Genetically engineered immunostimulatory bacterial strains and uses thereof

Abstract

To provide immunostimulatory bacteria and pharmaceutical compositions containing the bacteria.SOLUTION: The immunostimulatory bacteria provided herein contain one or more modalities that enhance the anti-tumor activity of the immunostimulatory bacteria. Among the immunostimulatory bacteria, provided herein are bacteria, such as Salmonella species, which are modified to be auxotrophic or are auxotrophic for adenosine and / or to contain plasmids encoding RNAi, such as shRNA and microRNA, that mediate gene disruption and / or expression of immune checkpoints, such as TREX1, VISTA and PD-L1, and genes that influence the immune system. The bacteria contain additional modifications to enhance their anti-tumor activity. Also provided are methods of inhibiting the growth or reducing the volume of a solid tumor by administering the pharmaceutical compositions.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications Benefit of priority is claimed to U.S. Provisional Patent Application No. 62 / 531,327, filed July 11, 2017, to Christopher D. Thanos and Laura Hix Glickman, entitled "ENGINEERED IMMUNOSTIMULATORY BACTERIAL STRAINS AND USES THEREOF." Benefit of priority is also claimed to U.S. Provisional Patent Application No. 62 / 648,380, filed March 26, 2018, to Christopher D. Thanos, Laura Hix Glickman and Justin Skoble, entitled "ENGINEERED IMMUNOSTIMULATORY BACTERIAL STRAINS AND USES THEREOF."

[0002] This application is related to U.S. application Ser. No. (Attorney Docket No. 62131.01701; U.S. Patent Application Publication No. 03 / 1701), filed on even date herewith, entitled "ENGINEERED IMMUNOSTIMULATORY BACTERIAL STRAINS AND USES THEREOF," which claims priority to U.S. Provisional Patent Application Ser. Nos. 62 / 531,327 and 62 / 648,380.

[0003] Where possible, the subject matter of each of these applications is incorporated by reference in its entirety.

[0004] INCORPORATION-BY-REFERENCE OF SEQUENCE LISTINGS PROVIDED ELECTRONICALLY An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic application was created on July 11, 2018, is 410 kilobytes in size, and is titled 1701SEQPC001.txt. [Background technology]

[0005] The field of cancer immunotherapy has made great progress, as evidenced by the clinical success of anti-CTLA4, anti-PD-1, and anti-PD-L1 immune checkpoint antibodies (see, e.g., Buchbinder et al. (2015) J. Clin. Invest. 125: 3377-3383; Hodi et al. (2015) J. Clin. Invest. 125: 3392-4000; and Chen et al. (2015) J. Clin. Invest. 125: 3384-3391). Tumors have evolved a complete immunosuppressive environment, which leads to multiple mechanisms for evading immune surveillance, reprogramming antitumor immune cells to suppress immunity, and persistent mutational resistance to current cancer therapies (see, e.g., Mahoney et al. (2015) Nat. Rev. Drug Discov. 14(8): 561-584). Designing immunotherapies that overcome immune tolerance and escape while simultaneously limiting the autoimmune-related toxicities of current immunotherapies presents a challenge to the field of immuno-oncology. Therefore, additional innovative immunotherapies and other treatments are needed. Summary of the Invention

[0006] Bacteria for anti-cancer therapy are provided that are modified to be immunostimulatory. The immunostimulatory bacteria provided herein offer a multifaceted approach to anti-tumor therapy. The bacteria provided herein, such as Salmonella species, can be fine-tuned to have potent anti-tumor activity. Bacteria provide a platform with many avenues for eliciting anti-tumor immunostimulatory activity. The bacteria contain plasmids encoding anti-cancer therapies such as RNAs, including microRNAs, shRNAs, and siRNAs, designed to suppress, inhibit, block, or otherwise silence immune checkpoint genes and products, as well as other targets. These targets play a role in immunosuppressive and immunostimulatory pathways, improving anti-tumor responses, such as stimulator of interferon genes (STING) and cGAS. Bacteria inherently stimulate the immune system, and bacterial infection induces immune and inflammatory pathways and responses, some of which are desirable for anti-tumor treatment and others that are undesirable. Modifying bacteria by deleting or modifying genes and products that cause undesired inflammatory responses and genes that induce desirable immunostimulatory anti-tumor responses can improve the anti-tumor activity of bacteria. Bacteria can also accumulate in tumor cells and tissues, replicating therein and lysing the cells. Bacteria can migrate from the site of administration and accumulate in other tumors and tumor cells, exerting an abscopal effect. All of these bacterial properties are utilized herein to ensure the production of immunostimulatory bacteria with multiple anti-tumor activities and properties that can act synergistically.

[0007] Compositions, uses thereof, and methods for modulating immune responses to treat diseases, such as cancer, are provided. The compositions include the immunostimulatory bacteria provided herein. Methods for treatment and uses of the bacteria for treatment are also provided. Subjects for treatment include humans and other primates, pets such as dogs and cats, and other animals such as horses.

[0008] Pharmaceutical compositions comprising the immunostimulatory bacteria and methods and uses thereof for treating diseases and disorders, particularly proliferative disorders such as tumors, including solid tumors, are provided.

[0009] Also provided are methods for inhibiting the growth or reducing the volume of solid tumors by administering the immunostimulatory bacteria or pharmaceutical compositions or using compositions for treatment, for example, by administering or using a composition comprising an effective amount, in a single dosage, of an attenuated Salmonella spp. to a human patient with a solid tumor cancer.

[0010] It should be understood that the RNAi and modification of bacteria and all of the plasmids described can be combined in any desired combination.Therefore, reference to immunostimulatory bacteria refers to the bacteria that comprises the RNAi of at least one target, and can have any or all of the modifications described herein.

[0011] The immunostimulatory bacterium comprises an aspartate-semialdehyde dehydrogenase (ASD) gene encoding an RNA (RNAi) that inhibits, suppresses, or blocks expression of an immune checkpoint or other target, wherein the inhibition, suppression, or blockage enhances an anti-tumor immune response in a subject; the RNA is encoded on a plasmid in the immunostimulatory bacterium; and - (asd - ) is provided.

[0012] For the purposes herein, RNAi includes all forms of double-stranded RNA, and can be used to silence the expression of target nucleic acid.RNAi includes shRNA, siRNA and microRNA.Any of these forms can be interchanged in the embodiments disclosed and described herein.Generally, RNAi is encoded on a plasmid in bacteria.Plasmids can contain other heterologous nucleic acids that code for the desired product that modulates or adds activity, or products to bacteria, or other such products that can modulate the immune system of the subject that will be treated with bacteria.Bacterial genes can also be added, deleted or destroyed.These genes can also code for the product that allows bacteria to grow and reproduce, or for the product that modulates the host's immune response to bacteria.

[0013] Also provided are immunostimulatory bacteria that are adenosine auxotrophic and comprise a sequence of nucleotides encoding an RNA (RNAi) that inhibits, suppresses, or blocks the expression of 3' repair exonuclease 1 (TREX1). Also provided are immunostimulatory bacteria that are adenosine auxotrophic and comprise a sequence of nucleotides encoding an RNA that inhibits, suppresses, or blocks the expression of VISTA (a gene encoding a V-domain Ig suppressor of T cell activation). Also provided are immunostimulatory bacteria that are adenosine auxotrophic and comprise a sequence of nucleotides encoding an RNA that inhibits, suppresses, or blocks the expression of programmed death ligand 1 (PD-L1).

[0014] Among these immunostimulatory bacteria are those of the genus Salmonella, including those Salmonella that contain nucleic acids encoding RNAs that inhibit, suppress, block, or silence the expression of 3' repair exonuclease 1 (TREX1) and / or VISTA.

[0015] Also provided are immunostimulatory bacteria comprising a sequence of nucleotides encoding an RNA that inhibits, suppresses or blocks the expression of 3' repair exonuclease 1 (TREX1) and a sequence of nucleotides encoding an RNA that inhibits, suppresses or blocks the expression of PD-L1.

[0016] Also provided are immunostimulatory bacteria comprising a sequence of nucleotides encoding an RNA that inhibits, suppresses or blocks the expression of VISTA, and a sequence of nucleotides encoding an RNA that inhibits, suppresses or blocks the expression of PD-L1.

[0017] Immunostimulatory bacteria such as S. typhimurium are provided that carry a plasmid encoding an RNAi, such as an miRNA or shRNA, that mediates gene blockade of one or more of TREX1, VISTSA, and PD-L1, as well as other such targets known to those skilled in the art and / or listed or exemplified herein. Bacterial species that carry such plasmids include, but are not limited to, strains of Salmonella, Shigella, Listeria, E. coli, and Bifidobacteria. For example, species include Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, and Salmonella enteritidis.

[0018] Examples of bacterial species include Salmonella, Shigella, E. coli, Bifidobacterium, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, Chlamydia, and Hemophilus influenzae. and strains of the genera Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, and Erysipelothrix, or attenuated or modified strains thereof of any of the preceding lists of bacterial strains.

[0019] Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix. For example, Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus sornnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelaemustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, and Agrobacterium tumerfacium.

[0020] Exemplary strains of Salmonella are exemplified herein, particularly Salmonella typhimurium strains such as the strain designated YS1646 (ATCC #202165) or VNP20009. Other strains include RE88, SL7207, χ8429, χ8431, and χ8468. Exemplary modified Salmonella strains provided herein are the immunostimulatory bacterial strains AST-104, AST-105, AST-106, AST-108, AST-109, AST-112, AST-113, AST-115, AST-117, AST-118, AST-119, AST-120, AST-121, AST-122, and AST-123. Sequences and descriptions of these are provided in the Detailed Description, Examples, and Sequence Listing. The immunostimulatory bacteria can be derived from an attenuated strain of bacteria or can be made attenuated based on the modifications described herein, such as deletion of asd, so that replication is restricted in vivo.

[0021] The immunostimulatory bacteria provided herein encode various genes and / or inhibitors of the expression of genes and / or gene products that contribute to a reduced anti-tumor immune response and / or products that stimulate the immune system, and are therefore immunostimulatory. The inhibition of TREX1 described herein is immunostimulatory because it inhibits PD-L1. Adenosine auxotrophy is also immunostimulatory. Inhibitory RNAs (RNAi), such as shRNAs, microRNAs, or siRNAs, targeted to block or inhibit the expression of TREX1, PD-L1, VISTA (a gene encoding a V-domain Ig suppressor of T cell activation), TGF-beta, and CTNNB1 (a gene encoding β-catenin), among others, or combinations thereof, as well as any shRNAs that inhibit or block the expression of other immunosuppressive genes whose expression is activated or enhanced by the tumor or tumor microenvironment (TME) are provided. Expression of these RNAs utilizes two independent immunostimulatory pathways, resulting in enhanced tumor colonization in a single therapy. The effect of this combination is enhanced by the strains provided herein that are adenosine auxotrophic, providing preferential accumulation in or recruitment into an adenosine-rich immunosuppressive tumor microenvironment. Depletion of adenosine in such a TME further enhances the immunostimulatory effect. Such combinations of features in any of the bacterial strains known or that can be genetically engineered for therapeutic administration provide similar immunostimulatory effects.

[0022] One of the targets is TGF-beta, which has three isoforms: 1, 2, and 3. One of the targets is TGF-beta, specifically isoform 1, but not isoforms 2 and 3. Toxicity is associated with isoforms 2 and 3. For example, cardiac valve toxicity is associated with the inhibition of isoform 2. Isoform 1 is present in most cancers (see, for example, the TCGA database). It is advantageous to inhibit only isoform 1. RNAi can be advantageously used for this purpose, since it can be designed to recognize targets very specifically. With regard to TGF-beta, specific inhibition of isoform 1 can be achieved by targeting a sequence that is unique to isoform 1 and not present in isoforms 2 or 3 (see, for example, the RNA for TGF-beta isoform 1 in Example 2), or by selecting a sequence that targets isoforms 1 and 3, but not 2. Immunostimulatory bacteria are also provided in which the plasmid encodes an shRNA or microRNA that specifically inhibits, suppresses or blocks expression of TGF-beta isoform 1, but not expression of TGF-beta isoform 2 or TGF-beta isoform 3; or in which the plasmid encodes an shRNA or microRNA that specifically inhibits, suppresses or blocks expression of TGF-beta isoforms 1 and 3, but not expression of isoform 2.

[0023] In addition, RNAi of PD-L1 provided by the immunostimulatory bacteria provided herein, such as miRNA or shRNA-mediated gene blockade, also improves colonization.Knockout of PD-L1 has been shown to promote S. typhimurium infection.For example, it has been observed that PD-L1 knockout mice have a bacterial load at least 10 times higher than wild-type mice, and PD-L1 is protective against S. typhimurium infection (see, for example, Lee et al. (2010) Immunol. 185:2442-2449).

[0024] The engineered immunostimulatory bacteria provided herein, such as S. typhimurium immunostimulatory bacteria, include multiple synergistic modalities to induce cold tumor immune reactivation and promote tumor antigen-specific immune responses while simultaneously inhibiting immune checkpoint pathways that tumors exploit to subvert and evade persistent anti-tumor immunity. Adenosine auxotrophy and enhanced vascular destruction are included in embodiments. This improvement in tumor targeting and enhanced vascular destruction via adenosine auxotrophy enhances potency while simultaneously localizing inflammation and limiting systemic cytokine exposure and autoimmune toxicity observed with other immunotherapy modalities.

[0025] Immunostimulatory bacteria are provided that target the TREX1 gene, such as those that are adenosine auxotrophic and / or encode double-stranded RNA, such as shRNA or miRNA, that inhibit its expression, and that may encode additional RNA, such as miRNA or shRNA, that targets and inhibits the expression of other checkpoint inhibitors. Among these bacteria are immunostimulatory bacteria that are adenosine auxotrophic. Methods and uses for treating tumors, including solid tumors and hematological malignancies, are provided. Among the methods and uses, the immunostimulatory bacteria are adenosine auxotrophic, and the uses and treatments involve targeting cd73 + and / or cd73 + / cd39 + Treat tumors.

[0026] The RNA is expressed under the control of a promoter recognized by the eukaryotic host cell transcription machinery, such as an RNA polymerase II (RNAP II) and RNA polymerase III (RNAP III) promoter. The RNAP III promoter is generally constitutively expressed in eukaryotic hosts; it may regulate the RNAP II promoter. The RNA, such as miRNA and shRNA, is provided on a plasmid that is stably expressed by the bacterium. Exemplary of such a bacterium is a globally attenuated Salmonella strain, such as an adenosine auxotroph, that is attenuated by passage or other methods or based on the modifications described herein. Exemplary of a bacterium is a Salmonella strain. Exemplary of a Salmonella strain is a modified S. typhimurium strain that contains an asd mutation to select for antibiotic non-use. These strains may also contain an asd mutation.

[0027] Promoters that are expressed in the tumor microenvironment (TME), such as promoters that are expressed in hypoxic conditions or in conditions where the pH is less than 7, can be selected for the environment of the tumor cells.

[0028] Bacterial strains are provided that contain miRNA or shRNA against the TREX1 or VISTA gene. The TREX1 or VISTA gene can be under the control of an RNAP III promoter, such as the H1 promoter. Knocking down TREX1 induces vascular destruction, which increases colonization and reduces immunosuppression. The bacterial strains provided herein can contain miRNA or shRNA that inhibit the expression of other checkpoint inhibitors, including, but not limited to, PD-L1. Strains containing multiple RNAs, such as miRNAs or shRNAs, generally contain different promoters for each RNA. For example, the bacteria can be genetically modified S. typhimurium strains that contain miRNA or shRNA against the PD-L1 gene under the control of the U6 promoter and miRNA or shRNA against TREX1 under the control of the H1 promoter. Genetically modified S. typhimurium strains that contain miRNA or shRNA against the SIRP-α gene under the control of the H1 promoter are also provided. An exemplary bacterium, such as an S. typhimurium strain, may include an miRNA or shRNA against the β-catenin gene under the control of an RNAP III promoter, such as the H1 promoter, and / or an miRNA or shRNA against the VISTA gene under the control of an RNAP III promoter, such as the H1 promoter. Various combinations of miRNAs or shRNAs, which may be directed against other immune checkpoint targets, such as adenosine auxotrophy, miRNAs or shRNAs against TREX1, and / or RNAs that inhibit, suppress, or block PD-L1 or one or both of TREX1 and PD-1 or VISTA, may be included in the engineered immunostimulatory bacterium.

[0029] Immunostimulatory bacteria that are cGAS agonists are provided. An exemplary example of such bacteria is S. typhimurium, which is a cGAS agonist and / or a stimulator of interferon genes (STING) agonist. These can be administered in applications and methods such as radiation therapy and chemotherapy in which cytosolic DNA is produced or accumulated. STING activates innate immunity in response to sensing nucleic acids in the cytosol. Downstream signaling is activated through the binding of cyclic dinucleotides (CDNs) synthesized by bacteria or by the host enzyme cGAS in response to binding to cytosolic dsDNA. Bacterial and host-produced CDNs have different phosphate bridge structures, which distinguish their ability to activate STING. CDNs are synthesized by bacteria or by the host enzyme cGAS in response to binding to cytosolic dsDNA. IFN-β is the signature cytokine of active STING.

[0030] The plasmids in any of the bacteria described and listed above and herein include plasmids encoding RNAi and other heterologous nucleic acids. Plasmids can exist as many or few copies. This can be controlled by selecting elements such as replication origins. Low, high, and medium copy number plasmids and replication origins are well known to those skilled in the art and can be selected. In the immunostimulatory bacteria embodiments herein, plasmids can exist at low to medium copy numbers, such as about 150 or less copies, to low copy numbers, such as less than about 25 or about 20 or 25 copies. Exemplary origins are those derived from pBR322, p15A, pSC101, pMB1, colE1, colE2, pPS10, R6K, R1, RK2, and pUC.

[0031] Contemplated plasmids can include RNAi constructs that inhibit, suppress, or block expression of immune checkpoints or other targets and their products, such as nucleic acid sequences encoding the listeriolysin O (LLO) protein (cytoLLO) without a signal sequence, CpG motifs, DNA nuclear targeting sequences (DTS), deletions of genes encoding flagellin subunit(s), and retinoic acid-inducible gene-I (RIG-I) binding elements.

[0032] The immunostimulatory bacteria provided herein express aspartate-semialdehyde dehydrogenase, which, when administered to a subject for treatment, allows growth in DAP-supplemented media but limits replication in vivo. - (asd - Such bacteria may be self-limiting and may be advantageous for treatment. The bacteria may be aspartate-semialdehyde dehydrogenase (asd) because all or part of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (asd) is disrupted or deleted, resulting in the endogenous asd not being expressed. - In other embodiments, the gene encoding asd may be contained on a plasmid for expression in vivo.

[0033] Any of the immunostimulatory bacteria provided herein can generally contain a nucleic acid containing a CpG motif or CpG island on a plasmid, where the motif is recognized by Toll-like receptor 9 (TLR9). Nucleic acids encoding CpG motifs or islands are abundant in prokaryotes, and therefore the CpG motif can be included in or be part of a bacterial gene and encoded on a plasmid. The bacterial gene encoding asd contains an immunostimulatory CpG.

[0034] The immunostimulatory bacteria provided herein can be auxotrophic for adenosine, or adenosine and adenine. Any of the bacteria herein can be adenosine trophic, which can advantageously increase anti-tumor activity because adenosine accumulates in many tumors and is immunosuppressive.

[0035] Where wild-type bacteria contain flagella, the immunostimulatory bacteria provided herein can be flagellin-deficient. These can be made flagellin-deficient by disrupting or deleting all or part of the gene(s) encoding flagella. For example, immunostimulatory bacteria are provided that have a deletion of the gene encoding one or both of the flagellin subunits fliC and fljB, thereby rendering the bacteria flagella-deficient.

[0036] The immunostimulatory bacteria provided herein can contain a nucleic acid encoding a listeriolysin O (LLO) protein, cytoLLO, that lacks a periplasmic secretion signal sequence, resulting in accumulation in the cytoplasm. This mutation is expressed as asd - LLO is advantageously combined with bacteria. LLO is a cholesterol-dependent pore-forming hemolysin derived from Listeria monocytogenes that mediates bacterial phagocytic escape. When autolytic strains are introduced into tumor-bearing hosts, such as humans, the bacteria are engulfed by phagocytic immune cells and enter the vacuole. In this environment, the absence of DAP prevents bacterial replication and leads to bacterial autolysis within the vacuole. Subsequent lysis releases the plasmid, and accumulated LLO forms pores in the cholesterol-containing vacuolar membrane, allowing delivery of the plasmid into the host cell cytosol.

[0037] The immunostimulatory bacteria can contain a DNA nuclear targeting sequence (DTS), such as the SV40 DTS, encoded on a plasmid.

[0038] The immunostimulatory bacteria can have a deletion or modification of the gene encoding endonuclease-1 (endA) such that endA activity is inhibited or eliminated. Exemplary of these are immunostimulatory bacteria that contain one or more of a CpG motif, an asd gene selection marker for plasmid maintenance, and a DNA nuclear targeting sequence.

[0039] The immunostimulatory bacteria can contain nucleic acids on a plasmid encoding two or more different RNA molecules that inhibit, suppress or block expression of an immune checkpoint, or RNA molecules that encode inhibitors of a metabolite that is immunosuppressive or in an immunosuppressive pathway.

[0040] The nucleic acid encoding an RNAi such as shRNA, miRNA or siRNA can include a transcription terminator downstream of the nucleic acid encoding the RNA.

[0041] In all embodiments, the RNAi encoded on a plasmid in the immunostimulatory bacteria can be a short hairpin RNA (shRNA) or a microRNA (miRNA).

[0042] Immunostimulatory agents include RNAi that inhibit, suppress, block, or silence the expression of immune checkpoints and other targets whose inhibition, blockage, or silencing is immunostimulatory, including 3' repair exonuclease 1 (TREX1), PD-1, PD-L1 (B7-H1), VEGF, TGF-beta isoform 1, beta-catenin, CTLA-4, PD-L2, PD-1, PD-2, IDO1, IDO2, SIRPα, CD47, VISTA (B7-H5), LIGHT, HVEM, CD28, LAG3, TIM3, TIGIT, galectin-9, CEACAM1, CD155, CD112, CD226, CD244 (2B4), B7-H2, and B7-H3. , ICOS, GITR, B7-H4, B7-H6, CD27, CD40 / CD40L, CD48, CD70, CD80, CD86, CD137 (4-1BB), CD200, CD272 (BTLA), CD160, CD39, CD73, A2a receptor, A2b receptor, HHLA2, ILT-2, ILT-4, gp49B, PIR-B, HLA-G, ILT-2 / 4, OX40 / OX-40L, BTLA, KIR, TIM1, TIM4 and STAT3, stabilin-1 (CLEVER-1), DNASE II and RNASE H2. For example, any of the immunostimulatory bacteria may contain RNA that inhibits, suppresses, or blocks expression of one or a combination of TREX1, PD-L1, VISTA, TGF-beta such as TGF-beta isoform 1 or isoforms 1 and 3, beta-catenin, SIRP-alpha, VEGF, RNASE H2, DNASE II, and CLEVER-1 / stabilin-1.

[0043] Immunostimulatory bacteria are provided in which the plasmid comprises a sequence of nucleotides encoding RNAs that inhibit, suppress, or block the expression of at least two targets, each RNA being expressed from a different promoter, exemplified by combinations of targets for inhibition, suppression, or blocking that are at least two selected from TREX1 and PD-L1, TREX1 and PD-1, TREX1 and VISTA, TREX1 and SIRP-alpha, PD-L1 and TGF-beta isoform 1, PD-L1 and beta-catenin, PD-L1 and VISTA, TGF-beta isoform 1 and VISTA, SIRP-alpha and VISTA, and TREX1 and RNASE H2.

[0044] Other combinations of RNAi include RNAi that inhibit, suppress or block expression of one or a combination of TREX1, PD-L1, VISTA, TGF-beta isoform 1, beta-catenin, SIRP-alpha, VEGF, RNASE H2, DNASE II, and CLEVER-1 / stabilin-1. Other combinations include those where the targets for inhibition, suppression or blocking are a combination of at least two selected from TREX1 and PD-L1, TREX1 and PD-1, TREX1 and VISTA, TREX1 and SIRP-alpha, PD-L1 and TGF-beta isoform 1, PD-L1 and beta-catenin, PD-L1 and VISTA, TGF-beta isoform 1 and VISTA, SIRP-alpha and VISTA, TREX1 and RNASE H2, VISTA and RNASE H2, and VISTA and DNASEH2, or TREX1 and SIRPα, or TREX1 and VISTA, or TREX1 and VEGF, or PD-L1 and beta-catenin, or PD-L1 and TGF-beta isoform 1, or PD-L1 and VEGF, or TREX and PD-1.

[0045] The immunostimulatory bacteria include CTLA-4, PD-L1 (B7-H1), PD-L2, PD-1, PD-2, IDO1, IDO2, SIRPα, CD47, VISTA (B7-H5), VEGF, TGF-beta, LIGHT, HVEM, CD28, LAG3, TIM3, TIGIT, galectin-9, CEACAM1, CD155, CD112, CD226, CD244(2B4), B7-H2, B7-H3, ICOS, GITR, B7-H4, It can also include nucleic acids encoding RNA that inhibit, suppress or block expression of another different immune checkpoint or target to be inhibited, suppressed or blocked selected from among any of B7-H6, CD27, CD40 / CD40L, CD48, CD70, CD80, CD86, CD137(4-1BB), CD200, CD272(BTLA), CD160, CD39, CD73, A2a receptor, A2b receptor, HHLA2, ILT-2, ILT-4, gp49B, PIR-B, HLA-G, ILT-2 / 4, OX40 / OX-40L, BTLA, KIR, TIM1, TIM4, STAT3, CLEVER-1, DNASE II and RNASE-H2. Exemplary of these are one of human PD-L1 (SEQ ID NO: 31), human beta-catenin (SEQ ID NO: 32), human SIRPα (SEQ ID NO: 33), human TREX1 (SEQ ID NO: 34), human VISTA (SEQ ID NO: 35), human TGF-beta isoform 1 (SEQ ID NO: 193), and human VEGF (SEQ ID NO: 194). The RNA can target or include a sequence in an immune checkpoint nucleic acid set forth in any of SEQ ID NOs: 1-30, 36-40, and 195-217.

[0046] The plasmid in any of the immunostimulatory bacteria can also encode a sequence of nucleotides that is an agonist of retinoic acid-inducible gene I (RIG-I) or a RIG-I binding element.

[0047] The immunostimulatory bacteria can comprise one or more deletions in genes, such as one or more of purI (purM), msbB, purD, flagellin (fliC / fljB), pagP, adrA, CsgD, and hilA. The immunostimulatory bacteria can be msbB. For example, the immunostimulatory bacteria can comprise a purI deletion, an msbB deletion, an asd deletion, and an adrA deletion, and can also comprise a CsgD deletion. Exemplary bacterial gene deletions are the following: one or more mutations in genes that alter lipopolysaccharide biosynthesis selected from among one or more of rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; and / or introducing a suicide gene and one or more mutations selected from one or more of sacB, nuk, hok, gef, kil, or phlA; and / or introducing a lysis gene and one or more mutations selected from one or both of hly and cly; and / or Mutations in one or more virulence factor(s) selected from among IsyA, pag, prg, iscA, virG, plc, and act; and / or one or more mutations that alter the stress response selected from among recA, htrA, htpR, hsp, and groEL; and / or Mutations in min that disrupt the cell cycle; and / or one or more mutations that disrupt or inactivate a regulatory function selected from among cya, crp, phoP / phoQ, and ompR.

[0048] The RNAi described includes shRNA and miRNA. Exemplary miRNA backbones into which RNA encoding a target or its complement is inserted are those based on miR-16-2 (SEQ ID NO: 248) or the miRNA backbone of SEQ ID NO: 249. The immunostimulatory bacteria can include miR-103 (SEQ ID NO: 252), where mature miR-103 includes the sequence: 5'-AGCAGCAUUGUACAGGGCUAUGA-3'.

[0049] RNAi can be expressed under the control of RNA polymerase III or RNA polymerase II promoter.Generally, shRNA is expressed under the control of RNAP III promoter; miRNA is expressed under the control of RNAP II promoter.Many RNAP III and II promoters are known and available to those skilled in the art.RNAP III promoters include, for example, U3, H1, U6, 7SK and 7SL; RNAP II promoters include virus promoters, cytomegalovirus SV40 promoters and adenovirus promoters.Many virus promoters, especially the latter promoters, are strong constitutive promoters.

[0050] The immunostimulatory bacteria can be a strain of Salmonella, Shigella, E. coli, Bifidobacterium, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, and Erysipelothrix, or an attenuated or modified strain thereof of any of the preceding lists of bacterial strains.

[0051] Exemplary immunostimulatory bacteria are those in which the plasmid contains one or more of a nucleic acid sequence encoding the listeriolysin O (LLO) protein (cytoLLO) without a signal sequence, a CpG motif, a DNA nuclear targeting sequence (DTS), a deletion of a gene encoding a flagellin subunit(s), and a retinoic acid-inducible gene-I (RIG-I) binding element.

[0052] If the plasmid contains two or more encoding RNAs that inhibit, suppress or block expression, each is separated by at least about 75 nucleotides, or by at least 75 nucleotides, up to about or at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 nucleotides (or base pairs), up to about or 1600 nucleotides (or base pairs), or between 75 and 1500 or 1600 nucleotides (or base pairs).

[0053] Other exemplary immunostimulatory bacteria include those that are adenosine auxotrophic and contain a deletion in the gene(s) encoding the flagellum; a deletion in endA; a plasmid encoding CytoLLO; a nuclear localization sequence; and an asd plasmid complementation system, encoding an RNA that inhibits, suppresses, or blocks expression of an immune checkpoint or other target, the inhibition, suppression, or blockage enhancing an anti-tumor immune response in a subject.

[0054] Such immunostimulatory bacteria include strains of the genus Salmonella, such as Salmonella Typhimurium strains, e.g., strains designated AST100, VNP20009, or attenuated Salmonella Typhimurium strains selected from among strains YS1646 (ATCC #202165), RE88, SL7207, χ8429, χ8431, and χ8468.

[0055] The immunostimulatory bacteria may comprise a plasmid encoding an shRNA encoded by a sequence of nucleotides set forth in any of SEQ ID NOs: 36-40 and 75-78, or an miRNA encoded by a sequence of nucleotides set forth in any of SEQ ID NOs: 214-217.

[0056] Any of the immunostimulatory bacteria may be grown and harvested in stationary phase. Methods for producing immunostimulatory bacteria include culturing by standard methods and harvesting in stationary phase.

[0057] A composition comprising an immunostimulatory bacterium is provided. Such a composition comprises the bacterium and a pharmaceutically acceptable excipient or vehicle. A single dose is therapeutically effective for treating a disease or disorder for which immune stimulation provides treatment. Exemplary of such stimulation is an immune response, including, but not limited to, one or both of a specific immune response and a non-specific immune response, both a specific response and a non-specific response, an innate response, a primary immune response, an adaptive immune response, a secondary immune response, a memory immune response, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression.

[0058] Pharmaceutical compositions containing any of the immunostimulatory bacteria are provided. Uses of these for treating cancer and methods for treating cancer are also provided. The methods and uses include treating a subject with cancer, which involves administering the immunostimulatory bacteria or pharmaceutical composition to a subject, such as a human. A method for treating a subject with cancer, which includes administering the immunostimulatory bacteria, is provided. The methods and uses include combination therapy in which a second anti-cancer agent or treatment is administered. The second anti-cancer agent is a chemotherapeutic agent that induces cytoplasmic DNA, or radiation therapy, or an anti-immune checkpoint inhibitor such as an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, or CAR-T cells, or other therapeutic cells such as stem cells, TIL cells, and cells modified for cancer therapy.

[0059] The immunostimulatory bacteria described herein against TREX1, such as Salmonella strains, that encode RNAi, such as miRNAs and shRNAs, are complementary to therapeutics that are genotoxic or target or adversely affect DNA, resulting in cytoplasmic DNA.

[0060] Administration can be by any suitable route, such as parenteral, and can include additional agents that can facilitate or enhance delivery. Administration can be oral or rectal, or by aerosol to the lung, or intratumoral, intravenous, intramuscular, or subcutaneous.

[0061] Cancers include, but are not limited to, solid tumors and hematological malignancies such as cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, uterus, head and neck, ovary, prostate, brain, pancreas, skin, bone, liver, bone marrow, blood, thymus, uterus, testes, cervix, or liver.

[0062] The immunostimulatory bacteria can be formulated into compositions for administration, such as suspensions. They can be dried and stored as powders. Combinations of the immunostimulatory bacteria with other anti-cancer agents are also provided.

