor-cell inoculation (PTCI). On Day 9, two more groups of mice were injected intravenously with 2 × 106NY-ESO-TCR modified CD8+T cells or NY-ESO-TCR modified CD8+T cells in combination with 1 × 107S1.0 bacteria. Mice were housed in Texas A&M University, Laboratory Animal Resources and Research Facility, and health status checked daily. The survival of tumor bearing mice was monitored over 60 days. Tumor growth was monitored every other day and tumor volumes were calculated using the formula: Tumor Volume (mm3) =0.5 × length × width. Mice were humanely euthanized if tumor size reached above 4000 mm3.

[0098] MC32-CEA Colon cancer, Panc02 pancreatic cancer and B16 melanoma models in C57BL / 6 mice. Wild-type C57BL / 6 (B6) Thy 1.1 mice (Jackson Laboratories) were subcutaneously injected with 1×106MC32-CEA colon cancer cells, Panc02 mouse pancreatic cancer cells, or B16-ovalbumin melanoma cancer cells in the right lateral flank on day 0. For each mouse tumor model, the mice were then divided into five different groups (n = 5 mice per group). The first 3 groups of mice were injected intravenously with either 1× PBS control, 1× 107SC or S1.0 bacteria on day 7 PTCI. For MC32-CEA colon cancer model, on Day 9, two more groups of mice were injected intravenously with 2 × 106CAR-CEA modified CD8+T cells only or CAR-CEA modified CD8+T cells in combination with S1.0 bacteria. For Panc02 pancreatic cancer model, on Day 9, two more groups of mice were i.v. injected with 2 × 106mesothelin- modified CAR-CD8 T cells only or mesothelin- modified CAR- CEA modified CD8+T cells in combination with 1 × 107S1.0 bacteria. For B16-Ova melanoma cancer model, on Day 9, two more groups of mice were i.v. injected with 2 × 106OT-1 CD8 T cells only or OT-1 CD8 T cells in combination with 1 × 107S1.0 bacteria. Housing and checking health status of the treated mice, monitoring tumor growth and survival of tumor-bearing mice were performed as the methods described above.

[0099] Pancreatic cancer model in NSG mice. Wild-type NSG mice were subcutaneously injected with 1×106AsPC-1 human pancreatic cancer cells in the right lateral flank on day 0. The mice were then divided into five different groups (n=5 mice per group). The first 3 groups of mice were i.v. injected with either 1× PBS control, 1 × 107SC or S1.0 bacteria on day 7 PTCI. On Day 9, two more groups of mice were injected intravenously with 2 × 106mesothelin- modified CAR-CD8 T cells in combination with 1 × 107S1.0 bacteria. Housing and checking health status of the treated mice, monitoring tumor growth and survival of tumor-bearing mice were performed as the methods described above.

[0100] B16-Ova melanoma tumor in CD8 and Batf3 knockout (KO) mice. CD8 and Batf3 knockout and control mice were intraperitoneally (IP) injected with 1×106B16-Ova melanoma tumor cells on day 0. For each tumor gene KO or control mouse model, the mice were divided into four two groups (n=10 mice per group). On day 14 PTCI, the two groups of mice were IP injected with 100 ^^l 1× PBS or 1 × 107S1.0 bacteria in PBS. Mice were housed in the University of Missouri, Laboratory Animal Resources and Research Facility. Checking health status of the treated mice, monitoring tumor growth and survival of tumor-bearing mice were performed as the methods described above.

[0101] Spatial multiplex immunohistochemical imaging analysis. Single cell recognition and quantitative biomarker analysis were performed on subcutaneous Panc02 mouse pancreatic cancer tissues, dissected from tumor-bearing C57BL / 6 mice (n=3) by using Canopy Biosciences' ChipCytometry™ platform with the CellScape™ instrument (Canopy Biosciences; a Brucker Company). A 14-plex assay panel was performed on four explanted tumor samples from the groups of 1) control (untreated sample), 2) CAR-T cell only, 3) S1.0, and 4) S1.0 combination with CAR-T cells. Gating and quantification of target cell population were performed, and the percentages and cell counts were determined using the CellScape™ system. Single cell segmentation was performed on high-resolution images allowing for the phenotyping of each individual cell by cell classification / thresholding. For further spatial analysis, QuPath software was utilized to segment the images into equal-sized regions to calculate cell densities for CD11b+monocytes, CD8+T cells, CD4+T cells, and their co-expression with PD-1 and Ki-67.

[0102] Multiphoton image analysis. Multiphoton microscopy was conducted from the Day 21 explanted B16-Ova melanoma tumors by using Brucella polyclonal antibody (bs-2229R; Bioss Antibodies Inc.) and secondary anti rabbit CF 560 Antibody (Cat# 20300-1; Biotium). MDSCs staining was performed by using the anti- Ly-6g antibody (Biolegend).

