Immunostimulatory bacteria engineered to colonize tumors, tumor-resident immune cells, and the tumor microenvironment

Abstract

Provided are delivery immunostimulatory bacteria that have enhanced colonization of tumors, the tumor microenvironment and / or tumor-resident immune cells, and enhanced anti-tumor activity. The immunostimulatory bacteria are modified by deletion of genes encoding the flagella or by modification of the genes so that functional flagella are not produced, and / or are modified by deletion of pagP or modification of pagP to produce inactive PagP product. As a result, the immunostimulatory bacteria are flagellin" and / or pagP' . The immunostimulatory bacteria optionally have additional genomic modifications so that the bacteria are adenosine and / or purine auxotrophs. The bacteria optionally are one or more of asct, purl' and msbB' . The immunostimulatory bacteria, such as Salmonella species, are modified to encode proteins that induce type I interferon (IFN) expression, or that are variants thereof that have increased activity to induce type I IFN expression, or that are variants thereof that result in constitutive expression of type I IFN. The bacteria can encode a modified Stimulator of Interferon Genes (STING) protein from a non-human species, that has lower NF-κΒ signaling activity, and, optionally, higher type I IFN pathway signaling activity, compared to human STING. The bacteria preferentially infect immune cells in the tumor microenvironment, or tumor-resident immune cells, and / or induce less cell death in immune cells than in other cells. Also provided are methods of inhibiting the growth or reducing the volume of a solid tumor by administering the immunostimulatory bacteria.

Description

[0001] IMMUNOS TIMUL AT ORY BACTERIA ENGINEERED TO COLONIZE TUMORS, TUMOR-RESIDENT IMMUNE CELLS, AND THE TUMOR

[0002] MICROENVIRONMENT RELATED APPLICATIONS

[0003] Benefit of priority is claimed to U.S. Provisional Application Serial No.

[0004] 62 / 962,140, filed January 16, 2020, entitled“Immunostimulatory Bacteria Engineered To Colonize Tumors, Tumor-Resident Immune Cells, And The Tumor

[0005] Microenvironment,” to Applicant Actym Therapeutics, Inc., and inventors

[0006] Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, Alexandre Charles Michel Iannello, and Haixing Kehoe.

[0007] Benefit of priority also is claimed to U.S. Provisional Application Serial No. 62 / 934,478, filed November 12, 2019, entitled“ Immunostimulatory Bacteria

[0008] Engineered To Colonize Tumors And The Tumor Microenvironment,” to Applicant Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, and Alexandre Charles Michel Iannello.

[0009] Benefit of priority also is claimed to U.S. Provisional Application Serial No. 62 / 828,990, filed April 03, 2019, entitled“Salmonella Strains Engineered To

[0010] Colonize Tumors And The Tumor Microenvironment,” to Applicant Actym

[0011] Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, and Alexandre Charles Michel Iannello.

[0012] Benefit of priority also is claimed to U.S. Provisional Application Serial No. 62 / 811,521, filed February 27, 2019, entitled,“Tumor-Targeting Microorganisms that Promote Immuno-Stimulation of the Tumor Microenvironment,” to Applicant Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, and Alexandre Charles Michel Iannello.

[0013] Where permitted, the subject matter of each of these applications is incorporated by reference in its entirety. The immunostimulatory bacteria provided in each of these applications can be modified as described in this application, and such bacteria are incorporated by reference herein. IN CORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

[0014] An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on February 27, 2020, is 603 kilobytes in size, and is titled 1706SEQPC.txt.

[0015] BACKGROUND

[0016] Tumors have evolved a profoundly immunosuppressive environment. They initiate multiple mechanisms to evade immune surveillance, reprogram anti-tumor immune cells to suppress immunity, and continually mutate resistance to the latest cancer therapies (see, e.g., Mahoney et al. (2015) Nat. Rev. Drug Discov. 14(8): 561 - 584). The field of cancer immunotherapy has made great strides, as evidenced by the clinical successes of anti-CTLA4, anti -PD- 1 and anti-PD-Ll 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.

[0017] 125:3384-3391). Designing immunotherapies that overcome immune tolerance and escape, while limiting the autoimmune-related toxicities of current immunotherapies, challenges the field of immuno-oncology. Hence, additional and innovative immunotherapies and other therapies are needed.