[0063] Also provided are shRNAs and miRNAs, such as nucleic acid molecules comprising the sequence of a nucleic acid set forth in any of SEQ ID NOS: 36-40 and 75-78. Plasmids comprising such DNA are also provided. Immunostimulatory bacteria, such as Salmonella, comprising the plasmids are provided.

[0064] Combination therapies for treating cancer and malignant tumors are provided. The immunostimulatory bacteria can be administered prior to or concurrently with other cancer therapies, including radiation therapy, chemotherapy, particularly genotoxic chemotherapy that targets cytoplasmic DNA, and immunotherapies such as anti-checkpoint inhibitor antibodies, including anti-PD-L1, anti-CTLA4, and other such immunotherapies.

[0065] cd73 +Also provided are methods and uses for treating a subject having a tumor in which the immunostimulatory bacteria is adenosine auxotroph; the subject may be tested for CD73 by testing a tumor biopsy or other body tissue or fluid sample. + The patient has or is identified as having a tumor that is

[0066] Methods for increasing colonization of immunostimulatory bacteria in a subject are provided, comprising administering the immunostimulatory bacteria to the subject; and inhibiting or suppressing expression of TREX1 and / or activity of the encoded product of TREX1 in the subject.

[0067] The terms and expressions employed are used as terms of description and not of limitation, and in the use of such terms and expressions there is no intention to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are contemplated. [Brief explanation of the drawings]

[0068] [Figure 1] Figure 1 shows a schematic diagram of the process used to delete the asd gene from strain YS1646. The asd gene from S. typhimurium strain YS1646 was deleted using the lambda-derived Red recombination system described by Datsenko and Wanner [Proc Natl Acad Sci USA 97:6640-6645 (2000)]. [Figure 2] Figure 2 shows the results of a human PD-L1 shRNA screening using qPCR and Western blot. HEK293 cells were co-transfected with a PD-L1 cDNA expression plasmid and various pEQU6 plasmids encoding different shRNAs targeting PDL1. Figure 2A shows the results of qPCR analysis to determine the level of mRNA knockdown. Figure 2B shows Western blot analysis of human PD-L1 shRNA. PD-L1 protein expression levels were measured using Western blotting and densitometry. [Figure 3] Figure 3 shows the results of a human TREX1 shRNA screen using qPCR and Western blot. HEK293 cells were co-transfected with a TREX1 cDNA expression plasmid and various pEQU6 plasmids encoding different shRNAs targeting TREX1. Figure 3A shows the results of qPCR analysis used to determine the level of mRNA knockdown. Figure 3B shows the results of Western blot analysis of human TREX1 shRNA. PD-L1 protein expression levels were measured using Western blotting and densitometry. [Figure 4] Figure 4 shows the results of a human beta-catenin shRNA screen using qPCR and Western blot. HEK293 cells were cotransfected with a beta-catenin cDNA expression plasmid and various pEQU6 plasmids encoding different shRNAs targeting beta-catenin. Figure 4A shows the results of qPCR used to determine the level of mRNA knockdown. Figure 4B shows the results of Western blot analysis of the human beta-catenin shRNAs. The level of beta-catenin protein expression was measured using Western blotting and densitometry. [Figure 5] Figure 5 shows the results of a human SIRP alpha shRNA screen using qPCR and Western blot. HEK293 cells were co-transfected with a SIRP alpha cDNA expression plasmid and various pEQU6 plasmids encoding different shRNAs targeting SIRP alpha. Figure 5A shows the results of qPCR used to determine the level of mRNA knockdown. Figure 5B shows the results of Western blot analysis of human SIRP alpha shRNAs. Western blotting and densitometry were used to measure the level of SIRP alpha protein expression. [Figure 6]Figure 6 shows the results of a human TGF-beta isoform 1 shRNA screen using qPCR. HEK293 cells were co-transfected with a TGF-beta isoform 1 cDNA expression plasmid and various pEQU6 plasmids encoding different shRNAs targeting TGF-beta. qPCR was used to determine the level of mRNA knockdown. [Figure 7] Figure 7 shows the results of a human VEGF shRNA screen using qPCR. HEK293 cells were co-transfected with a VEGF cDNA expression plasmid and various pEQU6 plasmids encoding different shRNAs targeting VEGF. qPCR was used to determine the level of mRNA knockdown. [Figure 8] Figure 8 shows the results of a human VISTA shRNA screen using qPCR and Western blot. HEK293 cells were co-transfected with a VISTA cDNA expression plasmid and various pEQU6 plasmids encoding different shRNAs targeting VISTA. Figure 8A shows the results of qPCR used to determine the level of mRNA knockdown. Figure 8B shows the results of Western blot analysis of human VISTA shRNA. Western blotting and densitometry were used to measure the level of VISTA protein expression. [Figure 9]Figure 9 shows the results of qPCR evaluation of combined gene knockdown by HuPD-L1 + HuTREX1 RNAi. HEK293 cells were co-transfected with a TREX1 cDNA expression plasmid, a PD-L1 cDNA expression plasmid, and a pEQU6-H1 plasmid encoding ARI-134 shRNA targeting PD-L1 and TREX1, or a pEQU6 plasmid encoding ARI-123 shRNA targeting PD-L1 alone, or a pEQU6 plasmid encoding ARI-114 shRNA targeting TREX1. Figure 9A shows the results of the qPCR used to determine the level of PD-L1 mRNA knockdown. Figure 9B shows the results of the qPCR used to determine the level of TREX1 mRNA knockdown. [Figure 10] Figure 10 shows the results of qPCR evaluation of combined gene knockdown by HuPD-L1 + HuSIRP alpha RNAi. HEK293 cells were co-transfected with a PD-L1 cDNA expression plasmid, a SIRP alpha cDNA expression plasmid, and a pEQU6-H1 plasmid encoding ARI-135 containing shRNA targeting PD-L1 and SIRP alpha, or a pEQU6 plasmid encoding ARI-123 shRNA targeting PD-L1 alone, or a pEQU6 plasmid encoding ARI-175 shRNA targeting SIRP alpha. Figure 10A shows the results of qPCR used to determine the level of PD-L1 mRNA knockdown. Figure 10B shows the results of qPCR used to determine the level of SIRP alpha mRNA knockdown. [Figure 11]Figure 11 shows the results of qPCR evaluation of combined gene knockdown by HuPD-L1 + Hu beta-catenin RNAi. HEK293 cells were co-transfected with a PD-L1 cDNA expression plasmid, a beta-catenin cDNA expression plasmid, and the pEQU6-H1 plasmid encoding ARI-136 containing shRNA targeting PD-L1 and beta-catenin, or the pEQU6 plasmid encoding ARI-123 shRNA targeting PD-L1 alone, or the pEQU6 plasmid encoding ARI-169 shRNA targeting beta-catenin. Figure 11A shows the results of qPCR used to determine the level of PD-L1 mRNA knockdown. Figure 11B shows the results of qPCR used to determine the level of beta-catenin mRNA knockdown. [Figure 12] Figure 12 shows the results of qPCR evaluation of combined gene knockdown by HuPD-L1 + HuVISTA RNAi. HEK293 cells were co-transfected with a PD-L1 cDNA expression plasmid, a VISTA cDNA expression plasmid, and a pEQU6-H1 plasmid encoding ARI-137 (SEQ ID NO: 213) containing shRNA targeting PD-L1 and VISTA, or a pEQU6 plasmid encoding ARI-123 (SEQ ID NO: 2) shRNA targeting PD-L1 alone, or a pEQU6 plasmid encoding ARI-195 (SEQ ID NO: 25) shRNA targeting VISTA. Figure 12A shows the results of the qPCR used to determine the level of PD-L1 mRNA knockdown. Figure 12B shows the results of the qPCR used to determine the level of VISTA mRNA knockdown. [Figure 13]Figure 13 shows the results of qPCR evaluation of combined gene knockdown by mouse TREX1 + mouse PD-L1 RNAi. HEK293 cells were co-transfected with a mouse TREX1 cDNA expression plasmid, a mouse PD-L1 cDNA expression plasmid, and a pEQU6-H1 plasmid encoding an shRNA targeting mouse TREX1 and mouse PD-L1 (named ARI-128), or a pEQU6 plasmid encoding an shRNA targeting mouse PD-L1 alone (named ARI-115), or a pEQU6 plasmid encoding an shRNA targeting mouse TREX1 (named ARI-108). Figure 13A shows the results of the qPCR used to determine the level of PD-L1 mRNA knockdown. Figure 13B shows the results of the qPCR used to determine the level of TREX1 mRNA knockdown. [Figure 14] Figure 14 shows the results of qPCR evaluation of combined gene knockdown by mouse PD-L1 + mouse SIRP alpha RNAi. HEK293 cells were co-transfected with a mouse PD-L1 cDNA expression plasmid, a mouse SIRP alpha cDNA expression plasmid, and a pEQU6-H1 plasmid encoding an shRNA targeting mouse PD-L1 and SIRP alpha (named ARI-129), or a pEQU6 plasmid encoding an shRNA targeting PD-L1 alone (named ARI-115), or a pEQU6 plasmid encoding an shRNA targeting SIRP alpha (named ARI-138). Figure 14A shows the results of the qPCR used to determine the level of PD-L1 mRNA knockdown. Figure 14B shows the results of the qPCR used to determine the level of SIRP alpha mRNA knockdown. [Figure 15]Figure 15 shows the results of qPCR evaluation of combined gene knockdown by mouse PD-L1 + mouse VISTA RNAi. HEK293 cells were co-transfected with a mouse PD-L1 cDNA expression plasmid, a mouse VISTA cDNA expression plasmid, and a pEQU6-H1 plasmid encoding an shRNA targeting PD-L1 and VISTA (named ARI-132), or a pEQU6 plasmid encoding an shRNA targeting PD-L1 alone (named ARI-115), or a pEQU6 plasmid encoding an shRNA targeting VISTA (named ARI-157). Figure 15A shows the results of qPCR used to determine the level of PDL1 mRNA knockdown. Figure 15B shows the results of qPCR used to determine the level of beta-catenin mRNA knockdown. [Figure 16] Figure 16 shows the results of qPCR evaluation of combined gene knockdown by mouse TREX1 + mouse SIRP alpha RNAi. HEK293 cells were co-transfected with a mouse TREX1 cDNA expression plasmid, a mouse VISTA cDNA expression plasmid, and a pEQU6-H1 plasmid encoding an shRNA targeting PD-L1 and VISTA (designated ARI-131), or a pEQU6 plasmid encoding an shRNA targeting TREX1 alone (designated ARI-108), or a pEQU6 plasmid encoding an shRNA targeting SIRP alpha (designated ARI-138). Figure 16A shows the results of qPCR used to determine the level of TREX1 mRNA knockdown. Figure 16B shows the results of qPCR used to determine the level of SIRP alpha mRNA knockdown. [Figure 17]Figure 17 shows the results of qPCR evaluation of combined gene knockdown by mouse PD-L1 + mouse beta-catenin RNAi. HEK293 cells were co-transfected with a mouse PD-L1 cDNA expression plasmid, a mouse beta-catenin cDNA expression plasmid, and a pEQU6-H1 plasmid encoding an shRNA targeting PD-L1 and VISTA (named ARI-133), or a pEQU6 plasmid encoding an shRNA targeting PD-L1 alone (named ARI-115), or a pEQU6 plasmid encoding an shRNA targeting beta-catenin (named ARI-166). Figure 17A shows the results of qPCR used to determine the level of PD-L1 mRNA knockdown. Figure 17B shows the results of qPCR used to determine the level of beta-catenin mRNA knockdown. [Figure 18] Figure 18 shows the results of qPCR evaluation of combined gene knockdown by mouse TREX1 + mouse VISTA RNAi. HEK293 cells were co-transfected with a mouse TREX1 cDNA expression plasmid, a mouse VISTA cDNA expression plasmid, and a pEQU6-H1 plasmid encoding an shRNA targeting PD-L1 and VISTA (designated ARI-130), or a pEQU6 plasmid encoding an shRNA targeting TREX1 alone (designated ARI-108), or a pEQU6 plasmid encoding an shRNA targeting VISTA (designated ARI-157). Figure 18A shows the results of qPCR used to determine the level of TREX1 mRNA knockdown. Figure 18B shows the results of qPCR used to determine the level of VISTA mRNA knockdown. [Figure 19]Figure 19 shows a comparison of microRNA- and shRNA-mediated knockdown of mouse PD-L1. HEK293 cells were co-transfected with a mouse PD-L1 cDNA expression plasmid and a pEQU6 plasmid encoding either a microRNA (ARI-201) or shRNA (designated ARI-115) targeting PD-L1. Figure 19A shows the results of qPCR used to determine the level of PD-L1 mRNA knockdown. Figure 19B shows the results of Western blot analysis. The level of PD-L1 protein expression was measured using Western blotting and densitometry. [Figure 20] Figure 20 shows a comparison of microRNA- and shRNA-mediated knockdown of mouse TREX1. HEK293 cells were co-transfected with a mouse TREX1 cDNA expression plasmid and a pEQU6 plasmid encoding a microRNA (designated ARI-203) or shRNA (designated ARI-108) targeting TREX1. Western blot was used to determine the level of mRNA knockdown. [Figure 21] Figure 21 shows the results of TREX1 knockdown by RNA Pol II expression of microRNA. HEK293 cells were co-transfected with a mouse TREX1 cDNA expression plasmid and a pEQU6 plasmid encoding a shRNA targeting mouse TREX1 (designated ARI-108) or a pEQ plasmid encoding a CMV promoter and a microRNA targeting mouse TREX1 (designated ARI-204). Figure 21A shows the results of qPCR used to determine the level of mouse TREX1 mRNA knockdown. Figure 21B shows the results of Western blot analysis. The level of mouse TREX1 protein expression was measured using Western blotting and densitometry. [Figure 22]Figure 22 shows the results of PD-L1 knockdown by RNA Pol II expression of microRNA. HEK293 cells were co-transfected with a mouse PD-L1 cDNA expression plasmid and either the pEQU6 plasmid encoding a shRNA targeting mouse PD-L1 (named ARI-115) or the pEQ plasmid encoding a microRNA targeting the CMV promoter and mouse TREX1 (named ARI-202). Figure 22A shows the results of qPCR used to determine the level of mouse PD-L1 mRNA knockdown. Figure 22B shows the results of Western blot analysis. The level of mouse PD-L1 protein expression was measured using Western blotting and densitometry. [Figure 23] Figure 23 shows the efficacy of systemically administered strain AST-104 in a CT26 colon tumor model. BALB / c mice were implanted with single CT26 (2 x 10 cells) subcutaneous flank tumors (n = 8 per group). Mice with established tumors were intravenously injected with 1 x 10 CFU of strain YS1646 containing either a plasmid control (strain AST-102) or a TREX1 shRNA plasmid (strain AST-104), or a PBS control, on the days indicated by the arrows. Spaghetti plots show tumor growth, with each line representing an individual mouse. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length x width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. Tumor growth inhibition (TGI)% was calculated as 1-(mean test tumor volume / mean control tumor volume) x 100. *p<0.05 vs. plasmid control, Student's t-test. [Figure 24]Figure 24 shows the correlation between strain AST-104-mediated cytokine changes and the STING signature. BALB / c mice were implanted with a single CT26 (2×10 cells) subcutaneous flank tumor (n=8 per group). Mice with established tumors were intravenously injected with 5×10 CFU of strain YS1646 containing either a plasmid control (strain AST-102) or a TREX1 shRNA plasmid (AST-104), or a PBS control. Six hours after the first dose, mice were bled, and systemic serum cytokines were tested using a Luminex 200 instrument (Luminex Corporation) and a mouse cytometric bead array (BD Bead Array, FACS Fortessa, FCAP software, BD Biosciences). Figure 24A shows the levels of pro-inflammatory cytokines. Figure 24B shows the levels of immunosuppressive cytokines. *p<0.05, **p<0.01, Student's t-test. [Figure 25] Figure 25 shows the efficacy of systemically administered strain AST-104 in the MC38 colon tumor model. C57Bl / 6 mice (6–8 weeks old) were implanted with a single MC38 (2 × 10 cells) subcutaneous flank tumor (n = 10 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of YS1646 strain containing either a plasmid control (strain AST-102) or a TREX1 shRNA plasmid (strain AST-104), or a PBS control, on the days indicated by the arrows. Spaghetti plots show tumor growth, with each line representing an individual mouse. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) x 100. *p<0.05 vs. plasmid control, Student's t-test. [Figure 26]Figure 26 shows the efficacy of AST-104 in a checkpoint-resistant B16.F10 melanoma model. C57Bl / 6 mice (6–8 weeks old) were implanted with a single B16.F10 (5 × 10 cells) subcutaneous flank tumor (n = 10 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of YS1646 strain containing either a plasmid control (AST-102) or a TREX1 shRNA plasmid (AST-104), or a PBS control, on the days indicated by the arrows. Spaghetti plots show tumor growth, with each line representing an individual mouse. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) x 100. *p<0.05 vs. plasmid control, Student's t-test. [Figure 27] Figure 27 shows the efficacy of systemically administered AST-105 (shPD-L1) in a CT26 tumor model. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2×105 cell) subcutaneous flank tumor (n=8 per group). Mice with established tumors were injected IV with 5×106 CFU of YS1646 strain containing either a plasmid control (AST-102) or a PD-L1 shRNA plasmid (AST-105), or a PBS control, on the days indicated by the arrows. Separate groups received 100 μg of anti-PD-L1 antibody (clone 10F.9G2, BioXCell) via IP injection every week, starting with the first IV injection. Spaghetti plots show tumor growth, with each line representing an individual mouse. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) × 100. *p<0.05 vs. plasmid control, Student's t-test. [Figure 28] Figure 28 shows results demonstrating that AST-105 induces a significant cytokine response over that observed with PD-L1 mAbs. BALB / c mice (6-8 weeks old) were implanted with a single CT26 (2x105 cells) subcutaneous flank tumor (n=8 per group). Mice with established tumors were injected IV with 5x106 CFU of YS1646 strain containing either a plasmid control (AST-102) or a PD-L1 shRNA plasmid (AST-105), or a PBS control, on the days indicated by the arrows. Separate groups received 100µg of anti-PD-L1 antibody IP (clone 10F.9G2 clone, BioXCell) every week, starting with the first IV injection. Mice were bled 6 hours after the first dose and systemic serum cytokines were tested by Luminex (BD Bead Array and Luminex 200) and mouse cytometric bead array (FACS Fortessa, FCAP software, all BD Biosciences). *p<0.05, **p<0.01, Student's t-test. [Figure 29]Figure 29 shows the effect of intratumoral administration of strains AST-104 and AST-105 on tumor volume in bilateral flank colon tumors. BALB / c mice (6–8 weeks old) were implanted with double CT26 (2×105 cells) subcutaneous flank tumors in the right and left flanks (n=10 per group). Mice with established tumors were intratumoral injected with 5×106 CFU of YS1646 strain containing either a plasmid control (AST-102), a TREX1 shRNA plasmid (AST-104), or a PD-L1 shRNA plasmid (AST-105), or a PBS control, into the right flank on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. Tumor growth inhibition (TGI)% was calculated as 1-(mean test tumor volume / mean control tumor volume) x 100. Plots show the mean tumor growth for each group ± SEM in the injected group (left graph) and the distal group (right graph). *p<0.05, ***p<0.001, Student's t-test. [Figure 30] Figure 30 shows the therapeutic effect of intratumoral AST-104 administration on bilateral flank colon tumors in mice. BALB / c mice (6–8 weeks old) were implanted with double CT26 (2×105 cells) subcutaneous flank tumors in the right and left flanks (n=10 per group). Mice with established tumors were intratumoral injected with 5×106 CFU of YS1646 strain containing either a plasmid control (AST-102), a TREX1 shRNA plasmid (AST-104), or an shPD-L1 plasmid (AST-105), or a PBS control, into the right flank on days 10 and 14 after tumor implantation. Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. The figure shows the overall survival rate of mice. **p<0.01, log-rank (Mantel-Cox) test. [Figure 31]Figure 31 shows the level of tumor establishment in injected and distant tumors after IT administration of AST-104. BALB / c mice (6–8 weeks old) were implanted with double CT26 (2×10 cells) subcutaneous flank tumors in the right and left flanks (n=10 per group). Mice with established tumors were injected IT into the right flank with 5×10 CFU of the YS1646 strain (AST-104) containing the TREX1 shRNA plasmid. 35 days after tumor implantation (12 days after the final AST-104 administration), three mice were sacrificed. The injected and distant tumors were homogenized (GentleMACs™, Miltenyi Biotec) and plated on LB plates to count the number of colony-forming units (CFU) per gram of tumor tissue. The figure shows the mean CFU per gram of tissue, ±SD. [Figure 32] Figure 32 shows that CpG-scrambled plasmids have immunostimulatory antitumor properties. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of YS1646 strain (AST-100), YS1646 strain (AST-103) containing a scrambled shRNA control plasmid, or PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 – (mean test tumor volume / mean control tumor volume) × 100. The figure shows the mean tumor growth for each group ± SEM. **p<0.01, Student's t-test. [Figure 33]Figure 33 shows the efficacy of AST-106 (microRNA TREX1) vs. AST-104 (shRNA TREX1). BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of YS1646 (AST-104) containing a TREX1 shRNA plasmid or YS1646 (AST-106) containing a TREX1 microRNA plasmid, or PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) x 100. The figure shows the mean tumor growth for each group ± SEM. *p<0.05, Student's t-test. [Figure 34] Figure 34 shows a schematic diagram of the process used to delete the fliC gene. The flic gene was deleted from the chromosome of S. typhimurium strain AST-101 (an asd deletion strain of YS1646) using the lambda-derived Red recombination system described by Datsenko and Wanner [Proc Natl Acad Sci USA 97:6640-6645 (2000)]. [Figure 35] Figure 35 shows that flagellin deletion strains grow normally in LB. The figure shows the growth of strains AST-108 ASD(pATI-shTREX1) and AST-112 ASD / FLG(pATI-shTREX1) in LB broth at 37°C, as measured by OD600 using a Spectramax 96-well plate reader (Molecular devices). [Figure 36]Figure 36 shows that flagellin knockout improves antitumor efficacy. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the asd / flgB / fliC knockout strain (AST-113) containing the pATI shTREX1 plasmid, or the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid, or PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 – (mean test tumor volume / mean control tumor volume) × 100. The figure shows the mean tumor growth for each group ± SEM. *p<0.05, Student's t-test. [Figure 37] Figure 37 shows that flagellin knockout mice exhibit an increased IFN-gamma signature. BALB / c mice (6-8 weeks old) were implanted with a single CT26 (2 x 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 x 10 CFU of the asd / flgB / fliC knockout strain (AST-113) containing the pATI shTREX1 plasmid, or the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid, or PBS control. Mice were bled 6 hours after the first injection, and systemic serum cytokines were tested using a Luminex 200 instrument (Luminex Corporation) and a mouse cytometric bead array (BD Bead Array, FACS Fortessa, FCAP software, all BD Biosciences). *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test. [Figure 38]Figure 38 shows that flagellin is not required for tumor establishment. BALB / c mice (6-8 weeks old) were implanted with a single CT26 (2 x 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 x 10 CFU of the asd / flgB / fliC knockout strain (AST-113) containing the pATI shTREX1 plasmid, or the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid, or PBS control. On day 35 after tumor implantation (12 days after the last dose of genetically modified Salmonella therapy), three mice per group were sacrificed, and tumors were homogenized (GentleMACs™, Miltenyi Biotec) and plated on LB plates to count the number of colony-forming units per gram of tumor tissue. The figure shows the mean colony-forming units (CFU) per gram of tissue, ± SD. [Figure 39] Figure 39 shows that cytoLLO-expressing strains grow normally in vitro. The figure shows the growth of strains AST-110 [YS1646 with the asd deletion containing (pATI-shTREX1)] and AST-115 [YS1646 with the asd deletion and cytoLLO expression cassette knock-in containing (pATI-shTREX1)] in LB broth at 37°C, as measured by OD using a Spectramax 96-well plate reader (Molecular devices). [Figure 40]Figure 40 shows that AST-115 (ASD knockout + CytoLLO knock-in strain harboring shTREX1 plasmid) exhibits robust single-dose efficacy in a murine CT26 tumor model. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of AST-115 [YS1646 (containing pATI-shTREX1) with an asd deletion and a cytoLLO expression cassette knock-in at the asd locus] or PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) x 100. The figure shows the mean tumor growth for each group ± SEM. **p<0.01, Student's t-test. [Figure 41] Figure 41 shows that strain YS1646 requires tumor microenvironment levels of adenosine for growth. Growth of strain YS1646 (purI- / msbB-) and the wild-type parent strain ATCC14028 in LB broth at 37°C, as measured by OD600 using a Spectramax 96-well plate reader (Molecular devices), is shown. [Figure 42] Figure 42 shows that ASD, FLG, and CytoLLO genetically engineered strains require high adenosine for growth. Growth of strains AST-117 (YS1646 Δasd containing a low-copy shTREX-1 plasmid), AST-118 (YS1646 Δasd / filC / fljB containing a low-copy shTREX-1 plasmid), and AST-119 (YS1646 Δasd:LLO containing a low-copy shTREX-1 plasmid) in LB broth at 37°C is shown, as measured by OD using a Spectramax 96-well plate reader (Molecular devices). [Figure 43]Figure 43 shows that strains with a low-copy origin of a replicating asd-encoding plasmid have superior growth kinetics to strains with a high-copy origin of a replicating asd-encoding plasmid. Growth of strains YS1646, AST-117 (YS1646 Δasd containing a low-copy shTREX-1 plasmid with a functional asd gene), AST-104 (YS1646 containing a low-copy pEQ shTREX-1 plasmid lacking the asd gene), and AST-110 (YS1646 Δasd containing a high-copy pATI-shTREX-1 plasmid with a functional asd gene) in LB broth at 37°C, as measured by OD using a Spectramax 96-well plate reader (Molecular devices), is shown. [Figure 44] Figure 44 shows that strains carrying a low-copy asd plasmid are more compatible with mouse tumor cells than strains carrying a high-copy asd plasmid. The intracellular growth of strains AST-117 (YS1646 Δasd containing a low-copy shTREX-1 plasmid carrying a functional asd gene) and AST-110 (YS1646 Δasd containing a high-copy pATI-shTREX-1 plasmid carrying a functional asd gene) is shown in B16F.10 mouse melanoma cells and CT26 mouse colon carcinoma cells. 5 x 10 cells in 24-well dishes were infected with S. typhimurium strains at an MOI of 5. Thirty minutes after infection, the medium was replaced with medium containing gentamicin to kill extracellular bacteria. At the indicated time points, cell monolayers were lysed by osmotic shock, and cell lysates were diluted and plated on LB agar to enumerate CFU. [Figure 45]Figure 45 shows that the asd gene complementation system results in plasmid retention in S. typhimurium-infected tumors in vivo. BALB / c mice (6-8 weeks old) were implanted with a single CT26 (2 x 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 x 10 CFU of either the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid or YS1646 (AST-104) containing the pEQ shTREX-1 plasmid lacking the asd gene. Three mice per group were sacrificed 35 days after tumor implantation (12 days after the last dose of genetically modified Salmonella therapy). Tumors were homogenized using a GentleMACs™ homogenizer (Miltenyi Biotec) and plated on LB agar plates or LB agar plates containing 50 μg / mL kanamycin. The figure shows the percentage of kanamycin-resistant CFU in tumor tissue homogenates ±SD. [Figure 46] Figure 46 shows the improved therapeutic efficacy of the asd gene complementation system and the strain (AST-110) containing a plasmid carrying shTREX1. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid or the asd knockout strain (AST-109) containing the pATI scrambled plasmid, or the YS1646 strain (AST-104) containing the pEQ-shTREX-1 plasmid lacking the asd gene, or PBS control, on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) × 100. The figure shows the mean tumor growth for each group, ± SEM. [Figure 47] Figure 47 shows that the strain containing the low-copy shTREX1 plasmid (AST-117) has superior antitumor properties compared to the strain containing the high-copy plasmid (AST-110). BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid with a high copy number replication origin or the asd knockout strain (AST-117) containing the pATI shTREX1 plasmid with a low copy number replication origin, or PBS control, on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1-(mean test tumor volume / mean control tumor volume) x 100. The figure shows the mean tumor growth for each group, ±SEM. *p<0.05, Student's t-test. [Figure 48]Figure 48 shows that the AST-117 low-copy plasmid strain colonizes tumors better than the AST-110 high-copy plasmid strain, with a higher tumor-to-spleen colonization ratio. BALB / c mice (6-8 weeks old) were implanted with a single CT26 (2 x 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 x 10 CFU of either the asd knockout strain (AST-110) containing a high-copy-number pATI shTREX1 plasmid with a replication origin or the asd knockout strain (AST-117) containing a low-copy-number pATI shTREX1 plasmid with a replication origin. Three mice per group were sacrificed 35 days after tumor implantation (12 days after the final dose of genetically modified Salmonella therapy). Tumors were homogenized using a GentleMACs™ homogenizer (Miltenyi Biotec) and plated on LB plates to count the number of CFU per gram of tumor tissue. Figure 48A shows the mean CFU per gram of tumor tissue ±SD. Figure 48B shows the tumor-to-spleen colonization ratio. [Figure 49]Figure 49 shows that strains grown to stationary phase were equally potent and less inflammatory than the same strains grown to logarithmic phase. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of YS1646 strain (AST-104) containing the pEQ-shTREX-1 plasmid harvested in logarithmic or stationary phase, or with PBS control, on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 – (mean test tumor volume / mean control tumor volume) × 100. Figure 49A shows the mean tumor growth for each group, ±SEM. *p<0.05, Student's t-test. Figure 49B shows the levels of TNF-alpha and IL-6. Mice were bled 6 hours after the first dose, and systemic serum cytokines were tested by Luminex (Luminex Corp.) and mouse cytometric bead array (FACS Fortessa, FCAP software, all BD Biosciences). **p<0.01, Student's t-test. [Figure 50] Figure 50 shows that the autolytic strain (AST-120) cannot grow in the absence of DAP. The figure shows the growth over time of the Δasd:cytoLLO strain (AST-120) containing the pEQU6-shTREX1 plasmid that does not contain the asd gene in LB broth alone or in LB broth supplemented with 50 μg / mL DAP, as measured by OD using a Spectramax 96-well plate reader (Molecular devices). [Figure 51]Figure 51 shows the antitumor activity of the autolytic strain (AST-120). BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the Δasd:cytoLLO strain (AST-120) containing the pEQU6-shTREX1 plasmid, which does not contain the asd gene, or with PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 – (mean test tumor volume / mean control tumor volume) × 100. The figure shows the mean tumor growth for each group, ± SEM. *p<0.05, Student's t-test. [Figure 52] Figure 52 shows that TREX1 expression is increased in several human tumor types. Analysis of relative gene expression of the TREX1 gene using the TCGA database was performed across a wide range of tumor types. Tumor types with significant upregulation of TREX1 compared to normal tissues are shown: prostate, breast, cervical, uterine, and bladder cancer (p-values: BRCA - 7.7e-16; PRAD - 9.4e-12; UCEC - 2.5e-05; BLCA - 3.7e-03; CESC - 7.7e-03) and some forms of kidney cancer (p-values: KIPAN - 8.9e-39; KIRC - 9.6e-35; KIRP - 5.8e-14; KICH - 4.9e-08). [Figure 53]Figure 53 shows that radiation therapy after administration of S. typhimurium strain AST-106 increases tumor establishment. BALB / c mice (6–8 weeks old) were inoculated subcutaneously with 1 × 105 murine TSA breast cancer cells in the right flank. Mice bearing established tumors were administered the following: IV injection of 5 × 106 CFU of AST-106 (YS1646 transformed with pEQU6-miTREX1) followed by 0 Gy 4 hours later (3 mice); 5 × 106 CFU of AST-106 followed by 20 Gy 4 hours later (3 mice); 20 Gy irradiation followed by 5 × 106 CFU of AST-106 4 hours later (3 mice); or PBS IV followed by 0 Gy irradiation (1 mouse). Focal radiation therapy was administered using a small animal radiation research platform (SARRP) device (XStrahl Life Sciences). Mice were sacrificed 24 hours later, and tumors were harvested and weighed. Tumors were homogenized in 10 mL of sterile PBS using M-tubes in a GentleMacs™ device (Miltenyi Biotec), then serially diluted 10-fold and plated onto LB agar plates containing kanamycin. The next day, colony-forming units (CFUs) were counted, and CFU per gram of tumor tissue was calculated. *p<0.05, Student's t-test. DETAILED DESCRIPTION OF THE INVENTION

[0069] overview A.Definition B. Overview of Immunostimulatory Bacteria C. Cancer immunotherapy 1. Immunotherapy 2. Adoptive Immunotherapy 3. Cancer vaccines and oncolytic viruses D. Bacterial cancer immunotherapy 1. Bacterial treatment 2. Comparison of immune responses to bacteria and viruses 3. Treatment with Salmonella a. Tumor-tropic bacteria. b. Salmonella enterica serovar vertifimurium C. Attenuation of the bacteria i.msbB-mutant ii.purI-mutant iii. Combinations of attenuating mutations iv. VNP20009 and other attenuated S. typhimurium strains v. Attenuated S. typhimurium genetically engineered to deliver macromolecules 4. Enhancing immunostimulatory bacteria to increase the therapeutic index ASD gene deletion B adenosine auxotrophy C. flagellin-deficient strain d. Salmonella genetically engineered to escape Salmonella-containing vacuoles (SCVs) e. Deletions in Salmonella genes required for biofilm formation f. Deletion in a gene in the LPS biosynthetic pathway g. SPI-1 gene deletion h. Endonuclease (endA) mutation to increase plasmid delivery i. RIG-I inhibition j.DNASE II inhibition k.RNase H2 inhibition l. Stabilin-1 / CLEVER-1 inhibition m. Bacterial culture conditions E. Construction of Exemplary Plasmids 1. Interfering RNA (RNAi) a.shRNA B microRNA 2. Origin of Replication and Plasmid Copy Number 3. CpG motifs and CpG islands 4. Plasmid Maintenance / Construct Selection 5. DNA nuclear targeting sequence F. Tumor-targeted immunostimulatory bacteria contain RNAi against exemplary immune target genes to stimulate anti-tumor immunity 1.TREX1 2.PD-L1 3. VISTA 4.SIRPα 5. β-catenin 6. TGF-β 7. VEGF 8. Additional Exemplary Checkpoint Targets G. Combining RNAI shRNAs against multiple immune targets within single therapy modalities and combination therapies 1. TREX1 and other targets 2. TREX1 and Radiation Therapy 3. TREX1 and immunogenic chemotherapy 4. Combination therapy using anti-checkpoint antibodies H. Pharmaceutical Production, Compositions, and Formulations 1.Manufacturing a. Cell bank production b. Manufacturing of APIs c. Manufacturing of drug products 2. Composition 3. Preparation a. Liquids, injections, emulsions b. Heat-stable dry formulation 4. Compositions for other routes of administration 5. Dosage and Administration 6. Packaging and Articles of Manufacture I. Methods of Treatment and Use 1. Cancer and tumors 2. Administration 3. Monitoring J. Working Example

[0070] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention(s) pertain. All patents, patent applications, published applications and literature, GenBank sequences, databases, websites and other publications mentioned throughout this disclosure are incorporated by reference in their entirety unless otherwise noted. In the event that there are multiple definitions for terms in this specification, those in this section shall prevail. When referring to a URL or other such identifier or address, it should be understood that such identifiers may change and specific information on the Internet may be overwhelmed, but equivalent information may be found by searching the Internet. Reference thereto evidences the availability and general dissemination of such information.