[0103] Metabolomics analysis. Metabolites were isolated from explanted tumor tissues of untreated control, SC and S1.0 treated B16-Ova melanoma tumor-bearing wild-type C57BL / 6 mice (n=5 per treatment group). The polar solvents were then dried and analyzed by mass spectrometry analysis. For each metabolite, the average expression values across three replicates of each treatment condition were calculated. Partial least squares discriminant analysis (PLSDA) was performed by using the metabolomics analysis software MetaboAnalyst (https: / / metaboanalyst.ca / ) to perform peak alignment and identification. Then, hierarchical clustering analysis was done on the z-normalized average expression values of metabolites to group the data based on their expression patterns across tumor conditions. The heatmap of metabolites group with higher expression values in the S1.0 treated tumors compared to the controls is plotted.

[0104] Histopathology analysis. After euthanasia, representative samples of the tumor mass from each mouse were collected and fixed by immersion in 10% neutral buffered formalin at room temperature for 24 hours and then stored in 70% ethanol before embedding in paraffin, sectioning at 5 μm and staining with hematoxylin and eosin (H&E). Bright field whole slide images of H&E- stained tissue sections were captured as digital files by scanning at 20 × using a 3DHistech Panoramic SCAN II FL scanner (Budapest, Hungary). The scanned images were evaluated by a board-certified veterinary pathologist in a blinded manner and scored on a scale from 0 = normal to 4 = extensive for local tissue invasion and abundance of lymphocytes, neutrophils, macrophages and necrosis. Statistical analysis of the histopathology ordinal data was performed by using the GraphPad Prism software (Dotmatics, https: / / www.graphpad.com).

[0105] scRNA-seq analysis. The scRNA-seq analysis was performed on tumor tissues derived from B16-Ova melanoma tumor-bearing wild-type C57BL / 6 mice (n=5 per treatment group). The tumor was dissociated by using MACS tumor dissociation kit (Miltenyi biotec) and viable single cell suspension were analysed and run on 10× Genomics platform. Raw sequencing reads from single- cell RNA sequencing (scRNA-seq) data were processed using the Cell Ranger 7.1.0 pipeline (10x Genomics). Reads were aligned to the mm10 reference genome, and gene expression matrices were generated. All groups were pooled together and then QC filtered and preprocessed together to control bias. scRNA-seq data analysis was performed on R using the Seurat v4. Cells with < 500 reads and expressing > 15 percent mitochondrial genes expression were filtered out. Genes expressing in < 3 cells were filtered out. The filtered data was then library size normalized and scaled to 10000 total, followed by log(X+1) transformation suing the ‘NormalizeData’ function. The top 2000 highly variable genes (HVG) were selected using ‘FindVariableFeatures’ function for principal componentanalysis (PCA) was performed using the 2000 HVGs. Uniform Manifold Approximation and Projection (UMAP) was performed using the top 15 principal components to project the data onto a two-dimensional space for visualization. Cells were then clustered using the Louvain algorithm with a resolution of 0.8 and 22 cell clusters were identified.

[0106] Annotation. Significant marker genes for each cluster were identified by performing differential analysis using the Wilcoxon rank sum test using the ‘FindAllMarkers’ function. Each individual cluster was annotated using the marker genes and canonical cell type markers from PanglaoDB.

[0107] Differential expression, enrichment analysis and cell scoring. Differentially expressed gene (DEG) analysis was performed using the Wilcoxon rank-sum test for each cell type and across all four treatment conditions. Significant DEGs were selected by an adjusted P value cut-off of 0.05. Top 100 DEGs for each cell type were used for gene list enrichment analysis with the KEGG_2019_Mouse database using Enrichr. Cell scores for gene sets were calculated using the AUCell for gene sets. Gene sets obtained from the KEGG Mouse Pathway Database and T-cell exhaustion markers were retrieved. Gene expression and cell proportion were visualized in R using Seuratv4’s inbuilt plotting functions, ggplot2 and dittoSeq.