[0018] SUMMARY

[0019] Provided are bacteria modified to be immunostimulatory for anti-cancer therapy. Immunostimulatory bacteria, as provided herein, provide a multi-faceted approach to anti-tumor therapy. Bacteria provide a platform in which there are numerous avenues for eliciting anti-tumor immunostimulatory activity. As provided herein, bacteria, such as species of Salmonella, are fine-tuned to have potent anti tumor activity by increasing their ability to accumulate in or target tumors, tumor- resident-immune cells, and / or the tumor microenvironment (TME). This is achieved by modifications that, for example, alter the type of cells that they can infect

[0020] (tropism), their toxicity, their ability to escape the immune system, such as escaping inactivation by complement, and / or the environments in which they can replicate. The immunostimulatory bacteria also can encode, for example, products that enhance or invoke an immune response and other therapeutic / anti-cancer products. The immunostimulatory bacteria provided herein, by virtue of their improved colonization of tumors / the tumor microenvironment / tumor-resident immune cells, and their resistance to complement and other anti -bacterial immune responses, can be administered systemically.

[0021] Bacteria by their nature stimulate the immune system; bacterial infection induces immune and inflammatory pathways and responses, some of which are desirable for anti-tumor treatment, and others, are undesirable. Modification of the bacteria by deleting or modifying genes and products that result in undesirable inflammatory responses, and adding or modifying genes that induce desirable immunostimulatory anti-tumor responses, improves the anti-tumor activity of the bacteria.

[0022] Bacteria accumulate in tumor cells and tissues, and by replicating therein can lyse cells. Bacteria migrate from the sites of administration and can accumulate in other ( e.g ., distal / metastatic) tumors and tumor cells to provide an abscopal effect.

[0023] The bacteria provided herein are modified so that they preferentially infect and accumulate in tumor-resident immune cells, tumors, and the tumor microenvironment, and deliver their plasmids that encode the therapeutic anti-cancer proteins and products. Herein, these properties of that bacteria are exploited to produce

[0024] demonstrably immunostimulatory bacteria with a plurality of anti-tumor activities and properties that can act individually and synergistically.

[0025] The genomes of the bacteria provided herein are modified to increase accumulation in tumors and in tumor-resident immune cells, and also in the tumor microenvironment. This is effected herein by deleting or disabling genes responsible for infection or invasion of non-tumor cells, such as epithelial cells, and / or decreasing the cytopathogenicity of the bacteria, particularly to immune cells and tumor-resident immune cells.

[0026] Upon accumulation in the tumor-resident immune cells, proteins encoded on plasmids under control of eukaryotic regulatory signals, are expressed, and secreted into the TME. Immunostimulatory bacteria provided herein encode proteins that have anti-cancer activity, such as by modulating the anti-tumor immune response. Bacteria provided herein encode proteins that lead to expression of type I interferon (IFN). Such proteins include STING (Stimulatory of Interferon Genes) and other

[0027] immunostimulatory proteins that are part of a cytosolic DNA / RNA sensor pathway leading to expression of type I IFN, and also variants of these proteins that increase expression of type I IFN or that result in constitutive expression of IFN. For example, the immunostimulatory proteins include constitutively active variants of cytosolic DNA / RNA sensors, such as those with gain-of-function mutations.

[0028] Provided are compositions, uses thereof and methods that modulate immune responses for the treatment of diseases, including for the treatment of cancer. The compositions contain immunostimulatory bacteria provided herein. Methods of treatment and uses of the bacteria for treatment also are provided. The subjects for treatment include humans and other primates, pets, such as dogs and cats, and other animals, such as horses, cows and other farm and zoo animals.

[0029] Provided are pharmaceutical compositions containing the immunostimulatory bacteria, and methods and uses thereof for treatment of diseases and disorders, particularly proliferative disorders, such as tumors, including solid tumors and hematologic malignancies.

[0030] Also provided are methods of inhibiting the growth or reducing the volume of a solid tumor by administering the immunostimulatory bacteria or pharmaceutical compositions or using the compositions for treatment. For example, provided are methods of administering or using a composition that contains, for a single dosage, an effective amount of an immunostimulatory bacterium, such as a Salmonella species, to a subject, such as a human patient, having a solid tumor cancer.

[0031] Provided are immunostimulatory bacteria that encode immunostimulatory proteins that are constitutively active proteins that stimulate or evoke expression of type I IFN. The immunostimulatory bacteria also can encode other anti-tumor therapeutics, such as RNAi, and cytokines and chemokines, and, other modifications of the bacteria and the plasmids described herein, can be combined in any desired combination.

[0032] Provided are immunostimulatory bacteria that have enhanced colonization of tumors, the tumor microenvironment and / or tumor-resident immune cells, and enhanced anti-tumor activity. The immunostimulatory bacteria are modified by deletion of genes encoding the flagella, and / or modification of the genes so that functional flagella are not produced, and / or deletion of pagP or modification of pagP to produce inactive PagP product. As a result, the immunostimulatory bacteria are flagellin (fliC / fljR ) and / or pagP . Alternatively, or additionally, the

[0033] immunostimulatory bacteria can be pagP / msbB .