[0071] As used herein, a therapeutic bacterium is a bacterium that, when administered to a subject, such as a human, confers an effect on a therapy, such as a cancer or anti-tumor therapy.

[0072] As used herein, immunostimulatory bacteria are therapeutic bacteria that, when introduced into a subject, accumulate in immune-privileged tissues and cells, such as tumors, and replicate and / or express immune-stimulating or immune-stimulating products. Immunostimulatory bacteria are attenuated in the host primarily due to their reduced toxicity or pathogenicity, and / or due to the encoded product having reduced toxicity or pathogenicity, due to their inability to replicate and / or express products outside of immune-privileged environments. The immunostimulatory bacteria provided herein are modified to encode a product(s) or exhibit characteristics or properties that confer immunostimulatory properties. Such products, properties, and characteristics include at least one of the following: shRNAs that target, block, or inhibit checkpoint genes, or genes that encode such inhibitors, or genes that encode metabolic products that are immunosuppressive or in immunosuppressive pathways. These include coding siRNAs such as shRNAs that target or inhibit the expression of TREX1, modifications that render the bacteria auxotrophic for adenosine, and / or inhibitors or blockers of immune checkpoint genes or their products, such as shRNAs that block or inhibit PD-L1.

[0073] As used herein, the strain designations VNP20009 (see, e.g., WO 99 / 13053; see also U.S. Pat. No. 6,863,894) and YS1646 and 41.2.9 are used interchangeably and each refer to the strain deposited with the American Type Culture Collection and assigned accession number 202165. VNP20009 is an engineered, attenuated strain of Salmonella typhimurium that contains deletions of msbB and purI and was generated from the wild-type strain ATCC 14028.

[0074] As used herein, the strain designations YS1456 and 8.7 are used interchangeably and each refers to the strain deposited with the American Type Culture Collection and assigned accession number 202164 (see U.S. Patent No. 6,863,894).

[0075] As used herein, an origin of replication is a sequence of DNA on a chromosome, plasmid, or virus from which replication begins. For small DNA molecules, including bacterial plasmids and small viruses, a single origin is sufficient.

[0076] The replication origin determines the copy number of the vector, which depends on the replication origin selected. For example, if the expression vector is derived from the low-copy-number plasmid pBR322, it can be approximately 25-50 copies / cell, and if it is derived from the high-copy-number plasmid pUC, it can be 150-200 copies / cell.

[0077] As used herein, a medium copy number of a plasmid in a cell is about 150, or 150 or less, and a low copy number is 15-30, such as 20 or less. A low to medium copy number is less than 150. A high copy number is greater than 150 copies / cell.

[0078] As used herein, a CpG motif is a pattern of bases that includes an unmethylated central CpG (the "p" refers to a phosphodiester bond between consecutive C and G nucleotides) surrounded by at least one base adjacent to the central CpG (at the 3' and 5' positions). A CpG oligodeoxynucleotide is an oligonucleotide that is at least about 10 nucleotides in length and includes an unmethylated CpG. At least the C in the 5'CG3' is unmethylated.

[0079] As used herein, the RIG-I binding sequence refers to a 5' triphosphate (5' ppp) structure derived directly from a poly(dA-dT) sequence or synthesized from this sequence by RNA pol III, which can activate type I IFN through the RIG-I pathway by interacting with RIG-I. The RNA has at least four A ribonucleotides (AAAA); it can contain 4, 5, 6, 7, 8, 9, 10 or more. The RIG-I binding sequence is introduced into a plasmid in bacteria and transcribed into polyA.

[0080] As used herein, "modification" refers to alterations to the sequence of amino acids in a polypeptide or the sequence of nucleotides in a nucleic acid molecule, including deletions, insertions, and substitutions of amino acids or nucleotides, respectively. Methods for modifying polypeptides are routine for those skilled in the art, such as by using recombinant DNA methodologies.

[0081] As used herein, modifications to the bacterial genome or to a plasmid or gene include nucleic acid deletions, substitutions and insertions.

[0082] As used herein, RNA interference (RNAi) is a biological method in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules and inhibiting translation, thereby inhibiting expression of the targeted gene.

[0083] As used herein, the RNA molecules that act through RNAi are called inhibitory based on their silencing of the expression of targeted gene.Silencing expression means that the expression of targeted gene is reduced or suppressed or inhibited.

[0084] As used herein, RNAi-mediated gene silencing is said to inhibit, suppress, block or silence the expression of targeted gene.Targeted gene comprises the nucleotide sequence corresponding to the sequence in inhibitory RNA, and thus inhibitory RNA silences the expression of mRNA.

[0085] As used herein, inhibiting, suppressing, blocking or silencing a targeted gene refers to a method of altering the expression, such as translation, of a targeted gene, thereby reducing the activity or expression of the product encoded by the targeted gene. Reduction includes complete or partial knockout, and is therefore performed in relation to the immunostimulatory bacteria and administration provided herein.

[0086] As used herein, small interfering RNA (siRNA) is a small piece of double-stranded (ds) RNA, usually about 21 nucleotides long, with a 3' overhang (2 nucleotides) at each end, which can be used to bind to messenger RNA (mRNA) at specific sequences and promote its degradation, thereby "interfering" with protein translation.By doing so, based on the nucleotide sequence of their corresponding mRNA, siRNA can prevent the production of specific proteins.This process is called RNA interference (RNAi), also called siRNA silencing or siRNA knockdown.

[0087] As used herein, short hairpin RNA or short hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn, which can be used to silence the expression of target genes through RNA interference (RNAi).The expression of shRNA in cells is typically achieved by delivering plasmid or through viral or bacterial vectors.

[0088] As used herein, the tumor microenvironment (TME) refers to the cellular environment in which a tumor resides, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). Conditions present include, but are not limited to, increased angiogenesis, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure, and metabolites or metabolic changes such as elevated levels of adenosine, which are indicative of tumor growth.

[0089] As used herein, human type I interferon (IFN) is a subgroup of interferon proteins that regulate the activity of the immune system. All type I IFNs bind to specific cell surface receptor complexes, such as the IFN-α receptor. Type I interferons include, among others, IFN-α and IFN-β. IFN-β proteins are produced by fibroblasts and have antiviral activity that is primarily involved in the innate immune response. Two types of IFN-β are IFN-β1 (IFNB1) and IFN-β3 (IFNB3).

[0090] As used herein, the description that nucleic acid or the RNA that it encodes targets a gene means that it inhibits, suppresses or silences the expression of the gene by any mechanism.Generally, such nucleic acid comprises at least the complementary part of the targeted gene, and this part is sufficient to form a hybrid with the complementary part.

[0091] As used herein, a "deletion," when referring to a nucleic acid or polypeptide sequence, refers to the deletion of one or more nucleotides or amino acids compared to a sequence such as a target polynucleotide or polypeptide or native or wild-type sequence.

[0092] As used herein, "insertion," when referring to a nucleic acid or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids within a target, native, wild-type, or other related sequence. Thus, a nucleic acid molecule that contains one or more insertions contains one or more additional nucleotides within the linear length of the sequence compared to the wild-type sequence.

[0093] As used herein, "addition" to nucleic acid and amino acid sequences describes the addition of nucleotides or amino acids onto either end compared to another sequence.

[0094] As used herein, " substitution " or " replacement " refers to the replacement of one or more nucleotides or amino acids with alternative nucleotides or amino acids in a native, target, wild-type or other nucleic acid or polypeptide sequence, without changing the length of the molecule (as described in the number of residues).Therefore, one or more substitutions in a molecule do not change the number of amino acid residues or nucleotides of the molecule.Amino acid replacement can be expressed in terms of the number of amino acid residues along the length of a polypeptide sequence, compared to a specific polypeptide.

[0095] As used herein, the phrase "at a corresponding position," or a nucleotide or amino acid position that "corresponds to" a nucleotide or amino acid position in a disclosed sequence as shown in a sequence listing, refers to a nucleotide or amino acid position identified in an alignment with the disclosed sequence using a standard alignment algorithm, such as the GAP algorithm, to maximize identity. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide. Generally, to identify corresponding positions, amino acid sequences are aligned to obtain the highest order match (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAM J Applied Math 48:1073).

[0096] As used herein, sequence alignment refers to the use of homology to align two or more sequences of nucleotides or amino acids. Typically, two or more sequences related by 50% or more identity are aligned. An aligned set of sequences refers to two or more sequences aligned at corresponding positions and can include aligned sequences from RNA, such as ESTs, and other cDNAs aligned with genomic DNA sequences. Related or variant polypeptide or nucleic acid molecules may be aligned by any method known to those skilled in the art. Such methods typically maximize matches, including manual alignment, using numerous available alignment programs (e.g., BLASTP), and others known to those skilled in the art. By aligning polypeptide or nucleic acid sequences, those skilled in the art can identify similar portions or positions using conserved and identical amino acid residues as a guide. Furthermore, those skilled in the art can also use conserved amino acid or nucleotide residues as a guide to find corresponding amino acid or nucleotide residues between human and non-human sequences. Corresponding positions can also be based on structural alignment, for example, by using computer-simulated protein structure alignments. In other instances, corresponding regions can be identified. Those skilled in the art can also use conserved amino acid residues as a guide to find corresponding amino acid residues between human and non-human sequences.

[0097] As used herein, the "property" of a polypeptide, such as an antibody, refers to any characteristic exhibited by the polypeptide, including, but not limited to, binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, thermotolerance, and tolerance to pH conditions. A change in the property can alter the "activity" of the polypeptide. For example, a change in the binding specificity of an antibody polypeptide can alter the ability to bind an antigen and / or various binding activities, such as affinity or avidity, or the in vivo activity of the polypeptide.

[0098] As used herein, "activity" or "functional activity" of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. Such activities can be empirically determined. Exemplary activities include, but are not limited to, the ability to interact with a biomolecule, for example, through antigen binding, DNA binding, ligand binding, or dimerization, or enzymatic activity, such as kinase activity or proteolytic activity. With respect to antibodies (including antibody fragments), activities include, but are not limited to, the ability to specifically bind a particular antigen, the affinity of antigen binding (e.g., high or low affinity), the avidity of antigen binding (e.g., high or low avidity), the on-rate, the off-rate, effector functions such as antigen neutralization or clearance, the ability to promote virus neutralization, and in vivo activities such as the ability to prevent infection or invasion by, or promote clearance of, pathogens, or penetrate specific tissues, fluids, or cells within the body. Activity can be assessed in vitro or in vivo using recognized assays such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays measuring binding or dissociation rates, immunohistochemistry and immunofluorescence histology and microscopy cell-based assays, flow cytometry, and binding assays (e.g., panning assays).

[0099] As used herein, "binding," "bonding," or grammatical variations thereof, refers to the participation of a molecule with another molecule in any attractive interaction, resulting in a stable association in which the two molecules are in close proximity to one another. Bonding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and intermolecular interactions such as chemical compounds, including, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, e.g., drugs.

[0100] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic, such as those produced recombinantly, including any fragment thereof that contains at least a portion of the variable heavy and light chain regions of an immunoglobulin molecule sufficient to form an antigen-binding site and, when assembled, specifically bind to an antigen. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). For example, an antibody refers to an antibody comprising two heavy chains (which can be designated H and H') and two light chains (which can be designated L and L'), where each heavy chain can be a full-length immunoglobulin heavy chain or a sufficient portion thereof to form an antigen-binding site (e.g., heavy chains include, but are not limited to, VH, VH-CH1, and VH-CH1-CH2-CH3), and each light chain can be a full-length light chain or a sufficient portion thereof to form an antigen-binding site (e.g., light chains include, but are not limited to, VL and VL-CL). Each heavy chain (H and H') is paired with one light chain (L and L', respectively). Typically, an antibody minimally comprises all or at least a portion of a variable heavy (VH) chain and / or a variable light (VL) chain. An antibody can also comprise all or a portion of a constant region.

[0101] For purposes herein, the term antibody includes full-length antibodies and portions thereof, including antibody fragments such as anti-EGFR antibody fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies also include synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intracellular antibodies. The antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subsubclass (e.g., IgG2a and IgG2b).

[0102] As used herein, "nucleic acid" refers to at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically joined together by a phosphodiester bond. Nucleic acid analogs, such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof, are also included in the term "nucleic acid." Nucleic acids also include DNA and RNA derivatives containing, for example, nucleotide analogs or "backbone" bonds other than phosphodiester bonds, such as phosphotriester, phosphoramidate, phosphorothioate, thioester, or peptide bonds (peptide nucleic acids). The term also includes, as equivalents, derivatives, variants, and analogs of either RNA or DNA made from nucleotide analogs, single-stranded (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. With respect to RNA, the uracil base is uridine.

[0103] As used herein, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular materials, or culture medium if produced by recombinant technology, or substantially free of chemical precursors, or other chemicals if chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

[0104] As used herein, "operably linked" in reference to a nucleic acid sequence, region, element, or domain means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, such that the nucleic acids can be transcribed, translated, and express a functional fusion protein, with the leader peptide resulting in the secretion of the fusion polypeptide. In some instances, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) can be operably linked to a nucleic acid encoding a second polypeptide, such that the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript may result in the expression of one of two polypeptides. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce a fusion protein containing either the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter controls or mediates transcription of the nucleic acid.

[0105] As used herein, "synthetic," for example in connection with a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0106] As used herein, naturally occurring α-amino acid residues are those 20 α-amino acid residues found in nature, which are incorporated into proteins in humans with their cognate mRNA codons through specific recognition of a charged tRNA molecule.

[0107] As used herein, a "polypeptide" refers to two or more covalently joined amino acids. The terms "polypeptide" and "protein" are used interchangeably herein.

[0108] As used herein, "peptide" refers to a polypeptide that is from 2 to about 40 or 40 amino acids in length.

[0109] As used herein, "amino acid" refers to an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, the amino acids contained in the provided antibodies include the 20 naturally occurring amino acids (see the table below), unnatural amino acids, and amino acid analogs (e.g., amino acids in which the α-carbon has a side chain). As used herein, the amino acids present in the various amino acid sequences of the polypeptides appearing herein are identified according to their well-known three-letter or one-letter abbreviations (see the table below). Nucleotides present in various nucleic acid molecules and fragments are designated using standard single-letter designations commonly used in the art.

[0110] As used herein, "amino acid residue" refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide bonds. Amino acid residues described herein are generally in the "L" isomeric form. Residues in the "D" isomeric form can be substituted for any L-amino acid residue as long as the desired functional properties are retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide. In adherence to standard polypeptide nomenclature as set forth in J. Biol. Chem., 243:3557-59 (1968) and adopted in Federal Regulations 1.821-1.822 on Patents, Trademarks, and Copyrights, abbreviations for amino acid residues are set forth in the following table:

[0111] [Table 1] [Table 2]

[0112] All sequences of amino acid residues represented by formulas herein have a left-to-right orientation in the conventional direction from amino terminus to carboxyl terminus. The phrase "amino acid residue" is defined to include the amino acids listed in the Table of Correspondence above, including modified, non-natural, and unusual amino acids. A dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, or to an amino-terminal group such as NH2, or to a carboxyl-terminal group such as COOH.

[0113] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0114] Such substitutions can be made in accordance with the exemplary substitutions shown in the table below.

[0115] [Table 3]

[0116] Other substitutions are also permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.

[0117] As used herein, "naturally occurring amino acids" refers to the 20 L-amino acids that occur in polypeptides.

[0118] As used herein, the term "unnatural amino acid" refers to an organic compound that has a structure similar to a natural amino acid, but is structurally modified to mimic the structure and reactivity of a natural amino acid. Thus, unnatural amino acids include, for example, amino acids or amino acid analogs other than the 20 naturally occurring amino acids, including, but not limited to, D-stereoisomers of amino acids. Exemplary unnatural amino acids are known to those of skill in the art and include, but are not limited to, 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β-alanine / β-aminopropionic acid (Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid / piperidine acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2′-diaminopimelic acid (Dpm). , 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), allohydroxylysine (Ahyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), alloisoleucine (Aile), N-methylglycine, sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle), and ornithine (Orn).

[0119] As used herein, a DNA construct is a single- or double-stranded, linear or circular DNA molecule that contains segments of DNA linked and arranged in a manner not found in nature. DNA constructs exist as a result of human manipulation and include clones and other copies of engineered molecules.

[0120] As used herein, a DNA segment is a portion of a larger DNA molecule having specific attributes. For example, a DNA segment encoding a specific polypeptide is a portion of a long DNA molecule, such as a plasmid or plasmid fragment, that encodes the amino acid sequence of the specific polypeptide when read in the 5' to 3' direction.

[0121] As used herein, the term polynucleotide refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in units of nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The term nucleotide is used in reference to single- and double-stranded molecules where the context allows. When the term is applied to a double-stranded molecule, it is used to indicate the overall length and will be understood to be equivalent to the term base pair. Those skilled in the art will recognize that the two strands of a double-stranded polynucleotide may differ slightly in length and their ends may be staggered, and therefore, not all nucleotides within a double-stranded polynucleotide molecule may be paired. Such unpaired ends will generally not exceed 20 nucleotides in length.

[0122] As used herein, production by recombinant means by using recombinant DNA methods means the use of well known methods of molecular biology to express proteins encoded by cloned DNA.

[0123] As used herein, " expression " refers to the method by which polypeptide is produced by transcription and translation of polynucleotide.The expression level of polypeptide can be evaluated by any method known in the art, including, for example, the method of determining the amount of polypeptide produced from host cell.Such method can include, but is not limited to, ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay and Bradford protein assay to quantify polypeptide in cell lysate.

[0124] As used herein, a "host cell" is a cell used to house, maintain, reproduce, and / or propagate a vector. Host cells can also be used to express a polypeptide encoded by a vector. The nucleic acid contained in the vector is replicated when the host cell divides, resulting in amplification of the nucleic acid.

[0125] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to a vector typically includes vectors into which a nucleic acid encoding a polypeptide, or a fragment thereof, can be introduced by restriction digestion and ligation. Reference to a vector also includes vectors containing a nucleic acid encoding a polypeptide, such as a modified anti-EGFR antibody. A vector is used to introduce a nucleic acid encoding a polypeptide into a host cell to amplify the nucleic acid or to express / display the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but can be designed to integrate a gene or portion thereof into a chromosome of the genome. Vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles are well known to those skilled in the art. Vectors also include "viral vectors" or "viral vectors." Viral vectors are genetically engineered viruses that are operably linked to exogenous genes and transfer the exogenous genes into cells (as vehicles or shuttles).

[0126] As used herein, "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences and may also include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that, upon introduction into an appropriate host cell, results in expression of cloned DNA. Suitable expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic and / or prokaryotic cells and those that remain episomal or integrate into the host cell genome.

[0127] As used herein, "primary sequence" refers to the sequence of amino acid residues in a polypeptide or the sequence of nucleotides in a nucleic acid molecule.

[0128] As used herein, "sequence identity" refers to the number of identical or similar amino acids or nucleotide bases compared between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by aligning nucleic acid or protein sequences and identifying similar or identical regions. For purposes herein, sequence identity is generally determined by alignment, and identical residues are identified. Alignment can be local or global. Matches, mismatches, and gaps can be identified between the compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of the aligned sequences, allowing identical or similar features to be aligned. Generally, these can be internal and terminal gaps. When gap penalties are used, sequence identity can be determined using no penalty for terminal gaps (e.g., no penalty is applied to terminal gaps). Alternatively, sequence identity can be determined without considering gaps, as the number of identical positions / length of all aligned sequences x 100.

[0129] As used herein, "global alignment" refers to an alignment of two sequences from beginning to end, with each character in each sequence being aligned only once. Alignment is produced regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on "global alignment" means that 50% of the residues are the same in the entire alignment of two compared sequences, each 100 nucleotides long. It is understood that global alignment can also be used to determine sequence identity even when the lengths of the aligned sequences are not the same. Differences at the ends of the sequences will be taken into account in determining sequence identity unless "no penalty for end gaps" is selected. Generally, global alignment is used on sequences that share significant similarity over most of their length. An exemplary algorithm for performing global alignment is the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48: 443). Exemplary programs for performing global alignments are publicly available and include the global sequence alignment tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ) and the program available at deepc2.psi.iastate.edu / aat / align / align.html.

[0130] As used herein, "local alignment" refers to aligning two sequences, but only aligning the portions of the sequences that share similarity or identity. Thus, local alignment determines whether a subsegment of one sequence exists in another sequence. If there is no similarity, no alignment will be achieved. Local alignment algorithms include BLAST or the Smith-Waterman algorithm (Adv. Appl. Math. 2: 482 (1981)). For example, 50% sequence identity based on "local alignment" means that in the full sequence alignment of two compared sequences of any length, a 100-nucleotide-long region of similarity or identity has 50% of the residues that are the same in the region of similarity or identity.

[0131] For purposes herein, sequence identity can be determined by standard alignment algorithm programs using the default gap penalty established by each supplier. Default parameters for gap programs include: (1) the unary comparison matrix (including a value of 1 for identity and 0 for non-identity) and weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, as described in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Whether any two nucleic acid molecules have nucleotide sequences, or any two polypeptides have amino acid sequences, that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical," or other similar variations describing percentage identity, can be determined using known computer algorithms based on local or global alignments (see, e.g., wikipedia.org / wiki / Sequence_alignment_software, which provides links to many known, publicly available alignment databases and programs).Generally, for the purposes herein, sequence identity is determined using a computer algorithm based on global alignment, such as the Needleman-Wunsch global sequence alignment tool available from NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (implementing the Huang and Miller algorithm by William Pearson (Adv. Appl. Math. (1991) 12:337-357)); and the program from Xiaoqui Huang available at deepc2.psi.iastate.edu / aat / align / align.html. Typically, the full-length sequences of each compared polypeptide or nucleotide are aligned over the full length of each sequence in global alignment. Local alignment can also be used when the sequences being compared are substantially the same length.

[0132] Thus, as used herein, the term "identity" refers to a comparison or alignment between a test and a reference polypeptide or polynucleotide. In one non-limiting example, "at least 90% identical to" refers to a percentage identity of 90 to 100% with respect to the reference polypeptide or polynucleotide. For illustrative purposes, assuming that the test and reference polypeptides or polynucleotides are compared to a length of 100 amino acids or nucleotides, identity at a level of 90% or higher indicates that 10% or less (i.e., 10 out of 100) of the amino acids or nucleotides in the test polypeptide or polynucleotide differ from those in the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed throughout the entire length of the amino acid sequence, or they can be clustered at one or more positions of varying lengths up to the maximum allowable length, for example, 10 / 100 amino acid difference (approximately 90% identity). Differences can also be due to deletion or truncation of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. At levels of homology or identity greater than about 85-90%, depending on the length of the sequences being compared, results may be independent of the program and gap parameters set; such high levels of identity can be easily assessed and often do not rely on software.

[0133] As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from a cause or condition, including but not limited to, an infection, an acquired condition, or a genetic condition, and characterized by an identifiable symptom.

[0134] As used herein, "treating" a subject exhibiting a disease or condition means that the subject's symptoms are partially or totally alleviated, or remain unchanged following treatment.

[0135] As used herein, treatment refers to any effect that alleviates the symptoms of a disease or disorder. Treatment includes prevention, therapy and / or cure. Treatment also includes any pharmaceutical use of any immunostimulatory bacteria or compositions provided herein.

[0136] As used herein, prevention refers to the prevention of potential disease and / or the prevention of worsening of symptoms or progression of disease.

[0137] As used herein, "prevention" or prevention, and their grammatical equivalents, refer to a method of reducing the risk or likelihood of developing a disease or condition.

[0138] As used herein, a "pharmaceutically active agent" includes any therapeutic or bioactive agent, including, but not limited to, anesthetics, vasoconstrictors, dispersing agents, and conventional therapeutic agents, including small molecule drugs and therapeutic proteins.

[0139] As used herein, a "therapeutic effect" means an effect resulting from treatment of a subject that alters, typically improves or reverses, the symptoms of a disease or condition, or cures the disease or condition.

[0140] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a drug, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. It is thus the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or disorder.

[0141] As used herein, "therapeutic efficacy" refers to the ability of a drug, compound, material, or composition comprising a compound to produce a therapeutic effect in a subject to which the drug, compound, material, or composition comprising the compound is administered.

[0142] As used herein, "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a drug, compound, material, or composition containing a compound that, when administered to a subject, will have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom, or reducing the occurrence of a viral infection. The maximum preventive effect does not necessarily occur by administering a single dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more doses.

[0143] As used herein, amelioration of symptoms of a particular disease or disorder by treatment, such as by administration of a pharmaceutical composition or other therapeutic agent, refers to any decrease in symptoms, whether permanent or temporary, long-term or transient, that can result from or be associated with administration of the composition or therapeutic agent.

[0144] As used herein, " anticancer agent " refers to any agent that is destructive or toxic to malignant cells and tissues.For example, anticancer agent includes agent that kills cancer cells, or inhibits or impairs the growth of tumor or cancer cells in particular.An exemplary anticancer agent is a chemotherapeutic agent.

[0145] As used herein, "therapeutic activity" refers to the in vivo activity of a therapeutic polypeptide. Generally, a therapeutic activity is an activity associated with the treatment of a disease or condition.

[0146] As used herein, the term "subject" refers to animals, including mammals such as humans.

[0147] As used herein, a patient refers to a human subject.

[0148] As used herein, animals include, but are not limited to, primates, including humans, gorillas, and monkeys; rodents, such as mice and rats; poultry, such as chickens; ruminants, such as goats, cows, deer, and sheep; pigs and other animals. Non-human animals exclude humans as animals considered. Polypeptides provided herein are from any source, including animals, plants, prokaryotes, and fungi. Most polypeptides are of animal origin, including mammalian origin.

[0149] As used herein, a "composition" refers to any mixture, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0150] As used herein, a "combination" refers to any association between or among two or more items. The combination can be two or more individual items, such as two compositions or two collections, a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are generally functionally related or associated.

[0151] As used herein, combination therapy refers to the administration of two or more different therapeutic agents. The different therapeutic agents can be provided and administered separately, sequentially, intermittently, or in a single composition.

[0152] As used herein, a kit is a packaged combination for purposes including, but not limited to, activation, administration, diagnosis, and evaluation of a biological activity or property, and may include other elements such as additional reagents and instructions for using the combination or these elements.

[0153] As used herein, "unit dosage form" refers to physically discrete units suitable for human and animal subjects and individually packaged as known in the art.

[0154] As used herein, "single dosage formulation" refers to a formulation for direct administration.

[0155] As used herein, the term "multi-dose formulation" refers to a formulation that contains multiple doses of therapeutic agent, and can be administered directly to provide several single doses of therapeutic agent.Doses can be administered over minutes, hours, weeks, days, or months.Multi-dose formulations can allow for dose adjustment, dose pooling, and / or dose division.Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.

[0156] As used herein, an "article of manufacture" is a product that is manufactured and sold. As used throughout this application, this term is intended to encompass any composition provided herein that is contained in a package.

[0157] As used herein, "fluid" refers to any composition that can flow. Fluids therefore encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0158] As used herein, an isolated or purified polypeptide or protein (e.g., an isolated antibody or antigen-binding fragment thereof), or biologically active portion thereof (e.g., an isolated antigen-binding fragment), is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. A preparation can be determined to be substantially free if it appears to be free of readily detectable impurities as determined by standard analytical methods used by those skilled in the art to assess its purity, such as thin-layer chromatography (TLC), gel electrophoresis, and high-performance liquid chromatography (HPLC), or is sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as the enzymatic and biological activity, of the material. Methods for purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In such cases, further purification may increase the specific activity of the compound. As used herein, "cell extract" or "lysate" refers to a preparation or fraction made from lysed or disrupted cells.

[0159] As used herein, "control" refers to a sample that is substantially identical to the test sample, except that it may be from a healthy volunteer not treated with the test parameter or, if it is a plasma sample, not suffering from the condition of interest. A control may also be an internal control.

[0160] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide comprising "an immunoglobulin domain" includes one or more polypeptides that are immunoglobulin domains.

[0161] As used herein, the term "or" is used to mean "and / or" unless expressly indicated to refer only to alternatives or where the alternatives are mutually exclusive.

[0162] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Thus, "about 5 amino acids" means "about 5 amino acids" and also "5 amino acids."

[0163] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when said event or circumstance occurs and instances when said event or circumstance does not occur. For example, a moiety that may be variant means that the part is variant or non-variant.

[0164] Abbreviations for any protecting groups, amino acids and other compounds used herein follow their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 11(9):1726-1732), unless otherwise indicated.

[0165] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0166] B. Overview of Immunostimulatory Bacteria Modified bacteria are provided herein, referred to as immunostimulatory bacteria, which encode inhibitory RNAs, such as designed shRNAs and designed microRNAs, that accumulate and / or replicate in tumors, and that target genes whose inhibition, suppression or silencing results in tumor therapy when the RNA is expressed in the subject being treated.The bacterial strains that are used for modification are all suitable for therapeutic use.The modified immunostimulatory bacteria provided herein are used and used in methods for treating cancer.The bacteria are modified for such uses and methods.

[0167] The immunostimulatory bacteria provided herein are modified by deleting or modifying bacterial genes to attenuate their inflammatory response, and are modified to enhance anti-tumor immune responses in hosts treated with the bacteria.For example, a plasmid encoding an RNAi that inhibits checkpoint genes in the host can be contained in the bacteria, and the bacteria can be adenosine auxotrophic.Attenuation of inflammatory responses to the bacteria can be achieved by reducing TNF-alpha in the host and / or deleting the msbB gene, which knocks out the flagellin gene.For example, the bacteria can be modified to stimulate host anti-tumor activity by adding a plasmid encoding an RNAi that targets host immune checkpoints and by adding a nucleic acid with CpG.