[0108] Orthotopic tumor model and treatment with S1.0 and antibodies to co-inhibitory molecules. Six-week-old female albino C57BL / 6 were obtained from The Jackson Laboratory (Bar Harbor, ME). Orthotopic pancreatic ductal adenocarcinoma was established by surgically implanting KrasG12D; P53flox / flox; PDX-1-Cre; Luciferase [KPC-Luc] cells into the pancreas of 7-8 week old mice (n=10 mice / group) as described previously. Establishment of tumors was confirmed 7 days post- surgical implantation using IVIS (in vivo imaging system) bioluminescence imaging (IVIS Spectrum, Revvity, Waltham, MA) and mice were randomized for different treatment groups. Tumor bearing mice were treated with either S1.0 (5.0 × 107cfu / mouse) administered intravenously or 200 µg / mouse of anti-PD1 (^PD-1, clone: RMP1-14, BioXCell, Lebanon, NH) intraperitoneally (IP), or a combination of ^PD-1 and S1.0. Control mice received manufacture recommended rat Ig2a isotype control antibody (BioXCell, Lebanon, NH). Mice were treated with ^PD-1 or isotype antibody, IP, on days 8, 10, 13, 19, 22, and 25 (FIG. 4R). IVIS bioluminescence imaging with 150 mg / kg body weight (BW) of D-Luciferin (Promega) administered IP was used to monitor tumor growth two times a week for survival studies. To quantify tumor growth, regions of interest were manually drawn around tumor borders using Living Image software (Living Image 4.8.0, Revvity Health Sciences Inc., USA) and average tumor radiance (p / s / cm2 / sr) was determined. For determining the infiltrationof immune cells in the tumor with different treatments, mice were sacrificed on days 15 PTCI (FIG. 4R), tumor tissues and spleen were collected, and single-cell suspensions were prepared for flow cytometry analysis. All animal procedures were reviewed and approved by the MD Anderson Cancer Center Animal Care and Use Committee.

[0109] Fluorescent cytometric analysis. Immune cells isolated from tumor and spleen were stained with a panel of anti-mouse antibodies that included anti-CD45-violetFluorTM(104, Cytek Biosciences, CA, US), anti-CD3-APC / Cy7 (17A2), anti-CD4-Alexa Fluor 700 (RM4-4), anti- CD8a-BV570TM(53-6.7), anti-CD19-PE / DazzleTM594 (6D5), anti-CD11c-APC (N418), anti- CD11b- BV750TM(M1 / 70), anti-CD163-APC / FireTM810 (S15409I), anti-NK1.1-BV785TM(PK136), anti-FoxP3-Pacific BlueTM(MF-14) anti-CD25- BV605TM(PC61), anti-CD69- PerCP / Cy5.5 (H1.2F3), anti-Ly6C-PE / Cy7 (HK1.4), anti-Ly6G- BV650TM(1A8), anti-PD-1- BV421TM(RMP1-30), anti-MHC-II- cFluor® BYG750 (M5 / 114.15.2, Cytek Biosciences, CA, US), anti-F4 / 80- Alexa Fluor 647 (QA17A29), anti-Zap70- PE (1E7.2) anti-Pax5-Alex Fluor 488 (1H9), and anti-FcgRIV- BV711TM(9E9), which were purchased from Biolegend (San Diego, CA, USA) unless otherwise indicated. One to two million cells were stained in a volume of 100^^l and the concentration of each antibody in the cocktail was 0.05 ^g / sample. The data was acquired on Cytek Northern Lights spectral flow cytometer (Cytek Biosciences, CA, US) and analyzed using FlowJo version 10.10 (Tree Star Inc. OR, USA).

[0110] Quantitative real-Time (qRT-PCR) validation. Total RNA was isolated from fresh control and S1.0-treated and B16-Ova melanoma tumor tissue homogenate using Trizol (Invitrogen cat no 15596026) and reverse transcribed into cDNA using the Superscript™ III First-Strand Synthesis system (Invitrogen cat no 18091050) as reported previously. Gene expression was determined by qRT-PCR using the CFX duet Real-Time PCR Detection System (Bio-Rad, USA) and SYBR Green (Invitrogen cat no 11761100). The RT-PCR was performed to determine the listed genes using the mouse-specific primers. Melt curves were generated, and fold expression was calculated by using the 2−∆∆Ctmethod, which was normalized using β-actin as the endogenous control.

[0111] Amino acid uptake assay. The tumors from various experimental groups were weighed, chopped, and placed into gentleMACS M Tubes (Miltenyi Biotec, cat no: 130-093-236). A mixture of methanol, acetonitrile, and water (5:3:2, v / v / v) was added at a volume equivalent to five times the tumor weight, and the samples were homogenized using the gentleMACSTMDissociator (Miltenyi Biotec). The homogenate was then transferred to 1.5 ml Eppendorf tubes and centrifuged at 13,000 × g for 15 minutes. The resulting supernatant was collected in glass vials and dried under agentle stream of nitrogen gas. The dried extracts were reconstituted in 40 μl of water / acetonitrile (8 / 2, v / v), and 10 μl of the solution was transferred to a glass autosampler vial, followed by the addition of 35 μl sodium borate buffer (100 mM, pH 9.0). Subsequently, 10 μl of 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate (AQC, 10 mM in acetonitrile) derivatizing reagent (Cayman Chemical, MI, USA) was added. The vials were tightly capped, vortexed, and incubated at 55 °C for 15 minutes. After cooling to room temperature, 1 μL of the derivatized sample was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). LC separations were performed using an ACQUITY Premier UPLC System (Waters Corporation, MA USA) equipped with a C18ACQUITY UPLC BEH Column (2.1 mm × 150 mm, 1.7 μm, 130 Å, part no: 186002353) operated at 50 °C. The mobile phase consisted of water with 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B), with a non-linear gradient program as follows: 0−2 min, 3% B; 2−8 min, 3–40% B; 8−8.1 min, 96% B; 8.1−12 min, 96% B; 12−12.5 min, 3% B; 12.5−14 min, 3% B. The flow rate was 500 μL / min, and the injection volume was 1 μL. Detection was performed using a Xevo-XS Triple Quadrupole Mass Spectrometer (TQ-XS) (Waters Corporation, MA USA) operated in positive ion mode with multiple reaction monitoring (MRM) mass spectrometry.