[0168] Bacterial strains can be attenuated strains, or strains attenuated by standard methods or by modifications provided herein such that their ability to colonize is primarily restricted to immune-privileged tissues and organs, particularly immune and tumor cells, including solid tumors. Examples of bacteria include, but are not limited to, strains of the genera Salmonella, Shigella, Listeria, E. coli, and Bifidobacterium. Examples of bacterial species include Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, and Salmonella enteritidis. Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix. For example, Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamushi, Rickettsia mauseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia niger These include Bacillus subtilis, Bacillus anthracis, Bacillus subtilis, Bacillus pyogenes ...

[0169] Bacteria accumulate based on one or more characteristics, including diffusion, migration, and chemotaxis, in immune-privileged tissues or organs or environments, environments that provide nutrients or other molecules that are auxotrophic, and / or environments containing replicating cells that provide an environment for bacterial invasion and replication. Immunostimulatory bacteria and species that provide such therapeutic treatments provided herein include species of Salmonella, Listeria, and E. coli. The bacteria contain a plasmid encoding one or more short hairpin (sh)RNA construct(s) or other RNAi modalities, the expression of which inhibits or blocks the expression of targeted genes. The shRNA construct is expressed under the control of a eukaryotic promoter, such as an RNA polymerase (RNAP) II or III promoter. Typically, the RNAP III (also referred to as POLIII) promoter is constitutive, while the RNAP II (also referred to as POLII) promoter may be regulated. In some examples, the shRNA targets the gene TREX1 and inhibits its expression. In some embodiments, the plasmid encodes multiple RNAi molecules, such as shRNAs or microRNAs, that inhibit two or more checkpoint genes, such as shRNAs for inhibiting PD-L1, VISTA, SIRPα, CTNNB1, TGF-beta, and / or VEGF, and others known to those of skill in the art. When multiple shRNAs are encoded, the expression of each is under the control of a different promoter.

[0170] Among the bacteria provided herein are bacteria modified to be adenosine auxotrophic. This can be achieved by modifying or deleting genes involved in purine synthesis, metabolism, or transport. For example, disrupting the tsx gene in Salmonella species, such as Salmonella typhi, results in adenosine auxotrophy. Adenosine is immunosuppressive and accumulates at high concentrations in tumors; adenosine auxotrophy improves the antitumor activity of the bacteria, as bacteria selectively replicate in tissues rich in adenosine.

[0171] Bacteria modified to have a defective asd gene are also provided. These bacteria for in vivo use are modified to carry a functional asd gene on an introduced plasmid; this maintains selection for the plasmid so that an antibiotic-based plasmid maintenance / selection system is not required. Also provided is the use of asd-deficient strains that are genetically engineered to not contain a functional asd gene on a plasmid and thus autolyze within the host.

[0172] Also provided are bacteria modified so that they are unable to produce flagella. This can be achieved by modifying the bacteria with a deletion of a gene encoding a flagellin subunit. Modified bacteria lacking flagellin are less inflammatory and therefore better tolerated, and induce stronger anti-tumor responses.

[0173] Bacteria engineered to produce listeriolysin O, which improves plasmid delivery in phagocytic cells, are also provided.

[0174] Bacteria modified to carry low copy CpG-containing plasmids are also provided. The plasmids can further include other modifications and RNAi.

[0175] If the bacterium is a Salmonella species, the bacterium can also be engineered to grow in a manner that reduces expression of the virulence SPI-1 (Salmonella pathogenicity island-1) gene. In Salmonella, the genes responsible for virulence, invasion, survival, and extraintestinal spread are located in the Salmonella pathogenicity island (SPI).

[0176] The bacteria contain a plasmid encoding an RNAi, such as an shRNA or microRNA, that inhibits a checkpoint, such as PD-L1 or TREX1 alone, or TREX1 and one or more of a second immune checkpoint. The bacteria can be further modified for other desirable characteristics, including selection for maintaining the plasmid, particularly for preparing strains without antibiotics. The immunostimulatory bacteria may also encode therapeutic polypeptides, including anti-tumor therapeutic polypeptides and drugs.

[0177] Exemplary of the immunostimulatory bacteria provided herein are species of the genus Salmonella. Exemplary of bacteria for modification as described herein are genetically engineered strains of Salmonella typhimurium, such as strain YS1646 (ATCC Catalog #202165; see also WO 99 / 13053, also referred to as VNP20009), which are plasmid-engineered to complement the asd gene knockout and antibiotic-free plasmid maintenance.

[0178] Modified immunostimulatory bacterial strains that have been conferred adenosine auxotrophy are provided herein, as are pharmaceutical compositions comprising such strains formulated for administration to subjects, such as humans, for use in methods of treating tumors and cancer.

[0179] The genetically engineered immunostimulatory bacteria provided herein incorporate multiple synergistic modalities to induce immune reactivation in cold tumors and promote tumor antigen-specific immune responses, while simultaneously inhibiting immune checkpoint pathways that tumors exploit to subvert and evade persistent antitumor immunity. Improved tumor targeting via adenosine auxotrophy and enhanced vascular destruction improve potency while localizing inflammation and limiting systemic cytokine exposure and autoimmune toxicity observed with other immunotherapy modalities. An exemplary such modified bacterium is the S. typhimurium strain, including such modifications of strain YS1646, particularly the asd- strain.

[0180] For example, immunostimulatory bacteria are provided herein that provide shRNA-mediated gene blockade of PD-L1. Gene blockade of PD-L1 has been shown to improve tumor colonization in mice. For example, it has been shown that S. typhimurium infection in PD-L1 knockout mice leads to a bacterial burden 10 times higher than that in wild-type mice (see Lee et al. (2010) Immunol. 185:2442-2449). Thus, PD-L1 is protective against S. typhimurium infection. Provided herein are immunostimulatory bacteria, such as S. typhimurium, carrying plasmids capable of RNAi-mediated gene knockdown of TREX1, PD-L1, or PD-L1 and TREX1. Such bacteria provide anti-tumor effects due to the combination of two independent pathways, resulting in an enhanced and sustained anti-tumor immune response in a single treatment.

[0181] C. Cancer immunotherapy The immunosuppressive environment found within the tumor microenvironment (TME) is a driver of tumor initiation and progression. Cancer emerges after the immune system fails to control and contain the tumor. Multiple tumor-specific mechanisms create a tumor environment that forces the immune system to tolerate tumors and their cells instead of eliminating them. The goal of cancer immunotherapy is to aid the immune system's natural ability to eliminate tumors. Acute inflammation associated with microbial infections has been linked to spontaneous tumor regression, based on centuries of observation.

[0182] 1. Immunotherapy Several clinical cancer immunotherapies seek to disrupt the balance of immunosuppression against antitumor immunity. Strategies to stimulate immunity through direct administration of cytokines such as IL-2 and IFN-α have resulted in modest clinical responses in a minority of patients while simultaneously inducing severe systemic inflammation-associated toxicity (Sharma et al. (2011) Nat Rev Cancer 11:805-812). The immune system has evolved several checks and balances to limit autoimmunity, such as the upregulation of programmed cell death protein 1 (PD-1) on T cells and its binding to its cognate ligand, programmed death ligand 1 (PD-L1), expressed on both antigen-presenting cells (APCs) and tumor cells. Binding of PD-L1 to PD-1 interferes with the CD8+ T cell signaling pathway, impairing CD8+ T cell proliferation and effector function and inducing T cell tolerance. PD-1 and PD-L1 are two examples of numerous inhibitory "immune checkpoints," which function by downregulating immune responses. Other inhibitory immune checkpoints include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), signal-regulatory protein α (SIRPα), V-domain Ig suppressor of T-cell activation (VISTA), programmed death-ligand 2 (PD-L2), indoleamine 2,3-dioxygenase (IDO) 1 and 2, lymphocyte-activation gene 3 (LAG3), galectin-9, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and T-cell immunoglobulin and mucin domain-containing 3 (T These include IM-3, also known as hepatitis A virus cellular receptor 2 (HAVCR2), herpesvirus entry mediator (HVEM), CD39, CD73, B7-H3 (also known as CD276), B7-H4, CD47, CD48, CD80 (B7-1), CD86 (B7-2), CD155, CD160, CD244 (2B4), B- and T-lymphocyte attenuator (BTLA, or CD272), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, or CD66a).

[0183] Antibodies designed to block immune checkpoints, such as anti-PD-1 (e.g., pembrolizumab, nivolumab) and anti-PD-L1 (e.g., atezolizumab, avelumab, durvalumab), have been successful in preventing T cell anergy and permanently breaking immune tolerance. Clinical benefit has been demonstrated in only a small proportion of treated patients, and those who often exhibit autoimmune-related toxicity (see, e.g., Ribas (2015) N Engl J Med 373:1490-1492; Topalian et al. (2012) N Engl J Med 366:3443-3447). This is further evidence of the need for more effective and less toxic therapies provided herein.

[0184] Another checkpoint blockade strategy involves binding to and inhibiting costimulatory receptors on APCs, such as CD80 or CD86, and inhibiting the induction of CTLA-4 on T cells, which outcompetes costimulatory cluster differentiation 28 (CD28), which binds to the same receptor but with lower affinity. This blocks stimulatory signals from CD28 and simultaneously transmits inhibitory signals from CTLA-4, preventing T cell activation (see Phan et al. (2003) Proc. Natl. Acad. Sci. USA 100:8372-8377). Anti-CTLA-4 therapy (e.g., ipilimumab) has shown clinical success and durability in some patients, while also showing a higher incidence of severe immune-related adverse events (see, for example, Hodi et al. (2010) N Engl J Med 363:711-723; Schadendorf et al. (2015) J. Clin. Oncol. 33:1889-1894). Tumors have also been shown to develop resistance to anti-immune checkpoint antibodies, highlighting the need for more durable anti-cancer therapies, which are provided herein.

[0185] 2. Adoptive Immunotherapy In an attempt to revitalize cold tumors and make them more immunogenic, a type of immunotherapy known as adoptive cell therapy (ACT) involves various strategies that utilize immune cells and reprogram them to have anti-tumor activity (Hinrichs et al. (2011) Immunol. Rev. 240:40-51). Dendritic cell-based therapies introduce genetically engineered dendritic cells (DCs) with additional immune stimulatory properties. These therapies have been unsuccessful due to their inability to break immune tolerance to cancer (see, for example, Rosenberg et al. (2004) Nat. Med. 12:1279). A method known as GVAX, which uses whole-body irradiated tumor cells containing endogenous tumor antigens and granulocyte-macrophage colony-stimulating factor (GM-CSF) to stimulate DC recruitment, has similarly failed clinical application due to its inability to break tumor tolerance (Copier et al. (2010) Curr. Opin. Mol. Ther. 12:647-653). Sipuleucel-T (Provenge), a personalized autologous cell-based therapy, was FDA-approved in 2010 for castration-resistant prostate cancer. It utilizes APCs harvested from the patient, rearmed to express the prostatic acid phosphatase (PAP) antigen, stimulate T cell responses, and then reintroduced after lymphectomy. Unfortunately, its widespread adoption has been limited by the low observed objective response rates and high cost, limiting its use to early stages of prostate cancer (Anassi et al. (2011) PT. 36(4):197-202). Similarly, autologous T cell therapy (ATC) involves harvesting a patient's own T cells, reactivating them ex vivo to overcome tumor tolerance, and then reintroducing them into the patient after lymphectomy. ATC has had limited clinical success, primarily in melanoma, and has encountered significant safety and feasibility issues that limit its usefulness (Yee et al. (2013) Clin. Cancer Res. 19:1-3).

[0186] Chimeric antigen receptor T cell (CAR-T) therapy involves patient-derived T cells that are genetically engineered to express a fusion protein between the T cell receptor and an antibody Ig variable extracellular domain. This confers the antigen-recognition properties of an antibody with the cytolytic properties of an activated T cell (Sadelain (2015) Clin. Invest. 125:3392-400). Success has been limited to B-cell and hematopoietic malignancies, at the cost of fatal immune-related adverse events (Jackson et al. (2016) Nat. Rev. Clin. Oncol. 13:370-383). Tumors may mutate to avoid recognition by target antigens, including CD19 (Ruella et al., (2016) Comput Struct Biotechnol J. 14: 357-362) and EGFRvIII (O'Rourke et al. (2017) Sci Transl Med. Jul 19; 9:399), thereby facilitating immune escape. Furthermore, CAR-T therapies, approved in the context of hematologic malignancies, face a significant hurdle for their feasibility for treating solid tumors: overcoming the highly immunosuppressive nature of the solid tumor microenvironment. Several additional modifications to existing CAR-T therapies will be essential and potentially offer feasibility for solid tumors (Kakarla, et al. (2014) Cancer J. Mar-Apr; 20(2): 151-155). If the safety of CAR-T improves significantly and their efficacy is extended to solid tumors, the feasibility and costs associated with these labor-intensive therapies will continue to limit their widespread adoption.

[0187] 3. Cancer vaccines and oncolytic viruses Cold tumors lack T cell and dendritic cell (DC) infiltration and are non-T cell inflammatory (Sharma et al. (2017) Cell 9; 168(4):707-723). In an attempt to reactivate cold tumors and make them more immunogenic, another type of immunotherapy utilizes microorganisms that can accumulate naturally or engineered in tumors. These include viruses designed to stimulate the immune system, express tumor antigens, and subsequently activate and reprogram the immune system to reject the tumor. Viral-based cancer vaccines have largely failed clinically due to several factors, including pre-existing or acquired immunity to the viral vector itself and a lack of sufficient immunogenicity to present tumor antigens (Larocca et al. (2011) Cancer J. 17(5):359-371). The lack of adequate adjuvant activation of APCs also hampers other non-viral vector cancer vaccines, such as DNA vaccines. In contrast, oncolytic viruses preferentially replicate in dividing tumor cells rather than healthy tissue, and subsequent tumor cell lysis leads to immunogenic tumor cell death and further viral dissemination. The oncolytic virus talimogene laherparepvec (T-VEC), which uses a modified herpes simplex virus in combination with the DC-recruiting cytokine GM-CSF, is FDA-approved for metastatic melanoma (Bastin et al. (2016) Biomedicines 4(3):21). While it has demonstrated clinical benefit in some melanoma patients and less immunotoxicity than other immunotherapies, it suffers from limited intratumoral administration routes and manufacturing requirements, as well as a lack of distal tumor efficacy and broad applicability to other tumor types. Other oncolytic virus (OV)-based vaccines, including those utilizing paramyxoviruses, reoviruses, and picornaviruses, among others, have similar limitations in inducing systemic anti-tumor immunity (Chiocca et al. (2014) Cancer Immunol. Res. 2(4):295-300). Systemic administration of oncolytic viruses presents unique challenges.When administered intravenously, the virus is rapidly diluted, requiring high titers that can lead to liver toxicity. Furthermore, if pre-existing immunity is present, the virus is rapidly neutralized in the blood, and acquired immunity limits repeated dosing (Maroun et al. (2017) Future Virol. 12(4):193-213).

[0188] Among the limitations of virus-based vaccine vectors and oncolytic viruses, the greatest limitation may be the virus itself. Viral antigens have significantly higher affinity for human T cell receptors (TCRs) than tumor antigens (Aleksic et al. (2012) Eur J Immunol. 42(12):3174-3179). Furthermore, tumor antigens simultaneously presented by MHC-1 on the surface of highly active APCs with viral vector antigens will outcompete for binding to TCRs, resulting in very low antigen-specific antitumor immunity. The tumor-targeting immunostimulatory vectors provided herein, which do not themselves confer high-affinity T cell epitopes, can circumvent these limitations.

[0189] D. Bacterial cancer immunotherapy 1. Bacterial therapy The recognition that bacteria have anti-cancer activity dates back to the 1800s, when several physicians observed tumor regression in patients infected with Streptococcus pyogenes. William Coley first began researching the use of bacteria for the treatment of advanced cancers and developed a vaccine composed of S. pyogenes and Serratia marcescens that was successfully used to treat a variety of cancers, including sarcoma, carcinoma, lymphoma, and melanoma. Subsequently, several bacteria, including species of Clostridium, Mycobacterium, Bifidobacterium, Listeria such as L. monocytogenes, and Escherichia, have been investigated as sources of anti-cancer vaccines (see, e.g., WO 1999 / 013053; WO 2001 / 025399; Bermudes et al. (2002) Curr. Opin. Drug Discov. Devel. 5:194-199; Patyar et al. (2010) Journal of Biomedical Science 17:21; Pawelek et al. (2003) Lancet Oncol. 4:548-556).

[0190] Bacteria can infect animal and human cells, and some have the natural ability to deliver DNA into the cytosol of cells, making them candidate vectors for gene therapy. Bacteria are also suitable for therapeutics because they can be administered orally, propagate easily in vitro and in vivo, and can be stored and transported in lyophilized form. Bacterial genetic traits are easily manipulated, and the complete genomes of numerous strains have been fully characterized (Felgner et al. (2016) mbio 7(5):e01220-16). Consequently, bacteria have been used to deliver and express a variety of genes, including those encoding cytokines, angiogenesis inhibitors, toxins, and prodrug-converting enzymes. For example, Salmonella has been used to express immune stimulatory molecules like IL-18 (Loeffler et al. (2008) Cancer Gene Ther. 15(12):787-794), LIGHT (Loeffler et al. (2007) PNAS 104(31):12879-12883), and Fas ligand (Loeffler et al. (2008) J. Natl. Cancer Inst. 100:1113-1116) in tumors. Bacterial vectors are also less expensive, easier to produce, and can be rapidly removed with antibiotics if necessary, making bacterial delivery a preferable and safer alternative to viral delivery.

[0191] However, to be useful, the bacterial strain itself must not be pathogenic, or it must not become pathogenic after being modified for use as a therapeutic agent. For example, in cancer treatment, therapeutic bacterial strains must be attenuated or rendered sufficiently non-virulent so that they do not cause systemic disease and / or septic shock, but still maintain some level of infectivity and effectively colonize tumors. Genetically modified bacteria have been described for use as antitumor agents to induce direct tumorigenic effects and / or to deliver tumorigenic molecules (Clairmont, et al. (2000) J. Infect. Dis. 181:1996-2002; Bermudez, D. et al. (2002) Curr. Opin. Drug Discov. Devel. 5:194-199; Zhao, M. et al. (2005) Proc. Natl. Acad. Sci. USA 102:755-760; Zhao, M. et al. (2006) Cancer Res. 66:7647-7652). One of these is a bioengineered strain of Salmonella enterica serovar Typhimurium (S. Typhimurium). These bacteria preferentially accumulate in tumors at over 1,000-fold higher abundance than in healthy tissues and are homogeneously distributed in tumor tissue (Pawelek, J. et al. (1997) Cancer Res. 57:4537-4544; Low, KB et al. (1999) Nat. Biotechnol. 17:37-41). Preferential replication allows the bacteria to produce and deliver high concentrations of various anticancer therapeutic agents directly into tumors, while minimizing toxicity to healthy tissues.These attenuated bacteria are safe in mice, pigs, and monkeys when administered intravenously (Zhao, M. et al. (2005) Proc Natl Acad Sci USA 102:755-760; Zhao, M. et al. (2006) Cancer Res 66:7647-7652; Tjuvajev J. et al. (2001) J. Control Release 74:313-315; Zheng, L. et al. (2000) Oncol. Res. 12:127-135), and certain live attenuated Salmonella strains have been shown to be well tolerated after oral administration in human clinical trials (Chatfield, SN et al. (1992) Biotechnology 10:888-892; DiPetrillo, MD et al. (1999) Vaccine 18:449-459; Hohmann, EL et al. (1996) J. Infect. Dis. 173:1408-1414; Sirard, JC et al. (1999) Immunol. Rev. 171:5-26). The S. typhimurium phoP / phoQ operon is a typical bacterial two-component regulatory system consisting of a membrane-associated sensor kinase (PhoQ) and a cytoplasmic transcriptional regulator (PhoP: Miller, SI et al. (1989) Proc Natl Acad Sci USA 86:5054-5058; Groisman, EA et al. (1989) Proc Natl Acad Sci USA 86: 7077-7081).PhoP / phoQ is essential for virulence, and its deletion results in poor survival of the bacterium in macrophages and marked attenuation in mice and humans (Miller, S. I. et al. (1989) Proc Natl Acad Sci USA 86:5054-5058; Groisman, E. A. et al. (1989) Proc Natl Acad Sci USA 86: 7077-7081; Galan, J. E. and Curtiss, R. III. (1989) Microb Pathog 6:433-443; Fields, P. I. et al. (1986) Proc Natl Acad Sci USA 83:189-193). PhoP / phoQ-deficient strains have been used as effective vaccine delivery vehicles (Galan, JE and Curtiss, R. III. (1989) Microb Pathog 6:433-443; Fields, PI et al. (1986) Proc Natl Acad Sci USA 83:189-193; Angelakopoulos, H. and Hohmann, EL (2000) Infect Immun 68:213-241). Attenuated Salmonella have been used for targeted delivery of oncolytic proteins (Bermudes, D. et al. (2002) Curr Opin Drug Discov Devel 5:194-199; Tjuvajev J. et al. (2001) J Control release 74:313-315).

[0192] Bacterial-based cancer therapies have demonstrated limited clinical benefit. Examples include Clostridium novyi (Dang et al. (2001) Proc. Natl. Acad. Sci. USA 98(26):15155-15160; U.S. Patent Application Publication Nos. 2017 / 0020931, 2015 / 0147315; U.S. Patent Nos. 7,344,710, and 3,936,354), Mycobacterium bovis (U.S. Patent Application Publication Nos. 2015 / 0224151 and 2015 / 0071873), and Bifidobacterium bifidum (Kimura et al. (1980) Cancer Res. 40:2061-2068), Lactobacillus casei (Yasutake et al. (1984) Med Microbiol Immunol. 173(3):113-125), Listeria monocytogenes (Le et al. (2012) Clin. Cancer Res. 18(3):858-868; Starks et al. (2004) J. Immunol. 173:420-427; U.S. Patent Application Publication No. 2006 / 0051380), and Escherichia coli (U.S. Patent No. 9,320,787) have been investigated as potential anticancer therapeutic agents.

[0193] For example, Bacillus Calmette-Guerin (BCG) strains have been approved for the treatment of bladder cancer in humans, are more effective than intravesical chemotherapy agents, and are often used as first-line treatment (Gardlik et al. (2011) Gene therapy 18:425-431). Another approach utilizes the attenuated intracellular bacterium Listeria monocytogenes, which can induce potent CD8+ T cell priming to express tumor antigens in mice (Le et al. (2012) Clin. Cancer Res. 18(3):858-868). In a clinical trial of a Listeria-based vaccine incorporating the tumor antigen mesothelin in a prime-boost approach with the allogeneic pancreatic cancer-based GVAX vaccine, a median survival of 6.1 months was observed in patients with advanced pancreatic cancer, compared with a median survival of 3.9 months for patients treated with the GVAX vaccine alone (Le et al. (2015) J. Clin. Oncol. 33(12):1325-1333). These results were not replicated in a large Phase 2b study, possibly demonstrating the difficulties of attempting to induce immunity to low-affinity self-antigens such as mesothelin.

[0194] Bacterial strains can be modified as described and exemplified herein to express inhibitory RNA (RNAi), such as shRNA and microRNA, that inhibit or block TREX1 and / or PD-L1, and optionally one or more additional immune checkpoint genes. Strains can be attenuated by standard methods and / or by gene deletion or modification, and by altering or introducing genes that enable the bacteria to grow in vivo in primarily immune-privileged environments, such as the TME in tumor cells and solid tumors. Bacterial strains for modification as described herein can be selected from the genera Shigella, Listeria, E. coli, Bifidobacterium, and Salmonella, for example, Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, and Salmonella enteritidis. Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix.For example, Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamushi, Rickettsia mauseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia niger Examples of immunostimulatory bacteria include Bacillus subtilis, Bacillus subtilis, Bacillus thuringiensis, Bacillus subtilis ...

[0195] 2. Comparison of immune responses to bacteria and viruses Bacteria, like viruses, have the advantage of being innately immune-stimulating. It is known that bacteria and viruses contain conserved structures known as pathogen-associated molecular patterns (PAMPs) that are sensed by host cell pattern recognition receptors (PRRs). Recognition of PAMPs by PRRs triggers downstream signaling cascades that result in the induction of cytokines and chemokines, and the initiation of immune responses that result in pathogen clearance (Iwasaki and Medzhitov (2010) Science 327(5963):291-295). The manner in which PAMPs engage the innate immune system and from what type of infectious agent determines the appropriate adaptive immune response to block invading pathogens.

[0196] One class of PRRs known as Toll-like receptors (TLRs) recognizes PAMPs of bacterial and viral origin and are located in various compartments within the cell. TLRs bind to a variety of ligands, including lipopolysaccharide (TLR4), lipoproteins (TLR2), flagellin (TLR5), unmethylated CpG motifs in DNA (TLR9), double-stranded RNA (TLR3), and single-stranded RNA (TLR7 and TLR8) (Akira et al. (2001) Nat. Immunol. 2(8):675-680; Kawai and Akira (2005) Curr. Opin. Immunol. 17(4):338-344). For example, S. typhimurium host surveillance is largely mediated through TLR2, TLR4, and TLR5 (Arpaia et al. (2011) Cell 144(5):675-688). These TLR signals, via MyD88 and TRIF adaptor molecules, to mediate the induction of NF-kB, depend on pro-inflammatory cytokines such as TNF-α, IL-6, and IFN-γ (Pandey et al. (2015) Cold Spring Harb Perspect Biol 7(1):a016246).

[0197] Another category of PRRs is the nod-like receptor (NLR) family. These receptors reside in the cytosol of host cells and recognize intracellular PAMPs. For example, S. typhimurium flagellin was shown to activate the NLRC4 / NAIP5 inflammasome pathway, leading to the cleavage of caspase-1 and the induction of the pro-inflammatory cytokines IL-1β and IL-18, which then triggered cell death by pyroptosis in infected macrophages (Fink et al. (2007) Cell Microbiol. 9(11):2562-2570).

[0198] Engagement of TLR2, TLR4, TLR5, and inflammasomes induces pro-inflammatory cytokines that mediate bacterial clearance and primarily activates NF-κB-driven signaling cascades, leading to the recruitment and activation of neutrophils, macrophages, and CD4+ T cells, but not DCs and CD8+ T cells, which are essential for antitumor immunity (Lui et al. (2017) Signal Transduct Target Ther. 2:17023). To activate CD8+ T cell-mediated antitumor immunity, IRF3 / IRF7-dependent type I interferon signaling is important for DC activation, cross-presentation of tumor antigens, and CD8+ T cell priming (Diamond et al. (2011) J. Exp. Med. 208(10):1989-2003; Fuertes et al. (2011) J. Exp. Med. 208(10):2005-2016). Type I interferons (IFN-α, IFN-β) are signature cytokines induced by two distinct TLR-dependent and TLR-independent signaling pathways. The TLR-dependent pathway for IFN-β induction occurs after pathogen endocytosis, and TLRs 3, 7, 8, and 9 detect pathogen-derived DNA and RNA elements within endosomes. TLR7 and 8 recognize viral nucleosides and nucleotides, and synthetic agonists such as resiquimod and imiquimod have been clinically validated (Chi et al. (2017) Frontiers in Pharmacology 8:304). Synthetic dsRNAs such as polyinosinic polycytidylic acid (poly(I:C)) and poly(ICLC), analogs formulated with poly-L-lysine to resist RNase digestion, are agonists of the TLR3 and MDA5 pathways and potent inducers of IFN-β (Caskey et al. (2011) J. Exp. Med. 208(12):2357-66). TLR9 detection of endosomal CpG motifs present in viral and bacterial DNA can also induce IFN-β via IRF3.Furthermore, TLR4 has been shown to induce IFN-β through MyD88-independent TRIF activation of IRF3 (Owen et al. (2016) mBio.7:1 e02051-15). It was subsequently shown that the ability of TLR4 to activate DCs via type I IFNs is likely biologically irrelevant, as TLR4 activation of DCs was independent of type I IFNs (Hu et al. (2015) Proc. Natl. Acad. Sci. USA 112:45). Furthermore, TLR4 signaling has not been shown to directly recruit or activate CD8+ T cells.

[0199] One TLR-independent type I IFN pathway is mediated by host recognition of single-stranded (ss) and double-stranded (ds) RNA in the cytosol. These are sensed by RNA helicases, including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), and through the IFN-β promoter stimulator 1 (IPS-1) adaptor protein-mediated phosphorylation of the IRF-3 transcription factor, leading to IFN-β induction (Ireton and Gale (2011) Viruses 3(6):906-919). A synthetic RIG-I-binding element, in the form of an AA dinucleotide sequence in the U6 promoter transcription start site, has also been unintentionally found in common lentiviral shRNA vectors. Its subsequent deletion in the plasmid prevented confounding off-target type I IFN activation (Pebernard et al. (2004) Differentiation. 72:103-111).

[0200] The second type of TLR-independent type I interferon induction pathway is mediated through stimulator of interferon genes (STING), a cytosolic, ER-resident adaptor protein now recognized as a central mediator for sensing cytosolic dsDNA from infectious pathogens or aberrant host cell damage (Barber (2011) Immunol. Rev 243(1):99-108). STING signaling activates the TANK-binding kinase (TBK1) / IRF3 axis and the NF-kB signaling axis, leading to the induction of IFN-β and other pro-inflammatory cytokines and chemokines that potently activate innate and adaptive immunity (Burdette et al. (2011) Nature 478(7370):515-518). STING-mediated sensing of cytosolic dsDNA requires the host cell nucleotidyl transferase cyclic GMP-AMP synthase (cGAS), which directly binds to dsDNA and, in response, synthesizes the cyclic dinucleotide (CDN) second messenger cyclic GMP-AMP (cGAMP), which binds to and activates STING (Sun et al. (2013) Science 339(6121):786-791; Wu et al. (2013) Science 339(6121):826-830). Bacterial CDNs, such as c-di-AMP produced by intracellular Listeria monocytogenes, can also directly bind to murine STING, but only three of the five human STING alleles do. Unlike bacterially produced CDNs, in which two purine nucleosides are joined by a phosphate bridge using a 3'-3' linkage, the internucleotide phosphate bridge in cGAMP synthesized by mammalian cGAS is joined by a non-canonical 2'-3' linkage.These 2'-3' molecules bind to STING with 300-fold higher affinity than bacterial 3'-3' CDNs and are therefore more potent physiological ligands of human STING (see, e.g., Civilil et al. (2013) Nature 498(7454):332-337; Diner et al. (2013) Cell Rep. 3(5):1355-1361; Gao et al. (2013) Sci. Signal 6(269):pl1; Ablasser et al. (2013) Nature 503(7477):530-534).

[0201] The cGAS / STING signaling pathway in humans may have evolved over time to preferentially respond to viral pathogens over bacterial pathogens, which may explain why bacterial vaccines carrying host tumor antigens have been generated as insufficient CD8+ T cell priming vectors in humans. TLR-independent activation of CD8+ T cells by STING-dependent type I IFN signaling from conventional DCs is the primary mechanism by which viruses are detected, and the STING pathway involves TLR-dependent type I IFN production by plasmacytoid DCs, which is only operational when the virus is inactivated (Hervas-Stubbs et al. (2014) J Immunol. 193:1151-1161). Furthermore, for bacteria such as S. typhimurium, although TLR4-mediated induction of IFN-β is possible, CD8+ T cells are neither induced nor required for clearance or protective immunity (Lee et al. (2012) Immunol Lett. 148(2): 138-143). For many bacterial strains, including S. typhimurium, the lack of physiologically relevant CD8+ T epitopes, even from genetically identical strains, hinders both bacterial vaccine development and post-infection protective immunity (Lo et al. (1999) J Immunol. 162:5398-5406). Therefore, bacterial-based cancer immunotherapies are biologically limited in their ability to induce type I IFNs to recruit and activate CD8+ T cells, which are necessary for tumor antigen cross-presentation and durable antitumor immunity. Therefore, engineering the bacterial immunotherapies provided herein to induce viral-like TLR-independent type I IFN signaling rather than TLR-dependent bacterial immune signaling will preferentially induce CD8+ T cell-mediated antitumor immunity.

[0202] STING activates innate immunity in response to sensing nucleic acids in the cytosol. Downstream signaling is activated through the binding of CDNs synthesized by bacteria or the host enzyme cGAS in response to binding to cytosolic dsDNA. Bacterial and host-produced CDNs have distinct phosphate bridge structures that distinguish their ability to activate STING. IFN-β is the signature cytokine of active STING, and virus-induced type I IFN, rather than bacterial-induced IFN, is required for effective CD8+ T cell-mediated antitumor immunity. The immunostimulatory bacteria provided herein include those that are STING agonists.

[0203] 3. Treatment with Salmonella Salmonella is exemplary of a bacterial genus that can be used as a cancer therapeutic. The Salmonella exemplified herein are attenuated strains, or those modified for use as cancer therapeutics based on reduced virulence.

[0204] a. Tumor-tropic bacteria Several bacterial species have been demonstrated to replicate preferentially within solid tumors when injected from a distal site. These include, but are not limited to, species of Salmonella, Bifidobacterium, Clostridium, and Escherichia. The natural tumor-homing properties of bacteria, combined with the host's innate immune response to bacterial infection, are thought to mediate antitumor responses. This tumor tissue tropism has been shown to reduce tumor size to varying degrees. One factor contributing to the tumor tropism of these bacterial species is their ability to replicate in anoxic or hypoxic environments. Some of these naturally tumor-tropic bacteria have been further engineered to enhance their antitumor potential (reviewed in Zu et al. (2014) Crit Rev Microbiol. 40(3):225-235; and Felgner et al. (2017) Microbial Biotechnology 10(5):1074-1078).

[0205] b. Salmonella enterica serovar vertifimurium Salmonella enterica serovar typhimurium (S. typhimurium) is an exemplary bacterial species for use as an anticancer therapeutic. One approach to using bacteria to stimulate host immunity against cancer is via the Gram-negative, facultative anaerobe S. typhimurium, which preferentially accumulates in hypoxic and necrotic areas of the body, including the tumor microenvironment. S. typhimurium accumulates in these environments because it can utilize nutrients from necrotic tissue, the tumor vasculature is leaky, and it is more likely to survive in the immune-evasive tumor microenvironment (Baban et al. (2010) Bioengineered Bugs 1(6):385-294). S. typhimurium can grow under both aerobic and anaerobic conditions; therefore, it can colonize small tumors with low levels of hypoxia and large tumors with high levels of hypoxia.

[0206] S. typhimurium is a Gram-negative facultative pathogen transmitted via the fecal-oral route. This pathogen causes a localized gastrointestinal infection, but after oral ingestion, it can invade the bloodstream and lymphatic system and infect systemic tissues such as the liver, spleen, and lungs. Systemic administration of wild-type S. typhimurium overstimulates TNF-α induction, leading to cytokine cascades and septic shock, which can be fatal if left untreated. Consequently, pathogenic bacterial strains such as S. typhimurium must be attenuated to prevent systemic infection without completely suppressing their ability to effectively colonize tumor tissue. Attenuation is often achieved by mutating cellular structures capable of inducing an immune response, such as the bacterial outer membrane, or by limiting their ability to replicate in the absence of supplemental nutrients.

[0207] S. typhimurium is an intracellular pathogen that can be rapidly internalized by myeloid cells such as macrophages or induce internalization in non-phagocytic cells such as epithelial cells. Once inside the cell, it can replicate within Salmonella-containing vacuoles (SCVs) and also escape into the cytosol of some epithelial cells. Many of the molecular determinants of S. typhimurium virulence have been identified, and the genes are grouped into Salmonella pathogenicity islands (SPIs). The two best-characterized pathogenicity islands are SPI-1, which mediates bacterial invasion of non-phagocytic cells, and SPI-2, which is essential for replication within SCVs (Agbor and McCormick (2011) Cell Microbiol. 13(12): 1858-1869). Both of these pathogenicity islands encode macromolecular structures termed type III secretion systems (T3SS) that are capable of translocating effector proteins across the host membrane (Galan and Wolf-Watz (2006) Nature 444:567-573).

[0208] C. Attenuation of the bacteria Therapeutic bacteria for cancer treatment should be attenuated. Various methods for attenuating bacterial pathogens are known in the art. For example, auxotrophic mutations render bacteria unable to synthesize essential nutrients, and deletions / mutations in genes such as aro, pur, gua, thy, nad, and asd (US Patent Application Publication No. 2012 / 0009153) are widely used. The nutrients produced by the biosynthetic pathways involved in these genes are often unavailable to host cells, making bacterial survival a challenge. For example, the attenuation of Salmonella and other bacterial species can be achieved by deleting the aroA gene, which is part of the shikimate pathway and is involved in glycolysis for aromatic amino acid biosynthesis (Felgner et al. (2016) MBio 7(5):e01220-16). Thus, deletion of aroA results in aromatic amino acid auxotrophy and subsequent attenuation of the bacterium (U.S. Patent Application Publication Nos. 2003 / 0170276, 2003 / 0175297, 2012 / 0009153, 2016 / 0369282, WO 2015 / 032165, and WO 2016 / 025582). Similarly, other enzymes involved in the biosynthetic pathway for aromatic amino acids, including aroC and aroD, have been deleted to achieve attenuation (U.S. Patent Application Publication Nos. 2016 / 0369282; WO 2016 / 025582). For example, S. typhimurium strain SL7207 is an aromatic amino acid auxotroph (aroA mutant); strains A1 and A1-R are leucine-arginine auxotrophs. VNP20009 is a purine auxotroph (purI mutant). As shown herein, it is also auxotrophic for the immunosuppressive nucleoside adenosine.

[0209] Mutations that attenuate bacteria include, but are not limited to, mutations in genes that alter lipopolysaccharide biosynthesis, such as rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; mutations that introduce suicide genes, such as sacB, nuk, hok, gef, kil, or phlA; mutations that introduce lysis genes, such as hly and cly; IsyA, pag, prg, iscA, virG, There are also mutations in virulence factors, such as plc and act; mutations that alter stress responses, such as recA, htrA, htpR, hsp, and groEL; mutations that disrupt the cell cycle, such as min; and mutations that disrupt or inactivate regulatory functions, such as cya, crp, phoP / phoQ, and ompR (U.S. Patent Application Publication Nos. 2012 / 0009153, 2003 / 0170276, 2007 / 0298012; U.S. Patent No. 6,190,657; WO 2015 / 032165; Felgner et al. (2016) Gut microbes 7(2):171-177; Broadway et al. (2014) J. Biotechnology 192:177-178; Frahm et al. (2015) mBio 6(2):e00254-15; Kong et al. (2011) Infection and Immunity 79(12):5027-5038; Kong et al. (2012) PNAS 109(47):19414-19419). Ideally, genetic attenuation involves gene deletions rather than point mutations to prevent spontaneous compensatory mutations that may result in reversion to the virulent phenotype.

[0210] i.msbB-mutant The enzyme lipid A biosynthesis myristoyltransferase, encoded by the msbB gene in S. typhimurium, catalyzes the addition of a terminal myristyl group to the lipid A domain of lipopolysaccharide (LPS) (Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Thus, deletion of msbB alters the acyl composition of the lipid A domain of LPS, a major component of the outer membrane of Gram-negative bacteria. This modification significantly reduces the ability of LPS to induce septic shock, attenuates the bacterial strain, and reduces the potentially harmful production of TNFα, thus lowering systemic toxicity. S. typhimurium msbB mutants maintain their ability to preferentially colonize tumors across other tissues in mice, retain antitumor activity, and thus increase the therapeutic index of Salmonella-based immunotherapy (U.S. Patent Application Publication Nos. 2003 / 0170276, 2003 / 0109026, 2004 / 0229338, 2005 / 0225088, 2007 / 0298012).

[0211] For example, deletion of msbB in S. typhimurium strain VNP20009 results in the production of predominantly pentaacylated LPS, which is less toxic than native hexaacylated LPS and allows for systemic delivery without inducing toxic shock (Lee et al. (2000) International Journal of Toxicology 19:19-25). Other LPS mutations can be introduced into the bacterial strains provided herein, including Salmonella strains, that dramatically reduce virulence, thereby providing lower toxicity and allowing for the administration of higher doses.

[0212] ii.purI-mutant Immunostimulatory bacteria are used that can be attenuated by being made auxotrophic for one or more essential nutrients, such as purines (e.g., adenine), nucleosides (e.g., adenosine), or amino acids (e.g., arginine and leucine). In particular, in embodiments of the immunostimulatory bacteria provided herein, such as S. typhimurium, the bacteria are rendered auxotrophic for adenosine, which preferentially accumulates in the tumor microenvironment. Thus, the immunostimulatory bacterial strains described herein are attenuated because they require adenosine for growth and preferentially colonize the adenosine-rich TME, as discussed below.

[0213] The enzyme phosphoribosylaminoimidazole synthetase encoded by the purI gene (synonymous with the purM gene) is involved in the purine biosynthesis pathway. Therefore, disruption of the purI gene renders bacteria auxotrophic for purines. In addition to being attenuated, purI mutants are abundant in the tumor environment and have significant antitumor activity (Pawelek et al. (1997) Cancer Research 57:4537-4544). This colonization has been previously attributed to the high concentration of purines present in the interstitial fluid of tumors as a result of their rapid cell turnover. Because purI bacteria cannot synthesize purines, they require the external source adenine, which is thought to lead to their growth restriction in the purine-rich tumor microenvironment (Rosenberg et al. (2002) J. Immunotherapy 25(3):218-225). Although the VNP20009 strain was initially reported to contain a deletion of the purI gene (Low et al. (2003) Methods in Molecular Medicine Vol. 90, Suicide Gene Therapy:47-59), subsequent analysis of the entire VNP20009 genome demonstrated that the purI gene is not deleted but is disrupted by a chromosomal inversion (Broadway et al. (2014) Journal of Biotechnology 192:177-178). The entire gene is contained within two portions of the VNP20009 chromosome (one of which harbors an active transposase) flanking the insertion sequence.

[0214] It is shown herein that the purI mutant S. typhimurium strain is auxotrophic for the nucleoside adenosine, which is highly abundant in the tumor microenvironment. Therefore, when using VNP20009, it is not necessary to introduce any additional modifications to achieve adenosine auxotrophy. For other strains and bacteria, the purI gene may be disrupted as present in VNP20009, or may contain a deletion of all or part of the purI gene to prevent reversion to the wild-type gene.

[0215] iii. Attenuating mutation combinations Bacteria with multiple genetic attenuations using gene deletions in different regions of the chromosome are desirable for bacterial immunotherapy because they increase attenuation and simultaneously reduce the likelihood of reversion to a virulent phenotype through gene acquisition by homologous recombination with wild-type genetic material. Restoration of virulence through homologous recombination would require two separate recombination events to occur within the same organism. Ideally, the combination of attenuating mutations selected for use in immunotherapy agents would increase tolerability without reducing efficacy, resulting in an increased therapeutic index. For example, disruption of the msbB and purI genes in S. typhimurium strain VNP20009 has been used to target tumors, inhibit their growth, and induce low virulence in animal models (Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002; Bermudez et al. (2000) Cancer Gene Therapy: Past Achievements and Future Challenges, edited by Habib Kluwer Academic / Plenum Publishers, New York, pp. 57-63; Low et al. (2003) Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy:47-59; Lee et al. (2000) International Journal of Toxicology 19:19-25; Rosenberg et al. (2002) J. Immunotherapy 25(3):218-225; Broadway et al. (2014) J. Biotechnology 192:177-178;Loeffler et al. (2007) Proc. Natl. Acad. Sci. USA 104(31):12879-12883;Luo et al. (2002) Oncology Research 12:501-508).When VNP20009 (msbB- / purI-) was administered to mice bearing syngeneic or human xenograft tumors, the bacteria preferentially accumulated within the extracellular components of tumors at ratios exceeding 300–1000:1, reduced TNFα induction, tumor regression, and prolonged survival compared with control mice (Clairmont et al. (2000) J. Infect. Dis. 181:1996–2002). However, results from a phase 1 human clinical trial demonstrated that VNP20009 was relatively safe and well tolerated, but insufficient accumulation was observed in human melanoma tumors, demonstrating very little antitumor activity (Toso et al. (2002) J. Clin. Oncol. 20(1):142–152). High doses, required to demonstrate any antitumor activity, were not possible due to toxicity.

[0216] Therefore, further improvements are needed. The immunostimulatory bacteria provided herein address this challenge.

[0217] iv. VNP20009 and other attenuated S. typhimurium strains An exemplary therapeutic bacterium that can be modified as described herein is a strain designated VNP20009 (ATCC#202165, YS1646). The clinical candidate VNP20009 (ATCC#202165, YS1646) was attenuated at least 50,000-fold by deletion of both the msbB and purl genes for safety (Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002; Low et al. (2003) Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy:47-59; Lee et al. (2000) International Journal of Toxicology 19:19-25). Similar attenuated Salmonella strains are also contemplated. As mentioned above, deletion of msbB alters the composition of the lipid A domain of lipopolysaccharide, a major component of the Gram-negative bacterial outer membrane (Low et al. (1999) Nat. Biotechnol. 17(1):37-41). This prevents lipopolysaccharide-induced septic shock, attenuates the bacterial strain, reduces systemic toxicity, and simultaneously reduces the potentially harmful production of TNFα (Dinarello, CA (1997) Chest 112(6 Suppl):321S-329S; Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Deletion of the purI gene renders the bacterium auxotrophic for purines, further attenuating the bacterium and enriching it in the tumor microenvironment (Pawelek et al. (1997) Cancer Res. 57:4537-4544; Broadway et al. (2014) J. Biotechnology 192:177-178).

[0218] The accumulation of VNP20009 in tumors is due to a combination of factors, including the inherent invasiveness of the parent strain ATCC14028, its ability to replicate in hypoxic environments, and its requirement for high concentrations of purines present in tumor interstitial fluid. Given this, it follows that VNP20009, which can accumulate to pathologically high levels in the tumor microenvironment and contribute to an immunosuppressive tumor microenvironment, is also auxotrophic for the nucleoside adenosine (Peter Vaupel and Arnulf Mayer, Oxygen Transport to Tissue XXXVII, Advances in Experimental Medicine and Biology 876, chapter 22, pp. 177-183). When VNP20009 was administered to mice bearing syngeneic or human xenograft tumors, the bacteria preferentially accumulated within the extracellular components of the tumor at a ratio of over 300–1000 to 1, inhibiting tumor growth and demonstrating prolonged survival compared with control mice (Clairmont et al. (2000) J. Infect. Dis. 181:1996–2002). Results from a phase 1 clinical trial demonstrated that VNP20009 was relatively safe and well tolerated, but insufficient accumulation was observed in human melanoma tumors, demonstrating very little antitumor activity (Toso et al. (2002) J. Clin. Oncol. 20(1):142–152). High doses, which would be necessary to affect any antitumor activity, were not possible due to toxicity correlated with high levels of proinflammatory cytokines.

[0219] For example, the leucine-arginine auxotroph A-1 (Zhao et al. (2005) PNAS 102(3):755-760; Yu et al. (2012) Scientific Reports 2:436; U.S. Patent No. 8,822,194; U.S. Patent Application Publication No. 2014 / 0178341) and its derivative AR-1 (Yu et al. (2012) Scientific Reports 2:436; Kawagushi et al. (2017) Oncotarget 8(12):19065-19073; Zhao et al. (2006) Cancer Res. 66(15):7647-7652; Zhao et al. (2012) Cell Cycle 11(1):187-193; Tome et al. (2013) Anticancer Research 33:97-102; Murakami et al. (2017) Oncotarget 8(5):8035-8042; Liu et al. (2016) Oncotarget 7(16):22873-22882; Binder et al. (2013) Cancer Immunol Res. 1(2):123-133; aroA-mutant S. typhimurium strain SL7207 (Guo et al. (2011) Gene therapy 18:95-105; U.S. Patent Application Publication Nos. 2012 / 0009153, 2016 / 0369282, and 2016 / 0184456) and its obligately anaerobic derivative YB1 (Yu et al. (2012) Scientific Reports 2:436; Leschner et al. (2009) PLoS ONE 4(8): e6692; Yu et al. (2012) Scientific Reports 2:436); aroA- / aroD- mutant S. typhimurium strain BRD509, a derivative of the SL1344 (WT) strain (Yoon et al. (2017) European J. of Cancer 70:48-61); asd- / cya- / crp- mutant S. typhimurium strain χ4550 (Sorenson et al.(2010) Biology: Targets & Therapy 4:61-73) and other strains of S. typhimurium, such as phoP- / phoQ- S. typhimurium strain LH430 (WO 2008 / 091375), can be used for tumor-targeted delivery and therapy.

[0220] Although VNP20009 failed to demonstrate clinical benefit in studies involving patients with advanced melanoma, a maximum tolerated dose (MTD) was established, and treatment was safely administered to patients with advanced cancer. Therefore, this strain, as well as other similar genetically engineered bacterial strains, can be used as tumor-targeting therapeutic delivery vehicles. The modifications provided herein provide a strategy for enhancing efficacy by increasing the antitumor efficacy and / or safety and tolerability of therapeutic agents.

[0221] v. Attenuated S. typhimurium genetically engineered to deliver macromolecules Bacterial strains are engineered to deliver therapeutic molecules, such as RNAi that targets and is inhibitory to immune checkpoints and other such targets.

[0222] The use of VNP20009 in clinical trials of metastatic melanoma did not result in significant changes in metastatic tumor burden, but some evidence of tumor establishment was demonstrated. VNP20009 and other S. typhimurium strains have been used as vectors to deliver various genes, including those encoding cytokines, antiangiogenic factors, inhibitory enzymes, and cytotoxic polypeptides (U.S. Patent Application Publication No. 2007 / 0298012). For example, delivery of the cytokine-encoding LIGHT using VNP20009 inhibited primary tumor growth and lung metastasis of carcinoma cell lines in immunocompetent mice, with no significant toxicity observed (Loeffler et al. (2007) Proc. Natl. Acad. Sci. USA 104(31):12879-12883). In another study, VNP20009, expressing the E. coli cytosine deaminase gene, was administered to patients who also received the prodrug 5-fluorocytosine (5-FC) orally. Two of three patients demonstrated intratumoral bacterial colonization for at least 15 days after the initial injection, indicating that cytosine deaminase converted 5-FC to the anticancer drug 5-FU. No adverse reactions from Salmonella were observed, and direct IV administration of 5-FU resulted in lower tumor drug concentrations than did bacterial delivery of the cytosine deaminase gene (Nemunaitis et al. (2003) Cancer Gene Therapy 10:737-744).

[0223] In other examples, attenuated Salmonella expressing herpes simplex virus thymidine kinase (HSVTK) demonstrated a 2.5-fold reduction in B16 melanoma tumor size through ganciclovir-mediated tumor growth inhibition (Pawelek, J. et al. (1997) Cancer Res 57:4537-4544), and the C-terminal p53 peptide (Cp53) delivered using S. typhimurium was induced to express in MCF7 breast cancer cells, resulting in a reduction in the tumor cell population (Camacho et al. (2016) Scientific Reports 6:30591). S. typhimurium has also been used to target tumor expression of, for example, IFN-γ (Yoon et al. (2017) European J. of Cancer 70:48-61); SIINF antigen (Binder et al. (2013) Cancer Immunol Res. 1(2):123-133); Vibrio vulnificus flagellin B (Zheng et al. (2017) Sci. Transl. Med. 9, 9537); and truncated IL-2 (Sorenson et al. (2010) Biology: Targets & Therapy 4:61-73).

[0224] S. typhimurium has also been engineered to deliver the tumor-associated antigen (TAA) survivin (SVN) to APCs to prime adaptive immunity (U.S. Patent Application Publication No. 2014 / 0186401; Xu et al. (2014) Cancer Res. 74(21):6260-6270). SVN is an inhibitor of apoptosis protein (IAP) that prolongs cell survival, provides cell cycle control, and is overexpressed in all solid tumors but underexpressed in healthy tissues. This technology utilizes Salmonella pathogenicity island 2 (SPI-2) and its type III secretion system (T3SS) to deliver the TAA to the cytosol of APCs, which then activate and induce TAA-specific CD8+ T cells and antitumor immunity (Xu et al. (2014) Cancer Res. 74(21):6260-6270). Similar to Listeria-based TAA vaccines, this approach has shown promise in mouse models, but effective tumor antigen-specific T cell priming in humans has yet to be demonstrated.

[0225] In addition to gene delivery, S. typhimurium has also been used to deliver small interfering RNA (siRNA) and short hairpin RNA (shRNA) for cancer therapy. For example, attenuated S. typhimurium has been modified to express certain shRNAs, such as those targeting Stat3 and IDO1 (U.S. Patent Application Publication No. 2007 / 074272 and U.S. Patent No. 9,453,227). VNP20009 was transformed with an shRNA plasmid against the immunosuppressive gene indoramin deoxygenase (IDO), successfully silencing IDO expression in a murine melanoma model, resulting in tumor cell death and significant tumor infiltration by neutrophils (Blache et al. (2012) Cancer Res. 72(24):6447-6456). The combination of this vector with co-administration of PEGPH20, an enzyme that depletes extracellular hyaluronan, showed beneficial results in the treatment of pancreatic ductal adenocarcinoma tumors (Manuel et al. (2015) Cancer Immunol. Res. 3(9):1096-1107; U.S. Patent Application Publication No. 2016 / 0184456). In another study, S. typhimurium strains attenuated by phoP / phoQ deletion and expressing signal transducer and activator of transcription 3 (STAT3)-specific shRNA were found to inhibit tumor growth, reduce the number of metastatic organs, and extend the lifespan of C57BL6 mice (Zhang et al. (2007) Cancer Res. 67(12):5859-5864).In another example, S. typhimurium strain SL7207 has been used to deliver shRNA targeting CTNNB1, the gene encoding β-catenin (Guo et al. (2011) Gene therapy 18:95-105; U.S. Patent Application Publication Nos. 2009 / 0123426, 2016 / 0369282), while S. typhimurium strain VNP20009 has been utilized in the delivery of shRNA targeting STAT3 (Manuel et al. (2011) Cancer Res. 71(12):4183-4191; U.S. Patent Application Publication Nos. 2009 / 0208534, 2014 / 0186401, and 2016 / 0184456; WO 2008 / 091375; and WO 2012 / 149364. siRNA targeting the autophagy genes Atg5 and Beclin 1 has been delivered to tumor cells using S. typhimurium strains A1-R and VNP20009 (Liu et al. (2016) Oncotarget 7(16):22873-22882). Improvements to such strains, such as the immunostimulatory bacteria provided herein, are needed to more effectively stimulate immune responses and possess other advantageous properties.

[0226] Any of the above-mentioned bacteria can be modified as described herein, such as by adding additional shRNAs or microRNAs encoding nucleic acids targeting other checkpoints, such as TREX1. The bacteria can be modified to have a reduced inflammatory effect, as described herein, and thus be less toxic. Consequently, for example, higher dosages can be administered. These Salmonella strains, as well as other bacterial species known to those skilled in the art and / or listed above and herein, can be modified as described herein, such as by introducing adenosine auxotrophy and / or shRNA to inhibit TREX1 expression, and other modifications as described herein. An exemplary example is the S. typhimurium species described herein. In light of this, the S. typhimurium strain VNP20009 is shown to be adenosine auxotrophic.

[0227] 4. Enhancing immunostimulatory bacteria to increase the therapeutic index Enhancements to immunostimulatory bacteria that reduce toxicity and improve antitumor activity are provided herein. Exemplary of such enhancements are the following, which are described with respect to Salmonella, particularly S. typhimurium; those skilled in the art will recognize that similar enhancements can be made in other bacterial species and other Salmonella strains.

[0228] ASD gene deletion The asd gene in bacteria encodes aspartate-semialdehyde dehydrogenase. S. typhimurium asd mutants have an obligate requirement for diaminopimelic acid (DAP), essential for cell wall synthesis, resulting in lysis in DAP-deficient environments. When the asd gene is complemented in trans on a plasmid, this DAP auxotrophy can be used in vivo to select for the plasmid and maintain its stability without antibiotics. An antibiotic-free plasmid selection system would be advantageous, allowing 1) the use of antibiotics administered as a rapid clearance mechanism in adverse symptomatic cases and 2) the scale-up of antibiotic-free production when such use is normally avoided. The asd gene complementation system provides such selection (Galan et al. (1990) Gene 28:29-35). Using the asd gene complementation system to maintain plasmids in the tumor microenvironment is expected to enhance the potential of engineered S. typhimurium to deliver plasmids encoding genes or interfering RNA.

[0229] An alternative use for asd mutants of S. typhimurium is to exploit the DAP auxotrophy to deliver macromolecules to infected cells without the ability to persistently colonize host tumors, producing autolytic (or suicidal) strains. Deletion of the asd gene renders the bacteria auxotrophic for DAP when grown in vitro or in vivo. The example described herein provides an asd-deleted strain that is DAP auxotrophic and contains a plasmid suitable for delivering RNAi, such as shRNA or miRNA, resulting in a strain that does not contain the asd complementing gene and is defective for in vivo replication. This strain is propagated in vitro in the presence of DAP, grows normally, and is then administered as an immunotherapeutic agent to a mammalian host lacking DAP. The suicidal strain is capable of invading host cells but is unable to replicate due to the absence of DAP in mammalian tissues, and automatically lyses, delivering its cytosolic contents (e.g., plasmids or proteins). In the example provided herein, the asd gene deletion strain of VNP20009 was further engineered to express the LLO protein without its endogenous periplasmic secretion signal sequence, allowing it to accumulate in the cytoplasm of Salmonella. LLO is a cholesterol-dependent pore-forming hemolysin from Listeria monocytogenes that mediates bacterial escape from phagocytosis. When the autolytic strain is introduced into tumor-bearing mice, the bacteria are engulfed by phagocytic immune cells and enter Salmonella-containing vacuoles (SCVs). In this environment, the absence of DAP prevents bacterial replication and leads to bacterial autolysis in the SCV. Subsequent lysis of the suicidal strain will release the plasmid, allowing the accumulated LLO to form pores in the cholesterol-containing SCV membrane, enabling delivery of the plasmid into the host cell cytosol.

[0230] B adenosine auxotrophy Tryptophan and metabolites derived from the ATP / adenosine pathway are key drivers of the immunosuppressive environment within tumors. Adenosine, present in free form both inside and outside cells, is an effector of immune function. Adenosine reduces T cell receptor-induced activation of NF-κB and inhibits IL-2, IL-4, and IFN-γ. Adenosine reduces T cell cytotoxicity, increases T cell anergy, and enhances T cell differentiation into Foxp3+ or Lag-3+ regulatory (T-reg) T cells. Regarding NK cells, adenosine reduces IFN-γ production and suppresses NK cell cytotoxicity. Adenosine blocks neutrophil adhesion and extravasation, reduces phagocytosis, and attenuates superoxide and nitric oxide levels. Adenosine also reduces the expression of TNF-α, IL-12, and MIP-1α on macrophages, attenuates MHC class II expression, and increases the levels of IL-10 and IL-6. Adenosine immunomodulatory activity occurs after its release into the extracellular space of tumors and activation of adenosine receptors (ADRs) on the surface of target immune cells, cancer cells, or endothelial cells. High adenosine levels in the tumor microenvironment result in localized immunosuppression, which limits the immune system's ability to eliminate cancer cells.

[0231] Extracellular adenosine is produced by the continuous activity of the membrane-bound ectoenzymes CD39 and CD73 expressed on tumor stromal cells, which phosphohydrolyze ATP or ADP from dead or dying cells, along with adenosine production. CD39 converts extracellular ATP (or ADP) to 5'AMP, which is then converted to adenosine by 5'AMP. Expression of CD39 and CD73 on endothelial cells is increased under hypoxic conditions in the tumor microenvironment, resulting in increased adenosine levels. Tumor hypoxia may result from insufficient blood supply and a disrupted tumor vasculature, impairing oxygen delivery (Carroll and Ashcroft (2005) Expert. Rev. Mol. Med. 7(6):1-16). Hypoxia in the tumor microenvironment also inhibits adenylate kinase (AK), which converts adenosine to AMP and leads to extremely high extracellular adenosine concentrations. Extracellular concentrations of adenosine in the hypoxic tumor microenvironment have been measured at 10–100 μM, which is up to approximately 100–1000 times higher than the typical extracellular adenosine concentration of approximately 0.1 μM (Vaupel et al. (2016) Adv Exp Med Biol. 876:177–183; Antonioli et al. (2013) Nat. Rev. Can. 13:842–857). Because hypoxic regions in tumors are distal to microvasculature, localized concentrations of adenosine in some areas of the tumor may be higher than others.

[0232] By directly acting and inhibiting the immune system, adenosine can also control cancer cell growth and dissemination by acting on cancer cell proliferation, apoptosis, and angiogenesis. For example, adenosine can initially promote angiogenesis through stimulation of A2A and A2B receptors. Stimulating receptors on endothelial cells can regulate intercellular adhesion molecule 1 (ICAM-1) and E-selectin expression on endothelial cells, maintaining vascular integrity and promoting vascular growth (Antonioli et al. (2013 Nat. Rev. Can. 13:842-857)). Activating one or more of A2A, A2B, or A3 on various cells by adenosine can stimulate the production of angiogenic factors such as vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), or angiopoietin 2 (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857).

[0233] Adenosine can also directly regulate tumor cell proliferation, apoptosis, and metastasis through interactions with receptors on cancer cells. For example, studies have shown that it promotes tumor cell proliferation in some breast cancer cell lines, and that activation of A1 and A2A receptors, while activation of A2B receptors, has cancer growth-promoting properties in colon carcinoma cells (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Adenosine can also induce apoptosis in cancer cells, and various studies have correlated this activation with activation of the A3-mediated extrinsic apoptosis pathway or the A2A and A2B-mediated intrinsic apoptosis pathway (Antonioli et al. (2013)). Adenosine can promote tumor cell migration and metastasis by increasing cell motility, adhesion to the extracellular matrix, and the expression of cell-binding proteins and receptors, promoting cell migration and motility.

[0234] The extracellular release of adenosine triphosphate (ATP) occurs from stimulated immune cells and damaged, dead, or stressed cells. When stimulated by this extracellular release of ATP, the NLR family pyrin domain-containing 3 (NLRP3) inflammasome activates caspase-1, leading to the secretion of cytokines IL-1β and IL-18, activating innate and adaptive immune responses (Stagg and Smyth (2010) Oncogene 29:5346-5358). ATP is catabolized to adenosine by the enzymes CD39 and CD73. Activated adenosine acts as a highly immunosuppressive metabolite through a negative feedback mechanism and has pleiotropic effects on multiple immune cell types in the hypoxic tumor microenvironment (Stagg and Smyth (2010) Oncogene 29:5346-5358). The adenosine receptors A2A and A2B are expressed on various immune cells and stimulated by adenosine, promoting cAMP-mediated signaling changes that result in an immunosuppressive phenotype in T cells, B cells, NK cells, dendritic cells, mast cells, macrophages, neutrophils, and NKT cells. As a result, adenosine levels can accumulate in pathological tissues, such as solid tumors, exceeding their normal concentrations by 100 times, leading to overexpression of ectonucleotidases such as CD73. Adenosine has also been shown to promote tumor angiogenesis and progression. Therefore, genetically engineered bacteria that are adenosine auxotrophic exhibit enhanced tumor targeting and colonization.

[0235] Immunostimulatory bacteria such as Salmonella typhimurium can be rendered adenosine auxotrophic by deletion of the tsx genes (Bucarey et al. (2005) Infection and Immunity 73(10):6210-6219) or purD (Husseiny (2005) Infection and Immunity 73(3):1598-1605). In the Gram-negative bacterium Xanthomonas oryzae, knockout of the purD gene has been shown to result in adenosine auxotrophy (Park et al. (2007) FEMS Microbiol Lett 276:55-59). As exemplified herein, S. typhimurium strain VNP20009 is adenosine auxotrophic due to its purI deletion, and therefore, further modification to render it adenosine auxotrophic is not necessary. Thus, embodiments of the immunostimulatory bacterial strains provided herein are adenosine auxotrophic. Such auxotrophic bacteria selectively replicate in the tumor microenvironment, further increasing the accumulation and replication of the administered bacteria in the tumor and reducing adenosine levels in and around the tumor, thereby reducing or eliminating the immunosuppression caused by adenosine accumulation. Exemplary of such bacteria provided herein are engineered strains of S. typhimurium that contain purI / msbB mutations to provide adenosine auxotrophy.

[0236] C. flagellin-deficient strain Flagella are organelles on the surface of bacteria that consist of long filaments attached via hooks to rotary motors that can rotate in a clockwise or counterclockwise manner, providing a means for locomotion. Flagella in S. typhimurium are important for chemotaxis and for establishing infection via the oral route due to their ability to mediate motility across mucosal layers in the gastrointestinal tract. Flagella have been demonstrated to be essential for chemotaxis to and colonization of cylindrical tumors in vitro (Kasinskas and Forbes (2007) Cancer Res. 67(7):3201-3209), and motility has been shown to be important for tumor penetration (Toley and Forbes (2012) Integr Biol (Camb). 4(2):165-176). However, flagella are not essential for tumor colonization in animals when the bacteria are administered intravenously (Stritzker et al. (2010) International Journal of Medical Microbiology 300:449-456). Each flagellar filament consists of tens of thousands of flagellin subunits. The S. typhimurium chromosome contains two genes, fliC and fljB, which encode antigenically distinct flagellin monomers. Mutants defective in both fliC and fljB are nonmotile and virulent when administered via the oral route of infection but maintain virulence when administered parenterally.