[0112] Statistical Analysis and reproducibility. The statistical differences between two groups and among groups were determined using t-test and one-way ANOVA, respectively. Tumor growth curves were compared using a two-way ANOVA. Tukey correction was used for multiple comparisons test. Statistical significance for survival analysis was analyzed using the Kaplan–Meier method and compared among groups using the Wilcoxon and log-rank test when indicated. All tests were two-tailed with a considered significance level of p< 0.05. All the statistical analysis were calculated using Graphpad Prism version10 (GraphPad Software). Graphs show the mean ± standard error of the means (error bars) are shown unless otherwise indicated. All in vitro experiments were independently repeated at least three times. All in vivo mouse experiments were conducted with 5 mice per group (n=5 / group) unless otherwise indicated and were repeated at least twice. The in vivo goat safety experiment was performed with groups of pregnant does (n=7 / group) for each of the two doses. All the in vitro and in vivo independently repeated experiments produced consistent or similar results.

[0113] Other objects, features and advantages of the disclosure will become apparent from the foregoing figures, detailed description, and examples. It should be understood, however, that the figures, detailed description, and examples, while indicating specific embodiments of the disclosure, are given by way of illustration only and are not meant to be limiting. Additionally, it is contemplatedthat changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from the detailed description. In further embodiments, features from specific embodiments may be combined with features from other embodiments. For example, features from one embodiment may be combined with features from any of the other embodiments. In further embodiments, additional features may be added to the specific embodiments described herein.

Claims

CLAIMS What is claimed is:

1. A pharmaceutical composition comprising an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole.

2. The pharmaceutical composition of claim 1, wherein the attenuated bacterial strain of Brucella melitensis contains a mutation in one or more of vjbR, asp14, or mucR virulence genes.

3. The pharmaceutical composition of claim 1, wherein the attenuated bacterial strain of Brucella melitensis contains a deletion of one or more of vjbR, asp14, or mucR virulence genes.

4. The pharmaceutical composition of claim 1, wherein the attenuated bacterial strain of Brucella melitensis contains a first gene encoding for tryptophanase and a second gene encoding for toluene-4 monooxygenase.

5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a second therapeutic agent.

6. The pharmaceutical composition of claim 5, wherein the second therapeutic agent is an anti- cancer therapeutic agent.

7. The pharmaceutical composition of claim 5, wherein the second therapeutic agent is an auto- immune therapeutic agent.

8. The pharmaceutical composition of claim 5, wherein the second therapeutic agent is an anti- inflammatory therapeutic agent.

9. A method of treating disease in a patient, the method comprising the step of administering to the patient a pharmaceutical composition comprising an attenuated bacterial strain of Brucella melitensis engineered to produce 5-hydroxyindole.

10. The method of claim 9, wherein the attenuated bacterial strain of Brucella melitensis contains a deletion of one or more of vjbR, asp14, or mucR virulence genes.

11. The method of claim 9, wherein the attenuated bacterial strain of Brucella melitensis contains a first gene encoding for tryptophanase and a second gene encoding for toluene-4 monooxygenase.

12. A method of treating disease in a patient, the method comprising the step of administering a pharmaceutical composition comprising an attenuated bacterial strain of Brucella melitensis expressing an immunomodulatory factor to the patient.

13. The method of claim 12, wherein the immunomodulatory factor is one or more of a nanobody that interacts with and stimulates CCR7, a nanobody that interacts with and antagonizes an immunosuppressive protein, a cancer-associated antigen, and a neoantigen.

14. The method of claim 12, wherein the immunomodulatory factor is one or more of Hormad1, Mesothelin, NY-SEO-1, VGLL1, MAGE-A4, MUC1, WT1, and PRAME.

15. The method of claim 12, further comprising administering 5-hydroxyindole.

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