[0237] Flagellin is the major pro-inflammatory determinant of Salmonella (Zeng et al. (2003) J Immunol 171:3668-3674) and is directly recognized by TLR5 on the cell surface and by NLCR4 in the cytosol (Lightfield et al. (2008) Nat Immunol. 9(10):1171-1178). Both pathways lead to a pro-inflammatory response, resulting in the secretion of cytokines including IL-1β, IL-18, TNF-α, and IL-6. Attempts are being made to create more potent Salmonella-based cancer immunotherapy by increasing the pro-inflammatory response to flagellin by genetically engineering bacteria to secrete Vibrio vulnificus flagellin B, which induces greater inflammation than the flagellin-encoded fliC and fljB (Zheng et al. (2017) Sci. Transl. Med. 9(376):eaak9537).

[0238] With this in mind, Salmonella bacteria such as S. typhimurium have been genetically engineered to lack both flagellin subunits fliC and fljB, resulting in reduced pro-inflammatory signaling. For example, as shown herein, a Salmonella strain lacking msbB, which results in reduced TNF-alpha induction, is combined with a fliC and fljB knockout. This results in a Salmonella strain with a combination of reduced TNF-alpha induction and reduced TLR5 recognition. These modifications, combined with msbB-, fliC-, and fljB-, are transformed with an immunostimulatory plasmid that may contain CpG and / or inhibitory RNAi molecules, such as shRNA or miRNA, to target immune checkpoints such as TREX1, PD-L1, VISTA, SIRP-alpha, TGF-beta, beta-catenin, VEGF, and combinations thereof. The resulting bacteria have reduced pro-inflammatory signaling yet robust antitumor activity.

[0239] For example, provided herein, a fliC and fljB double mutant was constructed in an asd deletion strain of S. typhimurium VNP20009. VNP20009, whose virulence was attenuated by disrupting purI / purM, was also engineered to contain an msbB deletion, which results in the production of a lipid A subunit that is less toxigenic than wild-type lipid A. This results in reduced TNF-α production in a mouse model after intravenous administration compared to strains with wild-type lipid A. The resulting strain is exemplary of a strain in which bacterial inflammation has been attenuated by modifying lipid A to reduce TLR2 / 4 signaling and deleting flagellin subunits to reduce TLR5 recognition and inflammasome induction. Deletion of flagellin subunits, combined with LPS modification, allows for excellent tolerability within the host and directs an immunostimulatory response to delivery of RNA interference to desired targets in the TME, inducing anti-tumor responses and promoting adaptive immune responses against tumors.

[0240] d. Salmonella genetically engineered to escape Salmonella-containing vacuoles (SCVs) Salmonella species, such as S. typhimurium, are intracellular pathogens that primarily replicate in membrane-bound compartments called Salmonella-containing vacuoles (SCVs). It has been shown that S. typhimurium can escape and replicate in the cytosol in some epithelial cell lines at low frequencies. Because the lipid bilayer of the SCV is a potential barrier, Salmonella species engineered to escape SCVs with high efficiency may be more efficient in delivering macromolecules, such as plasmids. Provided herein are Salmonella species and methods that enhance the frequency of escape from SCVs. This is achieved by deleting a gene essential for Salmonella that induces SIF (Single Insect Filament) formation. These mutants exhibit increased frequency of escape from SCVs and are able to replicate in the cytosol.

[0241] For example, enhanced plasmid delivery has been demonstrated using a sifA mutant of S. typhimurium. The sifA gene encodes SPI-2, a T3SS-2 secreted effector protein that mimics or activates the RhoA family of host GTPases (Ohlson et al. (2008) Cell Host & Microbe 4:434-446). Other genes encoding secreted effectors involved in SIF formation can be targeted. These include, for example, sseJ, sseL, sopD2, pipB2, sseF, sseG, spvB, and steA. Enhancing escape of S. typhimurium by preventing SIF formation allows surviving bacteria to be released into the cytosol, where they can replicate.

[0242] Another approach to enhance S. typhimurium escape from SCVs and increase the delivery of macromolecules such as plasmids is to express a heterologous hemolysin that leads to pore formation or disruption of the SCV membrane. One such hemolysin is the listeriolysin O protein (LLO) from Listeria monocytogenes, encoded by the hlyA gene. LLO is a cholesterol-dependent pore-forming cytolysin secreted from L. monocytogenes and primarily responsible for escape from phagocytosis and entry into the host cell cytosol. Secretion of LLO from S. typhimurium can result in bacterial escape and replication in the cytosol. To prevent intact S. typhimurium from escaping from the SCV and replicating in the cytosol, the nucleotides encoding the signal sequence can be removed from the gene. In this way, active LLO is contained within the cytoplasm of S. typhimurium, and LLO is released only upon bacterial lysis. Provided herein, VNP20009, engineered to express cytoLLO and enhance delivery of plasmids for expressing interfering RNA against targets such as TREX1, can enhance the therapeutic potential of immunostimulatory bacteria.

[0243] e. Deletion in a Salmonella gene required for biofilm formation Bacteria and fungi can form multicellular structures called biofilms. Bacterial biofilms are encapsulated in a mixture of secreted cell wall-associated polysaccharides, glycoproteins, and glycolipids, as well as extracellular DNA, collectively referred to as extracellular polymeric substances (ECPS). These EPS protect bacteria from multiple insults, including detergents, antibiotics, and antimicrobial peptides. Bacterial biofilms colonize surfaces and are a significant cause of infection in prosthetic devices, such as injection ports and catheters. Biofilms form in tissues with ongoing infection, increasing bacterial persistence and shedding and potentially limiting the effectiveness of antibiotic therapy. Chronic persistence of bacteria in biofilms is associated with increased tumorigenesis, for example, in S. typhi infections of the gallbladder (Di Domenico et al. (2017) Int. J. Mol. Sci. 18:1887).

[0244] S. typhimurium biofilm formation is regulated by CsgD, which activates the csgBAC operon and increases the production of the curli subunits CsgA and CsgB (Zakikhani et al. (2010) Molecular Microbiology 77(3):771-786). CsgA is recognized as a PAMP by TLR2 and induces IL-8 production from human macrophages (Tukel et al. (2005) Molecular Microbiology 58(1):289-304). Furthermore, CsgD indirectly increases cellulose production by activating the adrA gene, which encodes diguanylate cyclase. The small molecule cyclic di-guanosine monophosphate (c-di-GMP) produced by AdrA is a ubiquitous second messenger found in almost all bacterial species. The AdrA-mediated increase in c-di-GMP enhances expression of the cellulose synthase gene bcsA, which in turn increases cellulose production via stimulation of the bcsABZC and bcsEFG operons. A reduction in the ability of immunostimulatory bacteria such as S. typhimurium to form biofilms can be achieved through deletion of genes involved in biofilm formation, such as csgD, csgA, csgB, adrA, bcsA, bcsB, bcsZ, bcsE, bcsF, bcsG, dsbA, or dsbB (Anwar et al. (2014) Plos One 9(8):e106095).

[0245] S. typhimurium can form biofilms in solid tumors as a defense against phagocytosis by host immune cells. Salmonella mutants unable to form biofilms are more rapidly taken up by host phagocytes and removed from infected tumors (Crull et al. (2011) Cellular Microbiology 13(8):1223-1233). This increase in intracellular localization within phagocytes can reduce the persistence of extracellular bacteria, enhancing the effectiveness of plasmid delivery and gene knockdown by RNA interference, as described herein. Immunostimulatory bacteria engineered to reduce biofilm formation will increase the rate of clearance from tumors / tissues, thereby increasing the tolerability of therapy and preventing the colonization of prosthetic devices in patients, thereby enhancing the therapeutic benefit of these strains. Adenosine mimetics can inhibit S. typhimurium biofilm formation, indicating that high adenosine concentrations in the tumor microenvironment may contribute to tumor-associated biofilm formation (Koopman et al. (2015) Antimicrob Agents Chemother 59:76-84). Provided herein are live, attenuated bacterial strains, such as S. typhimurium, that contain a purI disruption (and thus colonize adenosine-rich tumors) and that have biofilm formation blocked by deletion of one or more genes essential for biofilm formation, engineered to deliver a plasmid encoding an interfering RNA that stimulates a robust antitumor immune response.

[0246] The adrA gene encodes a diguanylate cyclase that produces c-di-GMP, essential for S. typhimurium biofilm formation. c-di-GMP binds to and is an agonist for the host cytosolic protein STING. STING agonists, such as those described above, have been pursued as vaccine adjuvants for anti-cancer treatments, and bacteria have been engineered to secrete cyclic dinucleotides for use in immunotherapy (Libanova 2012, Synlogic 2018 AACR poster). Although immunostimulatory bacteria with reduced c-di-GMP production via deletion of adrA may seem counterintuitive, bacterial mutants, such as S. typhimurium mutants (including adrA mutants) that are unable to form biofilms, have demonstrated reduced therapeutic potential in mouse tumor models (Crull et al. (2011) Cellular Microbiology 13(8):1223-1233). Furthermore, some human alleles of STING are insensitive to bacterially produced 3'3' CDN binding (Corrales et al. (2015) Cell Reports 11:1022-1023).

[0247] Bacterial strains described herein, such as S. typhimurium strains that have been genetically engineered to be adenosine auxotrophic, have their ability to induce pro-inflammatory cytokines reduced by modifying LPS and / or deleting flagellin and / or deleting genes essential for biofilm formation, and have been further engineered to deliver interfering RNA, promote robust anti-tumor immune responses.

[0248] f. Deletion in a gene in the LPS biosynthetic pathway LPS of Gram-negative bacteria is a major component of the outer leaflet of the bacterial membrane. It consists of three major moieties: lipid A, a nonrepeating core oligosaccharide, and the O antigen (or O polysaccharide). The O antigen is the outermost part of LPS and serves as a protective layer against bacterial permeability, but the sugar composition of the O antigen varies widely among bacterial strains. Lipid A and the core oligosaccharide are very scarce and are more typically conserved within strains of the same bacterial species. Lipid A is the portion of LPS that contains endotoxin activity. It is typically a disaccharide modified with multiple fatty acids. These hydrophobic fatty acid chains anchor LPS to the bacterial membrane, while the rest of the LPS protrudes from the cell surface. The lipid A domain is responsible for much of the toxicity of Gram-negative bacteria. Typically, LPS in the blood is recognized as a severe pathogen-associated molecular pattern (PAMP) and induces a severe pro-inflammatory response. LPS is a ligand for a membrane-bound receptor complex containing CD14, MD2, and TLR4. TLR4 can signal through the MyD88 and TRIF pathways to stimulate the transporter membrane protein NFκB pathway, leading to the production of proinflammatory cytokines such as TNF-α and IL-1β, resulting in endotoxic shock, which can be life-threatening. LPS in the cytosol of mammalian cells can directly bind to the CARD domains of caspases 4, 5, and 11, causing autoactivation and pyroptotic cell death (Hagar et al. (2015) Cell Research 25:149-150). The composition of lipid A and the toxic potential of lipid A variants are well documented. For example, monophosphorylated lipid A is less inflammatory than lipid A with multiple phosphate groups. The number and length of acyl chains on lipid A can also have a profound effect on the degree of toxicity. Standard lipid A from E. coli has six acyl chains, and this hexaacylation is highly toxic. S. typhimurium lipid A is similar to that of E. coli; it is a glucosamine disaccharide bearing four primary and two secondary hydroxyacyl chains (Raetz and Whitfield (2002) Annu Rev Biochem. 71:635-700).The msbB mutant of S. typhimurium, as described above, is unable to undergo terminal myristoylation of its LPS and primarily produces penta-acylated LPS, which is significantly less toxic than hexa-acylated lipid A. The modification of lipid A with palmitate is catalyzed by palmitoyltransferase (PagP). Transcription of the pagP gene is under the control of the PhoP / PhoQ system, which is activated, for example, by low concentrations of magnesium within the SCV. Therefore, the acyl content of S. typhimurium is variable and, in the case of wild-type bacteria, can be hexa- or penta-acylated. The ability of S. typhimurium to palmitate its lipid A increases its resistance to antimicrobial peptides secreted into the phagolysosome.

[0249] In wild-type S. typhimurium, expression of pagP results in heptaacylated lipid A. In msbB mutants (which cannot add the terminal acyl chain of lipid A), induction of pagP results in hexaacylated LPS (Kong et al. (2011) Infection and Immunity 79(12):5027-5038). Hexaacylated LPS has been shown to be the most pro-inflammatory. Other groups have sought to exploit this pro-inflammatory signal, for example by deleting pagP so that only hexaacylated LPS is produced (Felgner et al. (2016) Gut Microbes 7(2):171-177; Felgner et al. (2018) Oncommunelogy 7(2): e1382791), but this can lead to poor tolerability due to the TNF-α-mediated pro-inflammatory nature of LPS and, paradoxically, poor adaptive immunity (Kocijancic et al. (2017) Oncotarget 8(30):49988-50001). Provided herein is a live, attenuated strain of S. typhimurium that produces only pentaacylated LPS and contains a deletion of the msbB gene (preventing terminal myristoylation of lipid A as described above) and is further modified by a deletion of pagP (preventing palmitoylation). When further modified to express an interfering RNA against an immune checkpoint such as TREX1, the strain engineered to produce pentaacylated LPS will have reduced levels of pro-inflammatory cytokines, increased susceptibility to and tolerance of antimicrobial peptides, and increased anti-tumor immunity.

[0250] g. SPI-1 gene deletion As mentioned above, in Salmonella species such as S. typhimurium, pathogenesis involves a cluster of genes called the Salmonella pathogenicity island (SPI). SPI-1 mediates epithelial cell invasion. SPI-1 encodes a type 3 secretion system (T3SS) that contributes to the translocation of effector proteins into the host cell cytosol and may provide actin for Salmonella uptake. The SPI-1 T3SS is essential for crossing the gastrointestinal epithelial layer but is not critical for infection when bacteria are injected parenterally. Injection of some proteins and the needle complex itself can also induce inflammasome activation and phagocyte pyroptosis. This pro-inflammatory cell death can limit the initiation of a robust adaptive immune response by directly inducing antigen-presenting cell (APC) death, as well as altering the cytokine environment and preventing the generation of memory T cells. The SPI-1 genes comprise several operons, including sitABCD, sprB, avrA, hilC, orgABC, prgKJIH, hilD, hilA, iagB, sptP, sicC, iacP, sipADCB, sicA, spaOPQRS, invFGEABCIJ, and invH.

[0251] As exemplified herein, live, attenuated strains of S. typhimurium engineered to deliver interfering RNA-encoding plasmids containing purI, msbB, and asd gene deletions are further modified to delete SPI-1 genes, such as regulatory genes (e.g., hilA or invF) essential for expression of the SPI-1-associated type 3 secretion system (T3SS-1), T3SS-1 structural genes (e.g., invG or prgH), or T3SS-1 effector genes (e.g., sipA or avrA). This secretion system is responsible for injecting effector proteins into the cytosol of non-phagocytic host cells, such as epithelial cells, leading to bacterial uptake. In this example, the additional deletion of the hilA gene from therapeutic Salmonella typhimurium strains administered either intravenously or intratumorally focuses S. typhimurium infection on phagocytic cells that do not require the SPI-1 T3SS for uptake, thereby extending the lifespan of these phagocytic cells. The hilA mutation also reduces the amount of pro-inflammatory cytokines, increasing the tolerability of the therapy as well as the quality of the adaptive immune response.

[0252] h. Endonuclease (endA) mutation to increase plasmid delivery The endA gene (e.g., SEQ ID NO: 250) encodes an endonuclease (e.g., SEQ ID NO: 251) that mediates degradation of double-stranded DNA in the periplasm of Gram-negative bacteria. Mutations in the endA gene enable high yields of plasmid DNA, so the most common strains of laboratory E. coli are endA-. This gene is conserved with the bacterial species. To facilitate intact plasmid DNA delivery, the endA gene of genetically engineered immunostimulatory bacteria is deleted or mutated to prevent its endonuclease activity. An exemplary such mutation is the E208K amino acid substitution (Durfee, et al. (2008) J. Bacteriol. 190(7):2597-2606) or a corresponding mutation in the bacterial species of interest. The E208-containing endA is conserved with bacterial species, including Salmonella. Therefore, the E208K mutation can be used to eliminate endonuclease activity in other bacterial species, including Salmonella species. Those skilled in the art can introduce other mutations or deletions to eliminate endA activity. This mutation, deletion, or disruption of the gene to eliminate endA activity in the immunostimulatory bacteria herein, such as Salmonella, increases the efficiency of intact plasmid DNA delivery, resulting in increased expression of RNAs, such as shRNAs and / or miRNAs, that target one or more of the immune checkpoints encoded in the plasmid, thereby increasing RNAi-mediated knockdown of checkpoint genes and enhancing anti-tumor efficacy.

[0253] i. RIG-I inhibition One of the TLR-independent type I IFN pathways is mediated by host recognition of single-stranded (ss) and double-stranded (ds) RNA in the cytosol. These are sensed by RNA helicases, including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), which induce type I IFN induction through the IFN-β promoter stimulator 1 (IPS-1) adaptor protein-mediated phosphorylation of the IRF-3 transcription factor (Ireton and Gale (2011) Viruses 3(6):906-919). RIG-I recognizes dsRNA and ssRNA with 5'-triphosphates. This moiety can bind directly to RIG-I or can be synthesized by poly(dA-dT)-dependent RNA polymerase III (Pol III) from a poly(dA-dT) template (Chiu, YH et al. (2009) Cell 138(3):576-91). A poly(dA-dT) template containing two AA dinucleotide sequences occurs at the U6 promoter transcription start site in a common lentiviral shRNA cloning vector. Its subsequent deletion in the plasmid blocks type I IFN activation (Pebernard et al. (2004) Differentiation. 72:103-111). The RIG-I binding sequence can be included in the plasmids provided herein; its inclusion can increase immune stimulation, which increases the antitumor activity of the immunostimulatory bacteria herein.

[0254] j.DNase II inhibition Another nuclease responsible for exogenous and self-DNA degradation is the endonuclease DNase II, which resides in endosomal compartments and degrades DNA after apoptosis. The absence of DNase II (Dnase2a in mice) leads to the accumulation of endosomal DNA, which escapes into the cytosol and activates cGAS / STING signaling (Lan YY et al. (2014) Cell Rep. 9(1):180-192). Similar to TREX1, DNase II deficiency in humans results in autoimmune type I interferonopathy. In cancer, dead tumor cells engulfed by tumor-resident macrophages prevent cGAS / STING activation and potential autoimmunity via DNase II digestion of DNA in the endosomal compartment (Ahn et al. (2018) Cancer Cell 33:862-873). Thus, embodiments of the immunostimulatory bacterial strains provided herein encode RNAi, such as shRNA or miRNA, that can inhibit, suppress, or block the expression of DNase II and inhibit DNase II in the tumor microenvironment, resulting in the accumulation of endocytosed apoptotic tumor DNA in the cytosol and can act as potent cGAS / STING agonists.

[0255] k.RNase H2 inhibition While TREX1 and DNase II function to remove abnormal DNA accumulation, RNase H2 similarly functions to remove pathogenic accumulations of RNA:DNA hybrids in the cytosol. Similar to TREX1, RNase H2 deficiency also contributes to the autoimmune phenotype of Aicardi-Goutières syndrome (Rabe, B. (2013) J Mol Med. 91:1235-1240). Notably, loss of RNase H2 and the subsequent accumulation of RNA:DNA hybrids or genome-embedded ribonucleotide substrates have been shown to activate cGAS / STING signaling (MacKenzie et al. (2016) EMBO J. Apr15;35(8):831-44). Thus, embodiments of the immunostimulatory bacterial strains provided herein encode RNAi, such as shRNA or miRNA, that inhibit, suppress, or block the expression of RNAse H2, thereby inhibiting RNase H2 and resulting in tumor-derived RNA:DNA hybrids and their derivatives, which activate cGAS / STING signaling and anti-tumor immunity.

[0256] l. Stabilin-1 / CLEVER-1 inhibition Another molecule that is primarily expressed on monocytes and is involved in immunoregulation is stabilin-1 (also known by the gene names STAB1, CLEVER-1, and FEEL-1). Stabilin-1 is a type I transport membrane protein that is upregulated on endothelial cells and macrophages, particularly tumor-associated macrophages, after inflammation (Kzhyshkowska et al. (2006) J. Cell. Mol. Med. 10(3):635-649). When inflammation is activated, stabilin-1 acts as a scavenger, assisting wound healing and apoptotic body clearance and preventing tissue damage such as liver fibrosis (Rantakari et al. (2016) PNAS 113(33):9298-9303). Upregulation of stabilin-1 has been shown to directly inhibit antigen-specific T cell responses, and knockdown by siRNA in monocytes enhances their pro-inflammatory function (Palani, S. et al. (2016) J Immunol. 196:115-123). Accordingly, embodiments of immunostimulatory bacterial strains provided herein encode RNAi, such as shRNA or miRNA, that inhibit, suppress, or block expression of stabilin-1 / CLEVER-1 in the tumor microenvironment, thereby enhancing the pro-inflammatory function of tumor-resident macrophages.

[0257] m. Bacterial culture conditions Culture conditions for bacteria can affect their gene expression. It has been reported that S. typhimurium can induce rapid, proinflammatory, caspase-dependent cell death in macrophages, but not epithelial cells, within 30–60 minutes of infection through a mechanism involving SPI-1 and its associated T3SS-1 (Lundberg et al. (1999) Journal of Bacteriology 181(11):3433–3437). This cell death is now known to be mediated by activation of the inflammasome, which activates caspase-1, which then promotes the maturation and release of IL-1β and IL-18, initiating a novel form of cell death termed pyroptosis (Broz and Monack (2011) Immunol Rev. 243(1):174–190). This pyroptotic activity can be induced using log-phase bacteria, whereas stationary-phase bacteria do not induce this rapid cell death in macrophages. The SPI-1 gene is induced during logarithmic phase growth. Therefore, by harvesting S. typhimurium to be used therapeutically at stationary phase, rapid pyroptosis of macrophages can be prevented. Macrophages are important mediators of the innate immune system, and they can act to secrete cytokines that are important for establishing an appropriate anti-tumor response. Furthermore, limiting the secretion of pro-inflammatory cytokines such as IL-1β and IL-18 will improve the tolerability of the administered S. typhimurium therapy. The immunostimulatory S. typhimurium harvested at stationary phase provided herein will be used to induce anti-tumor responses.

[0258] E. Construction of Exemplary Plasmids The immunostimulatory bacteria provided herein are modified, containing bacterial genomes and modifications to express bacterial genes and plasmids encoding products that are expressed in the bacterium by including a bacterial promoter, or in the host by including a suitable eukaryotic promoter and other regulatory regions as needed.

[0259] To introduce the plasmid, bacteria are transformed using standard methods such as electroporation with purified DNA plasmids constructed using routine molecular biology tools (DNA synthesis, PCR amplification, digestion with DNA restriction enzymes, and ligation of compatible cohesive end fragments with ligase).

[0260] As discussed below, the plasmid encodes one or more short hairpin (sh)RNA construct(s) or other inhibitory RNA modalities, the expression of which inhibits or blocks the expression of the targeted gene. RNAi constructs, such as shRNAs or microRNAs, are expressed under the control of a eukaryotic promoter, such as an RNA polymerase (RNAP) II or III promoter. Typically, the RNAP III (also referred to as POLIII) promoter is constitutive, while the RNAP II (also referred to as POLII) promoter may be regulated. In some examples, the shRNA targets the gene TREX1 and inhibits its expression. In some embodiments, the plasmid encodes multiple shRNAs targeted to inhibit two or more checkpoint genes, such as shRNAs for inhibiting PD-L1, VISTA, SIRPα, CTNNB1, TGF-beta, and / or VEGF, and others known to those skilled in the art. When multiple RNAi constructs, such as shRNAs, are encoded, the expression of each is under the control of a different promoter.

[0261] Bacterial strains, such as Salmonella strains, including S. typhimurium, provided herein are modified or identified to be adenosine auxotrophic in the tumor microenvironment and to carry a plasmid containing a gene encoding shRNA or microRNA that can knock down the gene expression of TREX1, PD-L1, VISTA, SIRP-alpha, beta-catenin, TGF-beta, and VEGF. S. typhimurium can infect multiple cell types, including both tumor cells and macrophages. In cells infected with S. typhimurium, the plasmid is released and can be transcribed by RNA polymerase. The shRNA produced can then be processed and interfere with the expression of target mRNA genes.

[0262] 1. Interfering RNA (RNAi) The plasmid herein encodes an RNAi nucleic acid that targets checkpoints and other desired targets as described above. RNAi includes shRNA, siRNA, and microRNA. RNA interference (RNAi) allows sequence-selective suppression of gene expression in eukaryotic cells using small interfering RNA (siRNA), a short synthetic dsRNA molecule with a sequence homologous to the target gene. RNAi technology provides a powerful tool for depleting disease-related transcription.

[0263] a.shRNA Typically approximately 19–29 base pairs in length, siRNAs function by degrading specific host mRNA sequences, preventing their translation into their respective protein products and effectively silencing the expression of the targeted gene. Short hairpin RNAs (shRNAs), which contain tight hairpin loops, are widely used in RNAi. shRNAs contain two complementary RNA sequences, each 19–29 bp long, linked by a 4–15 nucleotide loop spacer. The RNA sequence that is complementary to the target gene sequence (and therefore identical to the mRNA sequence) is known as the "sense" strand, while the strand that is simultaneously complementary to the mRNA (and identical to the target gene sequence) is known as the "antisense" or "guide" strand. The shRNA transcript is processed by the RNase III enzyme known as Dicer into siRNA duplexes. The product is then loaded into the RNA-induced silencing complex (RISC) with Argonaute (Ago) proteins and other RNA-binding proteins. RISC then localizes the antisense or "guide" strand to its complementary mRNA sequence, which is then cleaved by Ago (U.S. Patent No. 9,624,494). The use of shRNA is more cost-effective and preferable to siRNA because high intracellular concentrations of siRNA are associated with off-target effects and the concentration of siRNA becomes diluted during cell division. On the other hand, the use of shRNA results in stable and long-term gene knockdown, eliminating the need for multiple rounds of transfection (Moore et al. (2010) Methods Mol. Bio. 629:141-158).

[0264] Targets of interest for RNAi, such as microRNA and siRNA / shRNA-mediated silencing, include, but are not limited to, developmental genes such as cytokines and their receptors, cyclin kinase inhibitors, neurotransmitters and their receptors, and growth / differentiation factors and their receptors; oncogenes such as BCL2, ERBA, ERBB, JUN, KRAS, MYB, and MYC; tumor suppressor genes such as BRCA1, BRCA2, MCC, and p53; and enzymes such as ACC synthase and oxidase, ATPase, alcohol dehydrogenase, amylase, catalase, DNA polymerase, RNA polymerase, kinase, lactase, and lipase (U.S. Patent Nos. 7,732,417, 8,829,254, 8,383,599, 8,426,675, and 9,624,494; U.S. Patent Application Publication No. 2012 / 0009153). Of particular interest are PD-1, PD-2, PD-L1, PD-L2, CTLA-4, IDO1 and 2, CTNNB1 (β-catenin), SIRPα, VISTA, RNASEH2, and DNASE Immune checkpoint targets include II, CLEVER-1 / stabilin-1, LIGHT, HVEM, LAG3, TIM3, TIGIT, galectin-9, KIR, GITR, TIM1, TIM4, CEACAM1, CD27, CD40 / CD40L, CD48, CD70, CD80, CD86, CD112, CD137 (4-1BB), CD155, CD160, CD200, CD226, CD244 (2B4), CD272 (BTLA), B7-H2, B7-H3, B7-H4, B7-H6, ICOS, A2aR, A2bR, HHLA2, ILT-2, ILT-4, gp49B, PIR-B, HLA-G, ILT-2 / 4, and OX40 / OX-40L.Other targets include MDR1, arginase 1, iNOs, IL-10, TGF-β, pGE2, STAT3, VEGF, KSP, HER2, Ras, EZH2, NIPP1, PP1, TAK1, and PLK1 (U.S. Patent Application Publication Nos. 2008 / 091375, 2009 / 0208534, 2014 / 0186401, 2016 / 0184456, 2016 / 0369282; WO 2012 / 149364, WO 2015 / 002969, WO 2015 / 032165, WO 2016 / 025582).

[0265] Bacteria are attractive vectors for tumor-targeted delivery of siRNA and shRNA. Salmonella species, for example, are known to express IDO (Blache et al. (2012) Cancer Res. 72(24):6447-6456; Manuel et al. (2015) Cancer Immunol. Res. 3(9):1096-1107; U.S. Patent Application Publication Nos. 2014 / 0186401, 2016 / 0184456; WO 2012 / 149364, WO 2015 / 002969); STAT3 (Manuel et al. (2011) Cancer Res. 71(12):4183-4191; Zhang et al. (2007) Cancer Res. 67(12):5859-5864; U.S. Patent Application Publication Nos. 2014 / 0186401 and 2016 / 0184456; WO 2008 / 091375, WO 2012 / 149364, WO 2015 / 002969, and WO 2015 / 032165; β-catenin (Guo et al. (2011) Gene therapy 18:95-105; WO 2015 / 032165) and CTLA-4 (U.S. Patent Application Publication Nos. 2014 / 0186401, 2016 / 0184456; WO 2012 / 149364, WO 2015 / 002969).

[0266] Expressed RNAi, such as shRNA, mediates long-term, stable knockdown of their target transcripts as long as the shRNA is transcribed. RNA Pol II and III promoters are used to drive the expression of shRNA constructs depending on the type of expression required. Consistent with their role in the production of abundant endogenous small RNAs in healthy cells, Pol III promoters (such as U6 or H1) drive high levels of constitutive shRNA expression, with clearly defined transcription start sites and termination signals (4-6 thymidines). Pol II promoter-driven shRNAs can be expressed in a tissue-specific manner and are transcribed as longer precursors with cap and poly(A) signals that mimic pri-miRNAs and are processed. Such artificial miRNAs / shRNAs are efficiently incorporated into RISC, contributing to more potent inhibition of target gene expression; this allows for low-level shRNA expression and prevents saturation of components in the RNAi pathway. An additional advantage of Pol II promoters is that a single transcript can simultaneously express several miRNAs and mimic shRNAs. This multiplexing strategy can be used to simultaneously knock down the expression of two or more therapeutic targets or to target several locations in a single gene product (see, e.g., U.S. Patent Application Publication No. 2009 / 0208534).

[0267] B microRNA MicroRNAs (miRNAs) are short, non-coding, single-stranded RNA molecules approximately 20-24 nucleotides in length. Naturally occurring miRNAs are involved in the post-transcriptional regulation of gene expression; miRNAs do not encode genes. miRNAs have been shown to regulate cell proliferation and survival, as well as cell differentiation. miRNAs inhibit translation or promote RNA degradation by binding to target mRNAs with which they share sequence complementarity. They affect mRNA stability and translation; miRNAs inhibit translation and / or promote RNA degradation by binding to target mRNAs with which they share sequence complementarity. miRNAs present in eukaryotes are transcribed by RNA Pol II into capped, polyadenylated, hairpin-containing primary transcripts known as primary miRNAs, or pri-miRNAs. These pri-miRNAs are cleaved by the enzyme Drosophila RNase III and its cofactor Pasha / DGCR8 into approximately 70 nucleotide-long precursor miRNA hairpins known as precursor miRNAs or pre-miRNAs, which are then transported from the nucleus to the cytoplasm and cleaved by Dicer RNase III into miRNAs with approximately 22 nucleotide-long sense and antisense strand products. * The mature miRNA is incorporated into an RNA-induced silencing complex (RISC), which recognizes and binds to the target mRNA, usually in the 3' untranslated region (UTR) via an imperfect base pairing with the miRNA, resulting in translation inhibition or destabilization / degradation of the target mRNA (see, e.g., Auyeung et al. (2013) Cell 152(4):844-85).

[0268] As described herein, regulating gene expression by RNA interference (RNAi) often uses short hairpin RNA (shRNA) to inhibit, block, or otherwise interfere with the expression of targeted genes. While advantageously used and as used herein, in some cases, shRNA is insufficient to serve as a substrate for small RNA biogenesis factors, and can be processed into a heterogeneous mixture of small RNAs, and their precursor transcripts can accumulate in cells, inducing sequence-independent nonspecific effects and causing in vivo toxicity. miRNA is also considered for use herein. miRNA-like scaffolds, or artificial miRNAs (amiRNAs), can be used to reduce sequence-independent nonspecific effects (Watanabe et al. (2016) RNA Biology 13(1):25-33; Fellmann et al. (2013) Cell Reports 5:1704-1713). In addition to an improved safety profile, amiRNAs are more easily transcribed by Pol II than shRNAs, allowing for regulated cell-specific expression. Artificial miRNAs (amiRNAs) can silence gene expression more effectively, and in some cases more potently, than shRNAs, without generating large amounts of inhibitory RNA (McBride et al. (2008) Proc. Natl. Acad. Sci. USA 105(15):5868-5873). This effect has been determined to be due to more efficient siRNA processing from pre-miRNA precursors rather than from shRNA transcription (Boden et al. (2004) Nucl Acid Res 32(3):1154-1158).

[0269] miRNAs have been shown to regulate several cellular processes, including cell proliferation and survival, intracellular signal transduction, cell metabolism, and cell differentiation. In 1993, the first miRNA was identified in C. elegans (Lee et al. (1993) Cell 75:843-854), and subsequently, mammalian miRNAs were identified (Pasquinelli et al. (2000) Nature. 408(6808):86-89).Over 17,000 miRNAs have been identified in 142 bacterial species, and over 1,900 miRNAs have been identified in humans, any of which may be involved in cancer (e.g., miR-15 and miR-16 in B-CLL, miR-125b, miR-145, miR-21, miR-155, and miR-210 in breast cancer, miR-155 and let-7a in lung cancer, miR-145 in gastric cancer, and miR-29b in liver cancer); viral infections (e.g., miR-122 and miR-155 in HCV infection, miR-28, miR-125b, miR-150, miR-223, and miR-382 in HIV-1 infection, and miR-21 and miR-223 in influenza virus infection); immune-related diseases (e.g., miR-122 and miR-155 in HCV infection, miR-28, miR-125b, miR-150, miR-223, and miR-382 in HIV-1 infection, and miR-21 and miR-223 in influenza virus infection); -145, miR-34a, miR-155 and miR-326, miR-146a in systemic lupus erythematosus, miR-144, miR-146a, miR-150, miR-182, miR-103 and miR-107 in type 1 diabetes, miR-200a, miR-200b, miR-429, miR-122, miR-451 and miR-27 in non-alcoholic fatty liver disease, miR-29c, miR-34a, miR-155, and miR-200b in nonalcoholic steatohepatitis; and neurodegenerative diseases (e.g., miR-30b, miR-30c, miR-26a, miR-133b, miR-184*, and let-7 in Parkinson's disease, and miR-29b-1, miR-29a, and miR-9 in Alzheimer's disease) (Li and Kowdley (2012) Genomics Proteomics Bioinformatics 10:246-253).

[0270] Research has shown that specific endogenous miRNAs are up-regulated or down-regulated in certain cancers.For example, miR-140 is down-regulated in non-small cell lung cancer (NSCLC), and its overexpression is found to suppress PD-L1 (Xie et al. (2018) Cell Physiol. Biochem. 46:654-663); miR-197 is down-regulated in platinum-based chemotherapy-resistant NSCLC, leading to chemotherapy resistance, tumorigenesis and metastasis (Fujita et al. (2015) Mol Ther 23(4):717-727); Some miRNAs are down-regulated in cancer cells and have been found to enable the expression of PD-L1, including miR-200, miR-34a and miR-138 (Yee et al. (2017) J. Biol. Chem. 292(50):20683-20693). Some miRNAs, such as miR-21, miR-17, and miR-221, are also upregulated in lung cancer (Xie et al. (2018 Cell Physiol. Biochem. 46:654-663).

[0271] MicroRNA-103 (miR-103) was identified as the most upregulated microRNA in endothelial cells as a result of genotoxic stress and DNA damage after irradiation. miR-103 was found to downregulate the TREX1, TREX2, and FANCF genes, and a decrease in TREX1 expression was identified as the primary mechanism by which miR-103 mediates cell death and angiogenesis inhibition (Wilson et al. (2016) Nature Communications 7:13597). Because loss of TREX1 leads to the accumulation of ds- and ss-DNA, defective DNA repair, and cytokine release, Wilson et al. investigated whether miR-103 regulates cytokine expression. The results showed that miR-103 expression significantly upregulated the pro-inflammatory chemokines IP-10, RANTES, and MIG, and the cytokines IL-15, IL-12, and IFN-γ, and this upregulation was due to a miR-103-mediated decrease in TREX1 levels. The study also revealed a significant increase in the costimulatory receptors CD40 and CD160, and a decrease in the number of PD-L1+ macrophages and neutrophils in 4T1 tumors. Thus, miR-103 regulation of TREX1 is a potent modulator of the immune TME. Other miRNAs that target TREX1 include miR-107 (U.S. Patent No. 9,242,000), miR-27a, and miR-148b (U.S. Patent No. 8,580,757). miRNA-103 can be used in the plasmids herein to inhibit TREX1.

[0272] Artificial miRNAs (amiRNAs) can be delivered to cells and used to silence targeted genes by generating microRNA-based siRNA or shRNA vectors (shRNAmirs). The miR-30a backbone is often used in mammals, where a primary miRNA transcript of approximately 200–300 bases is contained within the vector, with a miRNA hairpin positioned at the center of the fragment and the native miRNA stem sequence replaced with the desired siRNA / shRNA-encoding sequence. Viral promoters such as CMV, MSCV, and TLR promoters; cellular promoters such as EIF-1a; inducible chimeric promoters such as tet-CMV; and tissue-specific promoters can be used (Chang et al. (2013) Cold Spring Harbour Protoc; doi:10.1101 / pdb.prot075853). Other miRNAs that can be used include mir-16-2 (Watanabe et al. (2016) RNA Biology 13(1):25-33), miR-155 (Chung et al. (2006) Nuc Acids Res 34:e53), miR17-92 (Liu et al. (2008) Nuc Acids Res 36(9):2811-2824), miR-15a, miR-16, miR-19b, miR-20, miR-23a, miR-27b, miR-29a, miR-30b, miR-30c, miR-104, miR-132s, miR-181, miR-191, miR-223 (U.S. Patent No. 8,426,675), and Let-7. miRNAs (WO 2009 / 006450; WO 2015 / 032165).

[0273] shRNAmirs are limited by the low computational efficacy of shRNA sequences, especially when expressed under low- or single-copy conditions. Third-generation artificial miRNAs, such as miR-E (based on miR-30a) and miR-3G (based on miR-16-2), have been developed and have been found to exhibit more potent gene silencing compared to shRNAmirs in both Pol II and Pol III-based expression vectors due to enhanced processing and accumulation of precisely defined guide RNAs. miR-E, developed through the discovery of a conserved CNNC motif within the stem 3p flanking sequence to enhance miRNA processing, differs from endogenous miR-30a in three aspects: the stem of miR-E is not expanded and has a guide strand on the opposite strand; two conserved base pairs adjacent to the loop are mutated from CU / GG to UA / UA; and XhoI / EcoRI restriction sites have been introduced into the flanking region for shRNA cloning (Fellmann et al. (2013) Cell Reports 5:1704-1713). Although miR-E was found to be more potent than miR-30a, the symmetric processing of both the 3p and 5p strands of miR-30a does not favor guide strand delivery over passenger strand delivery, which is suboptimal. Furthermore, cloning into miR-E using oligos longer than 100 nt is expensive and time-consuming (Watanabe et al. (2016) RNA Biology 13(1):25-33).

[0274] The amiRNA designated miR-16-2 (see, e.g., Watanabe et al. (2016) RNA Biology 13(1):25-33, Figure 1) is a third-generation (3G) amiRNA scaffold alternative; it is expressed in several tissues, is naturally asymmetric (the mature strand originates only from the 5p or 3p arm of the stem), and its stem and loop segments are small and rigid, simplifying vector cloning. miR-3G is generated by cloning an approximately 175-bp fragment containing the native miR-16-2 stem and loop, as well as 35-bp flanking sequences on both sides of the stem, into a vector. miR-3G contains further modifications of miR-16-2 by introducing cloning sites such as MluI and EcoRI into the 5p and 3p arm flanking sequences, respectively, and by perfectly base-pairing the guide (antisense) and passenger (sense) strand stems to the guide strand except for a mismatch at position 1. The restriction sites allow for the generation of novel targeting constructs via 88-mer double-stranded DNA oligonucleotides without disrupting the predicted secondary structure of the miR-16-2 hairpin and flanking elements. Furthermore, two CNNC motifs and one of the GHG motifs (small RNA processing enhancers) are modified in the 3p flanking sequence of miR-16-2. An siRNA targeting the gene(s) of interest is then exchanged for the first 21 nucleotides of the mature 5p guide and 3p passenger sequences. Studies have determined that miR-E and miR-3G are similarly potent. Due to the small size of its expression cassette (approximately 175 nt compared to approximately 375 for miR-E), miR-3G offers an attractive RNAi system and a simplified, cost-effective, single-step cloning method for its production. Similar to shRNAs, microRNAs can be delivered in vivo using bacteria as vectors. For example, it was shown that attenuated S. typhimurium can be used as a vector to orally deliver a plasmid expressing miRNA for CCL22 in mice exhibiting inflammation.Downregulation of CCL22 gene expression using this method was successful both in vitro and in vivo in a mouse model of atopic dermatitis (Yoon et al. (2012) DNA and Cell Biology 31(3):289-296). For purposes herein, miRNA16-2 can be used to generate miRNAs to be used in place of shRNAs. The sequence for the shRNA can be used to design miRNAs.

[0275] DNA encoding an RNAi that blocks and / or inhibits and / or targets any of the selected target genes, such as any immune checkpoint described herein or known to those skilled in the art, is inserted into a microRNA scaffold, such as the microRNA scaffold set forth in SEQ ID NO: 249 and shown below. Any suitable microRNA scaffold known to those skilled in the art can be used; generally, such scaffolds are based on naturally occurring microRNAs and modified to express RNAi. An exemplary such scaffold is one based on miR-16-2 (SEQ ID NO: 248). The sequence of the modified microRNA scaffold is: 5'-CCGGATC AACGCCCTAG GTTTATGTTT GGATGAACTG ACATACGCGT ATCCGTC NNNNNNNNNNNNNNNNNNNNNNN GTAG TGAAATATAT ATTAAAC NNNNNNNNNNNNNNNNNNNNN TACGGTAACGCG GAATTCGCAA CTATTTTATC AATTTTTTGC GTCGAC-3' (SEQ ID NO: 249) where N represents complementary antisense and sense nucleotide sequences, typically 18-26, e.g., 19-24, 19-22, or 19-20 base pairs in length, that target the gene to be silenced and are inserted before and after the microRNA loop. RNAs such as ARI-205 (SEQ ID NO: 214) and ARI-206 (SEQ ID NO: 215) are exemplary constructs based on the microRNA backbone of SEQ ID NO: 249 and encode 21 and 22 base pair homologous sequences, respectively. ARI-207 (SEQ ID NO: 216) and ARI-208 (SEQ ID NO: 217) are exemplary constructs based on the microRNA backbone of SEQ ID NO: 249 and encode 19 base pair homologous sequences. Another example is a construct designated ARI-201, which is the microRNA construct ARI-205, in which N is replaced with a sequence of nucleotides that targets mouse PD-L1. The construct designated ARI-202 represents the microRNA construct ARI-206, where N is replaced with a sequence targeting mouse PD-L1. Those skilled in the art can readily construct microRNAs for inclusion in plasmids as described and exemplified herein using the miR-16-2 backbone, or other suitable backbones known to those skilled in the art.

[0276] 2. Origin of Replication and Plasmid Copy Number Plasmids are autonomously replicating, extrachromosomal, circular, double-stranded DNA molecules maintained in bacteria using an origin of replication. Copy number influences plasmid stability; a high copy number generally provides a plasmid with better stability in the event of random partitioning during cell division. High plasmid numbers generally result in a slower growth rate, potentially allowing cells with fewer plasmids to dominate the culture due to faster growth. The origin of replication also determines a plasmid's reciprocity, i.e., its ability to replicate with another plasmid within the same bacterial cell. Plasmids that utilize the same replication system cannot coexist in the same bacterial cell; they are said to belong to the same reciprocity group. Introducing a new origin in the form of a second plasmid from the same reciprocity group mimics the results of replicating a resident plasmid. Therefore, any further replication is prevented until the two plasmids are segregated into different cells and have generated the correct pre-replicative copy number.

[0277] [Table 4]

[0278] Many bacterial replication origins are known to those skilled in the art. The origin can be selected to achieve the desired copy number. Replication origins contain sequences recognized as the initiation site for plasmid replication via DNA-dependent DNA polymerase (Solar et al. (1998) Microbiology And Molecular Biology Reviews 62(2):434-464). Different replication origins provide various plasmid copy levels within each cell, ranging from one to several hundred copies per cell. Commonly used bacterial plasmid replication origins include, but are not limited to, the pMB1-derived origin, which has a very high copy number, the ColE1 origin, p15A, pSC101, and pBR322, which have low copy numbers. Such origins are well known to those skilled in the art. The pUC19 origin results in a copy number of 500-700 copies per cell. The pBR322 origin has a known copy number of 15-20. These origins differ by only a single base pair. The ColE1 origin copy number is 15-20, while derivatives such as pBluescript have copy numbers in the range of 300-500. For example, the p15A origin in pACYC184 results in a copy number of approximately 10. The pSC101 origin provides a copy number of approximately 5. Other low copy number vectors from which origins can be derived include, for example, pWSK29, pWKS30, pWKS129, and pWKS130 (see Wang et al. (1991) Gene 100:195-199). Medium to low copy numbers are less than 150 or less than 100. Low copy numbers are less than 20, 25, or 30. One skilled in the art can identify plasmids with low or high copy numbers. For example, minipreps can be performed to experimentally determine whether the copy number is high or low. High copy plasmids should yield between 3 μg and 5 μg of DNA per ml of LB culture; low copy plasmids will yield between 0.2 μg and 1 μg of DNA per ml of LB culture.

[0279] The sequence of the bacterial plasmid, including the identity and sequence of the origin of replication, is well known (see, for example, snapgene.com / resources / plasmid_files / basic_cloning_vectors / pBR322 / ).

[0280] High-copy plasmids are chosen for heterologous expression of proteins in vitro because gene dosage increases relative to chromosomal gene and protein specific yield, and for therapeutic bacteria, the therapeutic dosage of the encoded therapeutic agent is even higher. However, for delivery of plasmids encoding RNA interference (RNAi), such as by S. typhimurium as described herein, high-copy plasmids would be ideally suited, although low copy numbers are shown herein to be more effective therapeutically.

[0281] The requirement for bacteria to maintain high copy plasmids can be problematic if the expressed molecules are toxic to the organism.The metabolic requirement for maintaining these plasmids can compromise in vivo replicative fitness.The optimal plasmid copy number for delivering interfering RNA can be determined by the mechanism of attenuation of the strain genetically engineered to deliver the plasmid.If necessary, those skilled in the art can select the appropriate copy number for a particular immunostimulatory species and strain of bacteria in light of the disclosures herein.It is shown herein that low copy number can be advantageous.

[0282] 3. CpG motifs and CpG islands Unmethylated cytidine-phosphate-guanosine (CpG) motifs are common in bacteria but not in vertebrate genomic DNA. Pathogenic DNA and synthetic oligodeoxynucleotides (ODNs) containing CpG motifs activate host defense mechanisms and trigger innate and acquired immune responses. Unmethylated CpG motifs contain a central unmethylated CG dinucleotide and flanking regions. Four distinct classes of CpG ODNs in humans have been identified based on differences in the structure and nature of the immune responses they induce. K-type ODNs (also called B-type) typically contain one to five CpG motifs on a phosphorothioate backbone. D-type ODNs (also called A-type) have a mixed phosphodiester / phosphorothioate backbone and contain a single CpG motif flanked by palindromic sequences that allow the formation of a stem-loop structure, as well as poly-G motifs at the 3' and 5' ends. C-type ODN has phosphorothioate backbone, and contains multiple palindrome CpG motifs, which can form stem-loop structure or dimer.P-class CpG ODN has phosphorothioate backbone, and contains multiple CpG motifs, with double palindrome, and can form hairpin at their GC-rich 3' end (Scheiermann and Klinman (2014) Vaccine 32(48):6377-6389).For the purpose herein, CpG is coded in plasmid DNA; can be introduced as motif or into gene.

[0283] Toll-like receptors (TLRs) are key receptors for sensing pathogen-associated molecular patterns (PAMPs) and activating innate immunity against pathogens (Akira et al. (2001) Nat Immunol. 2(8):675-680). TLR9, which does not naturally occur in mammalian DNA, recognizes hypomethylated CpG motifs in prokaryotic DNA (McKelvey et al. (2011) J Autoimmunity 36:76-86). Upon recognition of the CpG motif for phagocytosis of pathogens to endosomes in a subset of immune cells, it induces IRF7-dependent type I interferon signaling, activating innate and adaptive immunity.

[0284] Provided herein are immunostimulatory bacteria, such as Salmonella species, such as S. typhimurium, strains carrying plasmids containing CpG islands. These bacteria can activate TLR9 and induce type I IFN-mediated innate and adaptive immunity. The bacterial plasmids exemplified herein containing hypomethylated CpG islands can induce innate and adaptive anti-tumor immune responses in combination with RNAi encoded in the plasmid, such as RNAi targeting immune checkpoints, such as shRNA or miRNA targeting TREX1, thereby activating the TREX1-mediated STING pathway and achieving synergistic or enhanced anti-tumor activity. For example, the asd gene (SEQ ID NO: 48) encodes a high frequency of hypomethylated CpG islands. CpG motifs can be included in immunostimulatory bacteria in combination with any of the RNAi described or apparent in the description herein, resulting in enhanced or improved anti-tumor immune responses in treated subjects.

[0285] Immunostimulatory CpG can be typically included in a plasmid by including nucleic acid from a bacterial gene encoding a gene product, or by adding nucleic acid encoding a CpG motif. The plasmid herein can include a CpG motif. Exemplary CpG motifs are known (see, for example, U.S. Patent Nos. 8,232,259, 8,426,375 and 8,241,844). These have the general formula: (CpG) n where n is the number of repeats, for example, between 10 and 100, 10 and 20, 10 and 30, 10 and 40, 10 and 50, 10 and 75 base pairs in length.

[0286] Generally, at least one or two repeats are used; non-CG bases may be interspersed. Those skilled in the art are familiar with the general use of CpG motifs to induce immune responses by modulating TLRs, particularly TLR9.

[0287] 4. Plasmid Maintenance / Construct Selection Plasmid maintenance in a laboratory setting is consistently ensured by including an antibiotic resistance gene on the plasmid and using antibiotics in the growth medium. The use of asd deletion mutants complemented with a functional asd gene on the plasmid, as described above, allows for in vitro selection of the plasmid without the use of antibiotics and for in vivo selection of the plasmid. The asd gene complementation system provides such selection (Galaen et al. (1990) Gene 28:29-35). The use of the asd gene complementation system to maintain the plasmid in the tumor microenvironment enhances the potential of genetically engineered S. typhimurium to deliver plasmids encoding genes or interfering RNA.

[0288] RNA polymerase promoter The plasmid provided herein is designed to encode an interfering RNA that targets the immunological checkpoints described above.The RNA expression cassette comprises a promoter for transcription in human cells, such as an H1 promoter, a U6 promoter, or a CMV promoter.U6 and H1 are RNA polymerase III (RNAP III) promoters, which are responsible for producing and processing small RNAs.The CMV promoter is recognized by RNA polymerase II and allows for the expression of longer RNA stretches than RNAP III.The promoter precedes the interfering RNA, such as shRNA, siRNA, or miRNA, described above.

[0289] In eukaryotic cells, DNA is transcribed by three types of RNA polymerases: RNA Pol I, II, and III. RNA Pol I transcribes only ribosomal RNA (rRNA) genes, RNA Pol II transcribes DNA into mRNA and small nuclear RNAs (snRNAs), and RNA Pol III transcribes DNA into other small RNAs, such as ribosomal 5S rRNA (type I), transfer RNA (tRNA) (type II), and U6 snRNA (type III). shRNAs are typically transcribed in vivo under the control of eukaryotic type III RNA Pol III promoters, such as the human U6 promoter, which transcribes the U6 snRNA component of the spliceosome, and the H1 human promoter, which transcribes the RNA component of RNase P. The U6 and H1 promoters are structurally simple, have well-defined transcription start sites, and are more suitable than other Pol III or Pol II promoters for naturally driving the transcription of small RNAs. The U6 and H1 promoters do not possess the sequences necessary to transcribe anything downstream from the transcription start site (Makinen et al. (2006) J. Gene Med. 8:433-441). Therefore, they are the simplest promoters to use in expressing shRNAs.

[0290] Other promoters, such as type II Pol III tRNA promoters, have been successfully used to simultaneously express shRNAs, resulting in long dsRNA transcripts that can induce interferon responses. RNA Pol II promoters, such as the human cytomegalovirus (CMV) promoter, can also be used (U.S. Patent Nos. 8,202,846 and 8,383,599), but are often used to express long RNA stretches. Studies have shown that adding an enhancer from the CMV promoter near the U6 promoter can increase its activity, increase shRNA synthesis, and improve gene silencing (Xia et al. (2003) Nucleic Acids Res. 31(17):e100; Nie et al. (2010) Genomics Proteomics Bioinformatics 8(3):170-179). RNA Pol II promoters are typically avoided in shRNA transcription due to the generation of cytoplasmic DNA, which leads to a pro-inflammatory interferon response. In this case, cytoplasmic DNA-mediated interferon responses in cells from S. typhimurium-infected tumors, particularly in the context of TREX1 inhibition provided herein, have antitumor benefits. Prokaryotic promoters, including the T7, pBAD, and pepT promoters, can be used when transcription occurs in bacterial cells (Guo et al. (2011) Gene therapy 18:95-105; U.S. Patent Application Publication Nos. 2012 / 0009153, 2016 / 0369282; WO 2015 / 032165, 2016 / 025582).

[0291] RNA Pol III promoters are generally used to express constitutive shRNAs. For inducible expression, RNA Pol II promoters are used. Examples include the pBAD promoter, which can be induced by L-arabinose; tetracycline-inducible promoters such as TRE-tight, IPT, TRE-CMV, Tet-ON, and Tet-OFF; retroviral LTR; IPTG-inducible promoters such as LacI and Lac-O responsive promoters; LoxP-stop-LoxP promoters (U.S. Patent No. 8,426,675; International Publication No. 2016 / 025582); and the hypoxia-inducible promoter pepT (Yu et al. (2012) Scientific Reports 2:436). These promoters are well known. Exemplary of these promoters are human U6 (SEQ ID NO: 73) and human H1 (SEQ ID NO: 74).

[0292] [Table 5]

[0293] Tissue-specific promoters include the TRP2 promoter for melanoma cells and melanocytes; the MMTV promoter or WAP promoter for breast cancer and breast cancer cells; the Villin promoter or FABP promoter for intestinal cells; the RIP promoter for pancreatic beta cells; the keratin promoter for keratinocytes; the probasin promoter for prostate epithelium; the nestin promoter or GFAP promoter for CNS cells / cancer; the tyrosine hydroxylase S100 promoter or neurofilament promoter for neurons; the Clara cell secretory protein promoter for lung cancer; and the alpha myosin promoter in cardiac cells (U.S. Patent No. 8,426,675).

[0294] 5. DNA nuclear targeting sequence DNA nuclear targeting sequences (DTSs), such as the SV40 DTS, mediate the translocation of DNA sequences through the nuclear pore complex. This transport mechanism has been reported to depend on the binding of DNA-binding proteins containing nuclear localization sequences. The inclusion of DTSs on plasmids has been demonstrated to increase nuclear transport and expression (Dean, DA et al. (1999) Exp. Cell Res. 253(2):713-722), and their inclusion has been used to increase gene expression from plasmids delivered by S. typhimurium (Kong et al. (2012) PNAS 109(47):19414-19419).

[0295] Rho-independent or class I transcription terminators, such as the T1 terminator of the E. coli rrnB gene, contain DNA sequences that form secondary structures that result in the dissociation of the transcription elongation complex. The transcription terminator must be included in the plasmid to prevent expression of interfering RNAs by the S. typhimurium transcription machinery. This ensures that expression of the encoded interfering RNAs, such as shRNAs, microRNAs, and siRNAs, is restricted to the host cell transcription machinery.

[0296] Plasmids used for transformation of Salmonella, such as S. typhimurium, for the cancer therapies described herein contain all or part of the following features: 1) a CpG island, 2) a bacterial origin of replication, 3) an asd gene selectable marker for plasmid maintenance, 4) one or more human interfering RNA expression cassettes, 5) a DNA nuclear targeting sequence, and 6) a transcription terminator.

[0297] F. Tumor-targeted immunostimulatory bacteria contain RNAI against exemplary immune target genes to stimulate anti-tumor immunity The RNAi for any immune target can be coded in plasmid.These include but are not limited to any of those discussed in the present disclosure and any of those known to those skilled in the art.The following discussion describes exemplary targets.Plasmid can contain any RNAi for such target, including but not limited to shRNA, siRNA and microRNA.

[0298] 1.TREX1 In certain embodiments provided herein, the immunostimulatory bacteria encode inhibitory RNAs, such as shRNAs, that inhibit, block, or suppress TREX1 expression. The enzyme product encoded by TREX1, located upstream from cGAS, is a mediator of the type I interferon pathway. TREX1 encodes the major 3' DNA exonuclease (also known as DRNase III) in mammalian cells. Human TREX1 protein is catalytically as efficient as bacterial exonucleases (Mazur and Perrino (2001) J. Biol. Chem. 276:17022-17029). Immunostimulatory bacteria that inhibit TREX1 expression by processes other than RNA silencing are also contemplated herein.

[0299] For the immunostimulatory bacteria provided herein, such as those expressing shRNA against TREX1, loss of TREX1 activity and subsequent activation of cGAS / STING-induced vascular destruction enhances tumor colonization by S. typhimurium. The TREX1 gene encodes a 314-amino acid protein (Mazur et al. (2001) J.Biol.Chem 276:17022-17029), which exists as a homodimer and lacks endonuclease activity. TREX1 is one of several proteins involved in the repair of DNA damaged by exogenous genotoxic stress, including UV irradiation and DNA-damaging compounds. TREX1 can function as an editing exonuclease for DNA pol β by excising mismatched nucleotides from the 3' end (Mazur et al. (2001) J.Biol.Chem 276:17022-17029). ssDNA is degraded 3-4 times more efficiently than dsDNA (Lindahl et al. (2009) Biochem Soc Trans 37 (Pt 3), 535-538). Mutations at residues D18 and D200, often associated with autoimmune diseases, abolish dsDNA degradation by the TREX1 enzyme, reducing its ability to degrade ssDNA. The TREX1 enzyme translocates from the endoplasmic reticulum to the nucleus after DNA damage, indicating its involvement in the replication of damaged DNA. Activation and upregulation of the TREX1 promoter have been observed as a result of UVC exposure in mouse fibroblasts, and TREX1 null mouse cells have been demonstrated to be hypersensitive to UVC light (Tomicic et al. (2013) Bioch. Biophys. Acta 1833:1832-1843).

[0300] Mutations resulting in loss of TREX1 have been identified in patients with Aicardi-Goutières syndrome (AGS), a rare inherited disorder that shares phenotypic overlap with the autoimmune diseases systemic lupus erythematosus (SLE) and lupus pernio (Aicardi and Goutières, (2000) Neuropediatrics 31(3):113). TREX1 mutations are also associated with retinal vasculopathy associated with cerebral leukodystrophy. TREX1-mediated autoimmune diseases are associated with the inability of cells to prevent autoimmunity through degradation of ssDNA and dsDNA that accumulate in the cytoplasm. TREX1 null mice with inflammatory myocarditis suffer from circulatory failure, which is caused by chronic cytokine production (Morita et al. (2004) Mol Cell Biol 24 (15):6719-6727; Yang et al. (2007) Cell 131(5):873-886; Tomicic et al. (2013) Bioch. Biophys. Acta 1833(8):1832-1843). Therefore, TREX1 deficiency induces innate immunity and leads to inflammatory responses after cytoplasmic DNA accumulation (Wang et al. (2009) DNA Repair (Amst) 8:1179-1189). Because TREX1 is known to metabolize reverse-transcribed (RT) DNA, it was found that DNA material accumulating in the cytosol of TREX1-deficient cells is derived in part from endogenous retroelements that escape from the damaged nucleus (Stetson et al. (2008) Cell 134(4):587-598). During HIV infection, HIV RT DNA accumulates in the cytosol of infected T cells and macrophages, which would normally induce cGAS / STING activation of antiviral immunity. TREX1 digests this viral DNA, enabling HIV immune evasion (Yan et al. (2010) Nat. Immunol. 11(11):1005-1013).Thus, TREX1 acts as a negative regulator of STING, which may be used to evade detection by several retroviruses, such as murine leukemia virus (MLV), simian immunodeficiency virus (SIV), and many others (Hasan et al. (2014) Front. Microbiol. 4:393).

[0301] Like STING, TREX1 is expressed in most mammalian cell types, with macrophages and dendritic cells being key cytokine producers in TREX1 null mice (Ahn et al. (2014) J. Immunol. 193(9):4634-4642). Data indicate that TREX1 is responsible for degrading self-DNA that can leak from damaged nuclei into the cytosol, otherwise binding to and activating cGAS, leading to autoimmunity (Barber (2015) Nat. Rev. Immunol. 15(12):760-770). In support of this, TREX1-null mice lacking cGAS and TREX1-deficient cells are completely protected from type I interferon activation and lethal autoimmunity (Ablasser et al. (2014) J. Immunol. 192(12):5993-5997; Gray et al. (2015) J. Immunol. 195(5):1939-1943). In a negative feedback loop, type I interferons and type II IFNγ can also induce TREX1, thus limiting aberrant autoimmune activation (Tomicic et al. (2013) Bioch. Biophys. Acta 1833:1832-1843).

[0302] Lymphocytes from patients with Aicardi-Goutières syndrome containing mutated TREX1 were found to inhibit angiogenesis and neuroblastoma cell growth, an effect enhanced by the presence of IFN-α (Pulliero et al. (2012) Oncology Reports 27:1689-1694). The use of microRNA-103 has also been shown to inhibit TREX1 expression, disrupt DNA repair, and angiogenesis, reducing tumor growth in vivo (see U.S. Patent Application Publication No. 2014 / 0127284, Cheresh et al.).

[0303] TREX1 is a negative regulator of macrophage activation and proinflammatory function. TREX1 null macrophages were found to exhibit increased TNF-α and IFN-α production, high levels of CD86, increased antigen presentation to T cells, and impaired apoptotic T cell clearance (Pereira-Lopes et al. (2013) J. Immunol. 191:6128-6135). The inability to properly digest apoptotic DNA in TREX1 null macrophages generates large amounts of abnormal cytoplasmic DNA, which binds to cGAS, activates the STING pathway, and produces high levels of type I interferon (Ahn et al. (2014) J. Immunol. 193:4634-4642). However, not all cell types are sensitive to the immunostimulatory effects of Trex1 knockdown. Studies of individual cell types have found that dendritic cells, macrophages, fibroblasts, and keratinocytes produce type I IFN when Trex1 is knocked down, whereas B cells, cardiomyocytes, neurons, and microglia do not (Peschke et al. (2016) J. Immunol. 197:2157-2166). Therefore, inhibiting TREX1 function in phagocytes engulfed by S. typhimurium may enhance their pro-inflammatory activity and simultaneously drive the accumulation of cytoplasmic DNA from engulfed tumor cells, which then activates the cGAS / STING pathway. Using microRNA-103, inhibiting TREX1 expression, disrupting DNA repair, and angiogenesis, resulted in reduced tumor growth in vivo (see U.S. Patent Application Publication No. 2014 / 0127284, Cheresh et al.).

[0304] Studies have shown that cGAS and / or STING expression is inhibited in over one-third of colorectal cancers, and STING expression is also inhibited in many primary and metastatic melanomas and HPV +STING signaling remains intact in all tumor-resident APCs, which continuously sample the antigenic environment of the TME and target tumor antigens to CD8 + Batf3-lineage CD103 / CD8α cross-presenting to T cells + These APCs, including DCs, will also readily phagocytose S. typhimurium or be activated by type I IFN from neighboring macrophages that have phagocytosed S. typhimurium containing TREX1 gene knockdown.

[0305] Inactivating TREX1 enhances immune responses by allowing dsDNA to accumulate in the cytosol, which binds to the cyclic GMP-AMP (cGAMP) synthase (cGAS) enzyme, a cytosolic DNA sensor that induces the production of type I interferons and other cytokines via activation of the STING signaling pathway (Sun et al. (2013) Science 339(6121):786-791; Wu et al. (2013) Science 339(6121):826-830). Activating the STING pathway has been shown to induce potent innate and adaptive antitumor immunity (Corrales et al. (2015) Cell Reports 11:1018-1030).

[0306] Thus, embodiments of the immunostimulatory bacterial strains provided herein are administered to inhibit TREX1 and induce cGAS / STING activation in tumor-resident APCs, thereby activating these DCs and targeting host tumor antigens to CD8 + Cross-presentation to T cells induces local and systemic tumor regression and durable anti-tumor immunity (Corrales et al. (2015) Cell Reports 11:1018-1030; Zitvogel et al. (2015) Nat. Rev. Mol. Cell. Biol. 16:393-405).

[0307] The clinical activity of VNP20009 has been largely disappointing, in part due to its insufficient ability to establish in human tumors, a phenomenon not observed in mouse models (Nemunaitis et al. (2003) Cancer Gene Ther. 10(10):737-744; Toso et al. (2002) J. Clin. Oncol. 20(1):142-152; Heimann et al. (2003) J. Immunother. 26(2):179-180). The reason for the discrepancy in tumor establishment between humans and mice was later revealed to be that orthotopically implanted syngeneic mouse tumors were much more vascularized than human tumors. To more closely model the lack of human tumor angiogenesis in mice, we treated primary tumor models with VNP20009 and pretreated them with vascular disrupting agents to allow tumor establishment alone (Drees et al. (2015) J of Cancer 6(9):843-848; Drees et al. (2015) Anticancer Res. 35(2):843-849). Vascular disrupting agents such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA) have been shown to mediate tumor lysis in mice (but not humans) by directly binding to STING and inducing type I interferon signaling (Baguley (2003) Lancet Oncol. 4(3):141-148; Corrales and Glickman et al. (2015) Cell Reports 11(7):1018-1030). STING signaling induces the production of TNF-α and IFN-γ, cytokines that have been shown to directly promote vascular destruction by downregulating the αVβ3 integrin adhesion receptor on endothelial cells (Rueegg et al. (1998) Nat Medicine 4(4):408-414).Activation of STING induces the production of innate pro-inflammatory cytokines, such as TNF-α, IL-12p40, and IFN-γ, which are important for the activation of antitumor immunity (Burdette et al. (2011) Nature 478(7370):515-518).

[0308] Thus, the immunostimulatory bacteria provided herein, which express shRNA against TREX1, leading to loss of TREX1 and subsequent activation of cGAS / STING-induced vascular destruction, enhance tumor colonization by S. typhimurium.

[0309] 2.PD-L1 Programmed cell death protein 1 (PD-1) is an immunoinhibitory receptor involved in the negative regulation of immune responses. Its cognate ligand, programmed death-ligand 1 (PD-L1), is expressed on APCs and mediates CD8 activation upon binding to PD-1 on T cells. + PD-L1 expression is often associated with tumor aggressiveness and reduced survival in certain human cancers, resulting in the loss of T cell effector function and inducing T cell tolerance (Gao et al. (2009) Clin. Cancer Res. 15(3):971-979).

[0310] Antibodies designed to block immune checkpoints, such as anti-PD-1 (e.g., pembrolizumab, nivolumab) and anti-PD-L1 (e.g., atezolizumab, avelumab, durvalumab) antibodies, have been durably successful in preventing T cell anergy and breaking immune tolerance. Only a small proportion of treated patients, and those who often exhibit autoimmune-related toxicities, demonstrate clinical benefit (Ribas (2015) N. Engl. J. Med. 373(16):1490-1492; Topalian et al. (2012) N. Engl. J. Med. 366(26):2443-54). In addition to acquired toxicity, PD-1 / PD-L1 therapy often leads to resistance, and the combination of anti-CTLA-4 antibodies (e.g., ipilimumab) has had limited success in clinical trials and has been associated with significant additive toxicities. To limit the toxicity and enhance the potency of PD-L1 blockade, the immunostimulatory bacteria harboring shRNA against PD-L1 provided herein will synergize with TLR activation of immune cells to both activate and enhance anti-tumor immunity.

[0311] 3. VISTA Other nonredundant checkpoints in immune activation can exert synergistic effects with PD-1 / PD-L1 and CTLA-4, such as V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA). VISTA is primarily expressed on APCs, particularly tumor-infiltrating myeloid cells and myeloid-derived suppressor cells (MDSCs), and, to a lesser extent, on regulatory T cells (CD4+Foxp3+Tregs) (Wang et al. (2011) J. Exp. Med. 208(3):577-592). Similar to PD-L1, upregulation of VISTA directly suppresses T cell proliferation and cytotoxic function (Liu et al. (2015) PNAS 112(21):6682-6687). Monoclonal antibodies targeting VISTA have been shown to remodel the tumor microenvironment in mice, enhance APC activation, and strengthen antitumor immunity (LeMercier et al. (2014) Cancer Res. 74(7):1933-1944). Clinically, VISTA expression has been shown to be upregulated on tumor-resident macrophages after anti-CTLA-4 therapy in prostate cancer, demonstrating compensatory regulation of immune checkpoints (Gao et al. (2017) Nat. Med. 23(5):551-555). The majority of VISTA expression is located in intracellular compartments rather than on the surface of myeloid cells, which may limit the effectiveness of monoclonal antibody approaches (Deng et al. (2016) J. Immunother. Cancer 4:86). The ability to inhibit VISTA from within APCs using tumor-targeted bacteria containing shRNA against VISTA as provided herein will be more efficient and will completely inhibit the T cell suppressive function of VISTA, resulting in the activation of T cell-mediated anti-tumor immunity and tumor regression.

[0312] 4.SIRPα One mechanism by which tumor cells evade elimination is by preventing their phagocytosis by innate immune cells. Phagocytosis is inhibited by surface expression of CD47, which is widely expressed on hematopoietic and non-hematopoietic cells (Liu et al. (2015) PLoS ONE 10(9):e0137345). When CD47 binds to its receptor, signal-regulatory protein alpha (SIRPα), it initiates an inhibitory signal for phagocytosis. SIRPα is abundantly expressed on phagocytes, including macrophages, granulocytes, and DCs. Thus, the protein-protein interaction between CD47 and SIRPα represents another class of immune checkpoint specific to APCs, particularly tumor-resident macrophages. The effectiveness of CD47 in blocking phagocytosis is evidenced by the fact that it is often upregulated in various tumors, allowing them to avoid phagocytosis by APCs in the tumor microenvironment (Liu et al. (2015) Nat. Med. 21(10):1209-1215). Several methods have been investigated to block CD47 / SIRPα interaction, including the development of anti-CD47 or anti-SIRPα antibodies or antibody fragments, the use of small peptides that bind to either protein, or the knockdown of CD47 expression (U.S. Patent Application Publication Nos. 2013 / 0142786, 2014 / 0242095; WO 2015 / 191861; McCracken et al. (2015) Clin. Cancer Res. 21(16):3597-3601). For this reason, several monoclonal antibodies that directly target SIRPα in a process known as antibody-dependent cellular phagocytosis (ADCP) are in clinical development, either alone or in combination with tumor-targeting antibodies (e.g., rituximab, daratumumab, alemtuzumab, cetuximab) that can enhance phagocytosis of antibody-opsonized tumor cells (McCracken et al. (2015) Clin. Cancer Res. 21(16):3597-3601; Yanagita et al. (2017) JCI Insight 2(1):e89140).

[0313] The CD47 / SIRPα interaction also helps maintain red blood cell lifespan by preventing phagocytic elimination (Murata et al. (2014) J. Biochem. 155(6):335-344). Consequently, systemic therapies, such as anti-CD47 antibodies, that broadly disrupt this interaction result in anemic toxicity (Huang et al. (2106) J Thorac Dis. 126:2610-20). Systemic SIRPα-based therapies also carry the risk of adverse events, such as organ damage due to the generation of systemic hyperphagocytic self-eating macrophages. The use of tumor-targeted immunostimulatory bacteria containing shRNA against SIRPα, as provided herein, localizes CD47 / SIRPα disruption to the tumor microenvironment, eliminating these adverse events. Furthermore, inhibition of SIRPα in the context of bacterial activation of TLR-mediated pro-inflammatory signaling pathways would potently activate these macrophages, making them hyperphagocytic toward adjacent tumor cells (Bian et al. (2016) PNAS. 113(37): E5434-E5443).

[0314] 5. β-catenin Immune checkpoint pathways exemplify the multiple layers of regulation that exist to prevent immune overactivation and autoimmunity, and the challenges of subverting these pathways to promote antitumor immunity. One mechanism by which tumors have evolved to be refractory to checkpoint therapy is through a lack of T cell and dendritic cell (DC) infiltration, described as T cell-inflammable or "cold tumors" (Sharma et al. (2017) Cell 9;168(4):707-723). Intrinsic mechanisms have been identified in several tumors, leading to the elimination of antitumor T cells and resistance to immunotherapy. In melanoma, specifically, molecular profiling of checkpoint therapy-refractory tumors revealed an elevated β-catenin and its downstream target gene signature, which correlates with the lack of tumor-infiltrating lymphocytes (Gajewski et al. (2011) Curr. Opin. Immunol. 23(2):286-292).

[0315] CTNNB1 is an oncogene that encodes β-catenin, and can induce the expression of genes c-Myc and cyclin D1, leading to tumor growth. CTNNB1 mutations are associated with certain cancers. Gene silencing of CTNNB1 / β-catenin using S. typhimurium shRNA vectors can be used in cancer treatment (Guo et al. (2011) Gene therapy 18:95-105; U.S. Patent Application Publication No. 2012 / 0009153, U.S. Patent Application Publication No. 2016 / 0369282; International Publication No. 2015 / 032165). For example, shRNA silencing of CTNNB1 using S. typhimurium strain SL7207 as a delivery vector reduces tumor proliferation and growth in SW480 xenograft mice and decreases the expression of c-Myc and cyclin D1 compared to control cells (Guo et al. (2011) Gene therapy 18:95-105). Silencing of CTNNB1 for the treatment of hepatoblastoma can also be achieved using miRNAs, with or without antibody therapy against the immune checkpoints PD-1 and PD-L1 (WO 2017 / 005773). The use of siRNA or shRNA targeting CTNNB1 delivered via alternative vectors, such as liposomes, for the treatment of CTNNB1-associated cancers, including adenocarcinoma and squamous cell carcinoma, can also be effective (U.S. Patent Application Publication Nos. 2009 / 0111762 and 2012 / 0294929).

[0316] Elevated β-catenin signaling induces Batf3-lineage CD103 / CD8α + Direct inhibition of the chemokine CCL4 from DC recruitment results in antigen-stimulated tumor antigen-specific CD8 +It prevents them from priming T cells (Spranger et al. (2015) Nature 523(7559):231-235). β-catenin is a major downstream mediator of the WNT signaling pathway, a key embryonic developmental pathway that is also important for adult tissue regeneration, homeostasis, and hematopoiesis (Clevers et al. (2012) Cell 149(6):1192-1205). Excessive WNT / β-catenin signaling has been implicated in various cancers (Tai et al. (2015) Oncologist 20(10):1189-1198). Therefore, several strategies targeting WNT / β-catenin signaling have been pursued, but success has been hindered by a lack of specificity for the tumor microenvironment, resulting in off-target toxicity to intestinal stem cells, bone turnover, and hematopoiesis (Kahn (2014) Nat. Rev. Drug Dis. 13(7):513-532). The immunostimulatory bacteria provided herein overcome these challenges.

[0317] For example, the advantages of using immunostimulatory bacteria carrying shRNA against β-catenin provided herein are that they enhance chemokine-mediated infiltration of T cell-primed DCs and the transformation of cold tumors into a cellular inflammatory tumor microenvironment without the systemic toxicity of existing therapeutic modalities. Furthermore, bacterial activation of TLR innate immune signaling pathways synergizes with β-catenin inhibition, further promoting immune activation and antitumor immunity.

[0318] 6. TGF-β Transforming growth factor beta (TGF-β) is a pleiotropic cytokine with many roles in embryogenesis, wound healing, angiogenesis, and immune regulation. In mammalian cells, three isoforms exist: TGF-β1, TGF-β2, and TGF-β3; TGF-β1 is the most prevalent in immune cells (Esebanmen et al. (2017) Immunol Res. 65:987-994). The role of TGF-β as an immunosuppressant is perhaps its most prominent function. Its activation from a latent form in the tumor microenvironment exerts a broad range of immunosuppressive effects, particularly on DCs and their ability to tolerize antigen-specific T cells. TGF-β promotes Th1 CD4 + T cells can also be directly converted into immunosuppressive Tregs, further enhancing tumor tolerance (Travis et al. (2014) Annu Rev Immunol. 32: 51-82). Based on its tumor-specific immunosuppressive function and regardless of its known cancer cell growth and metastasis-promoting properties, inhibition of TGF-β is a target for cancer therapy. High TGF-β signaling has been demonstrated in several human tumor types, including CRC, HCC, PDAC, and NSCLC (Colak et al. (2017) Trends in Cancer 3:1). Systemic inhibition of TGF-β can result in unacceptable autoimmune toxicity, and the inhibition should be localized to the tumor microenvironment. Therefore, tumor-targeted immunostimulatory bacteria carrying RNAi, such as shRNA against TGF-β or shRNA against TGF-βRII, provided herein, disrupt tumor immune tolerance and stimulate anti-tumor immunity.

[0319] 7. VEGF Angiogenesis, or the development of new blood vessels, is an essential step for any tumor microenvironment to become established. Vascular endothelial growth factor (VEGF) is a mitogen important for endothelial proliferation and angiogenesis; inhibition of VEGF in the tumor microenvironment significantly reduces tumor vascularity, resulting in depletion of the tumor's blood supply (Kim et al. (1993) Nature 362(6423):841-4). This initial research led to the development of the monoclonal antibody inhibitor of VEGF, bevacizumab (Avastin; Genentech), which, in combination with chemotherapy, has become the standard of care for metastatic CRC. Systemic administration of bevacizumab also demonstrated significant toxicity in a phase II trial of NSCLC, including multiple deaths, most of which were due to bleeding. Thus, several next-generation antiangiogenic agents, such as the anti-VEGF receptor 2 antibody ramucirumab (Cyramza, Imclone) and the antiangiogenic tyrosine kinase inhibitor axitinib (Inlyta, Pfizer), have been evaluated but have failed to overcome systemic toxicity or significantly improve progression-free survival (Alshangiti et al. (2018) Curr Oncol. 25(Suppl 1):S45-S58). While the antitumor activity of anti-VEGF therapies shows some promise, systemic toxicity is clearly limiting. Therefore, therapies that target only the tumor microenvironment, such as the immunostimulatory tumor-targeted bacteria bearing shRNA against VEGF provided herein, deliver localized antiangiogenic therapy while simultaneously preventing systemic toxicity. This therapeutic modality has the added advantage of being taken up by myeloid cells, which primarily produce VEGF in the tumor microenvironment, which would have the greatest impact on tumor progression (Osterberg et al. (2016) Neuro-Oncology. 18(7):939-949).

[0320] 8. Additional Exemplary Checkpoint Targets Exemplary checkpoint targets for which RNAi, such as microRNAs and shRNAs, can be prepared or are exemplified herein include, but are not limited to, the following:

[0321] [Table 6] Other exemplary targets include, but are not limited to:

[0322] [Table 7]

[0323] G. Combining RNAI shRNAs against multiple immune targets within single therapy modalities and combination therapies Combinations of RNAi, such as shRNA or microRNA, that inhibit different targets in one bacterium are considered.These target combinations can be selected to act synergistically.The combination of RNAi that targets any two immune checkpoints can be introduced into the immunostimulatory bacterial host modified as described herein or into other therapeutic bacterial host.

[0324] 1. TREX1 and other targets To mitigate the induction of compensatory immune checkpoint pathways that are upregulated upon STING activation and can enhance antitumor immunity, the modified immunostimulatory bacteria provided herein contain short hairpin (sh)-RNA sequences against TREX1 in combination with shRNAs against other immune targets, including, but not limited to, PD-L1, VISTA, and SIRPα. Knocking down TREX1 and SIRPα in tumor-resident phagocytes can block the "don't eat me" interaction with CD47 on tumor cells and further enhance the susceptibility of the tumor microenvironment to S. typhimurium infection (Li et al. (2012) J Immunol 189(5):2537-2544), as provided herein. The combination of enhanced phagocytosis enabled by SIRPα inhibition and simultaneous knockdown of TREX1 promotes better delivery of cytosolic and stabilization of tumor DNA, which can more potently activate cGAS / STING signaling. In particular, the antitumor effect of CD47 / SIRPα blockade has been shown to require intact STING signaling, demonstrating the potential synergistic effect of combining TREX1-mediated STING activation with SIRPα inhibition (Liu et al. (2015) Nat. Med. 21(10):1209-1215). Knocking down TREX1 in combination with shRNA against PD-L1 provided herein enhances the pathogenesis and immunostimulatory properties of the modified S. typhimurium (Lee et al. (2010) J.Immunol. 185(4):2442-2449), resulting in a more inflammatory and immunogenic tumor microenvironment. Targeting shRNA against β-catenin and TGF-β also results in a more T cell inflammatory tumor microenvironment, providing a synergistic effect with shRNA against PD-L1, as provided herein.Combining localized checkpoint blockade with immune activation within the macrophage / myeloid compartment, particularly via the combination of shRNAs against TREX1 and VISTA provided herein, enhances the immune response by both promoting the presentation of tumor neoantigens by S. typhimurium-infected APCs and promoting the activation of tumor-specific T cells.

[0325] 2. TREX1 and Radiation Therapy The success of anti-cancer radiation therapy depends on the induction of type I interferon-dependent innate and adaptive immunity. TREX1 has been shown to attenuate anti-tumor immunity after high-Gy irradiation by degrading cytoplasmic DNA produced in damaged cancer cells, thereby blocking the type I interferon pathway mediated by cGAS and STING (Vanpouille-Box et al. (2017) Nature Communications 8:15618). Therefore, overexpression of TREX1 or knockout of cGAS / STING, which blocks activation of the IFN-I pathway, attenuates abscopal tumor responses upon irradiation. CD8 + To activate STING-mediated Batf3-DC priming of T cells and achieve maximal abscopal antitumor immunity, low doses of radiation that would not induce TREX1 were required (Vanpouille-Box et al. (2017) Nature Communications 8:15618). Downregulation of TREX1 has been shown to restore tumor cell sensitivity to ionizing radiation. For example, high-dose irradiation induced TREX1 expression and prevented the cytoplasmic accumulation of dsDNA, thereby inhibiting abscopal tumor regression (Vanpouille-Box et al. (2017) Nature Communications 8:15618). The immunostimulatory strains provided herein that block or inhibit TREX1 expression can reduce, eliminate, or blunt TREX1 expression during high-dose irradiation treatment, significantly expanding the therapeutic window.

[0326] Radiation therapy (RT) has an abscopal effect at low doses, but low doses are not always effective. However, at high doses, the abscopal effect is no longer observed. This is a known challenge with RT. It has been shown that radiation therapy promotes the upregulation of TREX1, which degrades cytosolic dsDNA, preventing IFN-β secretion following cGAS / STING signaling (see Vanpouille-Box et al. (2017) Nat. Commun. 8:15618). Therefore, the immunostimulatory bacteria provided herein can be administered together with RT to block the upregulation of TREX1. Administration of the immunostimulatory bacteria provided herein that encode shRNA or other products that inhibit TREX1 abolishes this response, thereby improving and complementing RT. Therefore, combination therapies are provided herein in which immunostimulatory bacteria that encode shRNA or other products that inhibit or reduce TREX1 expression are administered together with RT, before, together with, after, or intermittently with RT. The combination therapy of immunostimulatory bacteria and RT therapy can also include other anti-cancer therapies, such as administration of checkpoint inhibitors, and / or the inclusion of shRNA against other checkpoints, such as PD-L1, as described herein.

[0327] 3. TREX1 and immunogenic chemotherapy Induction of TREX1 was observed in mouse and human fibroblasts after DNA-damaging UV irradiation and in glioma and melanoma cells after treatment with the DNA alkylating agents nimustine, carmustine, and fotemustine and the topoisomerase I inhibitor topotecan. These tumor cells were resensitized to these anticancer therapies after TREX1 was knocked down with siRNA (Tomicic et al. (2013) Biochimica et Biophysica Acta 1833:1832-1843). TREX1 is induced only by damaging agents that efficiently induce AP-1, whereas agents that are weak inducers of Fos / Jun / AP-1, such as the methylating agent temozolomide and the topoisomerase II inhibitor etoposide, do not induce TREX1.

[0328] Independent studies have found that dsDNA accumulates and activates type I IFNs when treated with chemotherapies that stall DNA replication in the S phase, such as cisplatin, irinotecan, doxorubicin, and etoposide, but not with drugs that act in the M phase, such as vinorelbine and paclitaxel (Wilkinson R. presented at ESMO TAT Conference 2018). S-phase drugs likely result in the release of damaged DNA fragments that accumulate in the cytosol and upregulate TREX1. These chemotherapy agents include those that cause DNA strand breaks, such as nucleotide analogs, alkylating agents, platinum drugs, and intercalating agents (see, e.g., Swift et al. (2014) Int. J. Mol. Sci 15:3403-3431), which can induce TREX1 at levels sufficient to degrade DNA, thereby preventing activation of the type I interferon (IFN-I) pathway via cyclic GMP-AMP (cGAMP) synthase (cGAS) and its downstream adaptor stimulator of interferon genes (STING). Treatment using the immunostimulatory bacteria provided herein can be combined with chemotherapy drugs and additional checkpoint inhibitors. Thus, the immunostimulatory bacteria provided herein can be advantageously used in combination therapy with various anticancer drugs and treatments.

[0329] 4. Combination therapy using anti-checkpoint antibodies Therapeutic methods using the immunostimulatory bacteria provided herein can be combined with any other anti-cancer therapy, including checkpoint inhibitor therapy and other cancer treatments and chemotherapies discussed above.

[0330] H. Pharmaceutical Production, Compositions, and Formulations Provided herein are methods for producing pharmaceutical compositions and formulations comprising any of the immunostimulatory bacteria provided herein and a pharmaceutically acceptable excipient or additive. The pharmaceutical compositions can be used in the treatment of diseases, such as hyperproliferative diseases or conditions, such as tumors or cancer. The immunostimulatory bacteria can be administered as a single drug therapy or as a combination therapy with an additional drug or treatment. The compositions can be formulated for single-dose administration or multiple-dose administration. The drugs can be formulated for direct administration. The compositions can be provided as liquid or dry formulations.

[0331] 1.Manufacturing a. Cell bank production Because the active ingredients of the immunotherapeutics described herein consist of genetically engineered, self-replicating bacteria, selected compositions will be expanded into a series of cell banks that will be maintained for long-term storage and as starting material for manufacturing drug substances. The cell banks will be generated under current Good Manufacturing Practice (cGMP) in an appropriate manufacturing facility in accordance with Code of Federal Regulations (CFR) 21 part 211 or other relevant regulatory authorities. Because the active agents of the immunotherapeutics are live bacteria, the products described herein are, by definition, non-sterile and cannot be terminally sterilized. Care should be taken to ensure that aseptic procedures are used throughout the manufacturing process to prevent contamination. Therefore, all raw materials and solutions must be sterilized before use in the manufacturing process.

[0332] Master cell banks (MCBs) are created by serially isolating single colonies of selected bacterial strains to ensure that no contaminants are present in the starting material. A sterile culture vessel containing sterile medium (which can be a complex medium, such as LB or MSBB, or a defined medium, such as M9 supplemented with appropriate nutrients) is inoculated with a single, fully isolated bacterial colony, and the bacteria are allowed to replicate, for example, by incubating with shaking at 37°C. The bacteria are then prepared for cryopreservation by suspending them in a solution containing cryoprotectant(s).

[0333] Examples of cryoprotectants include proteins such as human or bovine serum albumin, gelatin, immunoglobulins, etc.; carbohydrates including monosaccharides (galactose, D-mannose, sorbose, etc.) and their non-reducing derivatives (e.g., methyl glucoside), disaccharides (trehalose, sucrose, etc.), cyclodextrins, and polysaccharides (raffinose, maltodextrin, dextran, etc.); amino acids (glutamic acid, glycine, alanine, arginine or histidine, tryptophan, tyrosine, leucine, phenylalanine, etc.); methylamines such as betaine; polyols such as trihydric or higher sugar alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; surfactants, e.g., Pluronic; or organic sulfur compounds such as dimethyl sulfoxide (DMSO), and combinations thereof. The cryoprotective solution may include one or more cryoprotectants in solution, which may also include buffers such as salts (e.g., sodium chloride, potassium chloride, magnesium sulfate, and / or sodium phosphate), tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and other such buffers known to those of skill in the art.

[0334] Suspension of bacteria in cryopreservation solution can be achieved by adding concentrated cryoprotectant(s) to the culture material to achieve a final concentration that preserves bacterial viability during the freeze-thaw process (e.g., glycerol at a final concentration of 0.5% to 20%), or by harvesting the bacteria (e.g., by centrifugation) and suspending them in a cryoprotectant solution containing the appropriate final concentration of cryoprotectant(s). The bacterial suspension in cryoprotectant solution is then filled into appropriate sterile vials (plastic or glass) equipped with a container closure system capable of maintaining complete closure under frozen conditions (e.g., butyl stoppers and crimp seals). The master cell bank vials are then frozen (either slowly using a rate-controlled freezer or quickly by placing them directly in a freezer). The MCB is then frozen and stored at a temperature that preserves long-term viability (e.g., at -60°C or below). The thawed master cell bank material is fully characterized to ensure identity, purity, and activity as regulated by appropriate authorities.

[0335] Working cell banks (WCBs) are generated in much the same way as master cell banks, except that the starting material is derived from an MCB. The MCB material can be transferred directly to a fermentation vessel containing sterile medium and expanded as described above. The bacteria are then suspended in a cryoprotectant solution, filled into the vessel, sealed, and frozen at -20°C or below. Multiple WCBs can be generated from the MCB material, and the WCB material can be used to generate additional cell banks (e.g., manufacturer's working cell banks, MWCBs). The WCBs are cryopreserved and characterized to ensure identity, purity, and activity. WCB material is typically the starting material used in the manufacture of drug substances for biologics, such as genetically engineered bacteria.

[0336] b. Manufacturing of APIs Drug substances are manufactured using sterile processes under cGMP as described above. Working cell bank material is typically used as the starting material for manufacturing drug substances under cGMP, although other cell banks (e.g., MCB or MWCB) can be used. Sterile processing is used to manufacture all cell therapies, including bacterial cell-based therapies. Bacteria from the cell bank are expanded by fermentation, which can be achieved by producing a preculture (e.g., in a shake flask) or by directly inoculating a fermenter. Fermentation is accomplished in a sterile bioreactor or flask, which can be disposable or reusable. Bacteria are harvested by concentration (e.g., by centrifugation, continuous centrifugation, or tangential flow filtration). The concentrated bacteria are purified from media components and bacterial metabolites by exchanging the media with a buffer (e.g., diafiltration). Bulk drug products are formulated as intermediates and stored (e.g., by freezing or drying) or processed directly into drug products. Drug substances are tested for identity, strength, purity, potency, and quality.

[0337] c. Manufacturing of drug products A drug product is defined as the final formulation of an active substance contained in its final container. Drug products are manufactured using a sterile process under cGMP. Drug products are manufactured from drug substances. The drug substances are thawed or reconstituted as needed and then formulated to the appropriate target strength. Because the active component of a drug product is a live, genetically engineered bacterium, strength is determined by the number of CFU contained within the suspension. The bulk product is diluted with a final formulation appropriate for storage and use, as described below. Containers are filled and sealed using a container closure system, and the drug product is labeled. The drug product is stored at the appropriate temperature, maintained stable, tested for identity, strength, purity, potency, and quality, and released for human use if it meets specific approval criteria.

[0338] 2. Composition Pharmaceutically acceptable compositions are prepared in consideration of the approval of regulatory authorities or other authorities, ...

Claims

1. 1. An in vitro method for identifying a subject having cancer for treatment with an immunostimulatory bacterium that is adenosine auxotrophic, comprising: Testing a previously obtained tumor biopsy sample to detect expression of cd73; and identifying samples that are cd73 positive and selecting subjects from whom said samples were obtained as candidates for treatment with an immunostimulatory bacterium that is adenosine auxotrophic. A method comprising:

2. cd73 ＋ 1. An immunostimulatory bacterium for use in treating a subject having cancer, including a tumor comprising the immunostimulatory bacterium comprises a plasmid encoding a therapeutic product for treating cancer; the immunostimulatory bacterium is a Salmonella spp.; the immunostimulatory bacterium is adenosine auxotrophic; The immunostimulatory bacteria are genomically modified so that the bacteria lack flagella; The wild-type bacterium contains flagella; The immunostimulatory bacteria may contain one or more deletions of the purI gene, thereby rendering the bacteria － (purM － ), msbB － , purD － , pagP － , adrA － , csgD － , and hilA － and The target is CD73 ＋ has been previously identified as having a tumor that is Immunostimulatory bacteria.

3. The tumor is CD73 ＋ / cd39 ＋ The immunostimulatory bacterium of claim 2, identified as:

4. Tumors were also tested for CD39; cd73 ＋ / cd39 ＋ Identifying the sample as a cd73 ＋ / cd39 ＋ selecting the subject from whom the sample was obtained as a candidate for treatment with the immunostimulatory bacteria that is adenosine auxotrophic; The method of claim 1.

5. The immunostimulatory bacterium of claim 2 or 3, wherein the genome of the immunostimulatory bacterium is modified so that the bacterium lacks flagella and is msbB − / pagP − .

6. Immunostimulatory bacteria are of the genotype fliC － / fljB － 6. The immunostimulatory bacterium of claim 5, wherein said bacterium has a cytoplasmic domain, such that said bacterium does not have a flagellum.

7. 5. The method of claim 4, wherein the immunostimulatory bacterium that is adenosine auxotrophic is a strain of the genus Salmonella.

8. 8. The method of claim 7, wherein the immunostimulatory bacterium that is adenosine auxotrophic is a Salmonella typhimurium strain.

9. 7. The immunostimulatory bacterium of any one of claims 2, 3, 5 and 6, wherein the parent strain of the immunostimulatory bacterium is an attenuated Salmonella typhimurium strain selected from among the strains designated YS1646 (ATCC#202165), RE88, SL7207, χ8429, χ8431 and χ8468, or is a wild-type Salmonella strain, or is the strain deposited as ATCC#14028.

10. 5. The method of any one of claims 1 and 4, wherein the immunostimulatory bacterium that is adenosine auxotrophic is a Salmonella, Shigella, Listeria, or E. coli species.

11. The immunostimulatory bacteria of any one of claims 2, 3, 5, 6 and 9, wherein the subject has cancer or a tumor or a metastasis or a hematological malignancy.

12. The immunostimulatory bacteria of any one of claims 2, 3, 5, 6 and 9, wherein the subject has cancer, including a solid tumor.

13. The immunostimulatory bacteria of any one of claims 2, 3, 5, 6 and 9, wherein the cancer is selected from lung cancer, head and neck cancer, gastric cancer, liver cancer, kidney cancer, breast cancer, colorectal cancer, prostate cancer, and chronic lymphoblastic leukemia.

14. 10. The method of claim 1 or 4, wherein the genome of the immunostimulatory bacterium that is adenosine auxotrophic is modified so that the bacterium lacks flagella.

15. 15. The method of claim 14, wherein the immunostimulatory bacterium that is adenosine auxotrophic is of the genus Salmonella.

16. Adenosine auxotrophic immunostimulatory bacteria express flagellin － (fliC － / fljB － 15. The method of claim 14, wherein the Salmonella genus is Salmonella spp., such that the bacterium does not have flagella.

17. 17. The method of any one of claims 1, 4, 7, 8 and 14-16, wherein the parent strain of the immunostimulatory bacterium that is adenosine auxotrophic is an attenuated Salmonella typhimurium strain selected from among the strains designated YS1646 (ATCC#202165), RE88, SL7207, χ8429, χ8431, and χ8468, or is a wild-type Salmonella strain, or is the strain deposited as ATCC#14028.

18. 18. The method of any one of claims 1, 4, 7, 8 and 14-17, wherein the cancer comprises a tumor or metastasis, or is a hematological malignancy.

19. The method of any one of claims 1, 4, 7, 8 and 14-17, wherein the subject has cancer, including a solid tumor.

20. 20. The method of any one of claims 1, 4, 7, 8 and 14-19, wherein the cancer is selected from lung cancer, head and neck cancer, gastric cancer, liver cancer, kidney cancer, breast cancer, colorectal cancer, prostate cancer, and chronic lymphoblastic leukemia.

21. cd73 ＋ 1. An immunostimulatory bacterium for treating a subject having cancer, including a tumor comprising the immunostimulatory bacterium comprises a plasmid encoding a therapeutic product for treating cancer; the immunostimulatory bacterium is a Salmonella spp.; the immunostimulatory bacterium is adenosine auxotrophic; the immunostimulatory bacterium is flagellin-deficient and the wild-type bacterium contains flagella; and The target is CD73 ＋ has been previously identified as having a tumor that is Immunostimulatory bacteria.

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