[0034] The immunostimulatory bacteria can be aspartate-semialdehyde

[0035] dehydrogenase ( as ), such as by virtue of disruption or deletion of all or a portion of the endogenous gene encoding aspartate-semialdehyde dehydrogenase ( asd ), whereby the endogenous asd is not expressed. The immunostimulatory bacteria can be modified to encode aspartate-semialdehyde dehydrogenase ( asd) on a plasmid under control of a bacterial promoter for growing the bacteria in vitro , so that bacteria will have limited replication in vivo.

[0036] The immunostimulatory bacteria optionally have additional genomic modifications so that the bacteria are adenosine or purine auxotrophs. The bacteria optionally are one or more of asd , purl and msbB . The immunostimulatory bacteria, such as Salmonella species, are modified to encode immunostimulatory proteins that confer anti-tumor activity in the tumor microenvironment, and / or are modified so that the bacteria preferentially infect immune cells in the tumor microenvironment or tumor-resident immune cells and / or induce less cell death in immune cells than in other cells. Also provided are methods of inhibiting the growth or reducing the volume of a solid tumor by administering the immunostimulatory bacteria.

[0037] Provided are methods of increasing tumor colonization of an

[0038] immunostimulatory bacterium, such as a Salmonella species, by modifying the genome of an immunostimulatory bacterium to be flagellin (fliC / fljR), whereby flagella are not produced, and / or to be pagP . In particular, the bacteria are flagellin adenosine auxotrophs, and also are as . The bacteria that are flagellin are derived from bacterial species that express flagella.

[0039] The bacteria also contain plasmids that encode therapeutic products, such as anti -turn or agents, proteins that increase the immune response of a subject, proteins the lead to constitutive or increased expression of immune stimulating proteins, such as type I interferon (IFN), including interferon-b. This includes encoding proteins that stimulate the immune system as part of a pathway that results in type I IFN

[0040] expression, and, in particular, by rendering such proteins constitutively active. The plasmids also can encode immunostimulatory proteins, such as cytokines, that increase the anti -turn or immune response in the subject. The bacteria contain plasmids that encode anti-cancer therapeutics, such as interfering RNA, including microRNA, shRNA, and siRNA, that are designed to suppress, inhibit, disrupt or otherwise silence immune checkpoint genes and products, and other targets that play a role in pathways that are immunosuppressive. The bacteria also can encode tumor antigens on the plasmids to stimulate the immune response against the tumors. The encoded proteins are expressed under the control of promoters recognized by eukaryotic, such as mammalian and animal, or viral, promoters. The bacteria can expresses one, two, or more of the therapeutic proteins / products, including combinations of the gain-of- function immunostimulatory proteins, and / or cytokines. These heterologous proteins are encoded on the plasmid under control of a promoter, such as an RNA polymerase II or III promoter, recognized by a eukaryotic host.

[0041] Provided are immunostimulatory bacteria containing a plasmid encoding a product under control of a eukaryotic promoter, where the genome of the

[0042] immunostimulatory bacterium is modified whereby the bacterium is flagellin / fljB ) and / or pagP~. The bacteria can be one or both of flagellin (fliC / fljB ) and pagP

[0043] . These immunostimulatory bacteria exhibit increased tumor / tumor microenvironment and tumor-resident immune cell colonization, and have increased anti-tumor activity.

[0044] Also provided are immunostimulatory bacteria containing a plasmid encoding a therapeutic product under control of a eukaryotic promoter, where the genome of the immunostimulatory bacterium is modified whereby the bacterium is pagP~ / msbB~. These bacteria also have increased colonization of tumors, tumor-resident immune cells, and the tumor microenvironment. Because of the resulting change in bacterial membranes and structure, the host immune response, such as complement activity, is altered so that the bacteria are not eliminated upon systemic administration. These bacteria also can be flagellin (fliC / fljBj and can comprise other modifications as described herein, including modifications that alter the cells that they can infect, resulting in accumulation in the tumor microenvironment, tumors and tumor-resident immune cells. Hence, the immunostimulatory bacteria provided herein can be systemically administered and exhibit a high level of tumor / tumor microenvironment and / or tumor-resident immune cell colonization. The immunostimulatory bacteria can be purl (purMj, and one or more of asc , purP , msbP , and one or both of flagellin

[0045] ( fliC / fljB ) and pagF . The immunostimulatory bacteria can be one or more of purl (purM ), msbB , purl) , flagellin (fliC / fljB ), pagP , adrA , csgD , qseC , hi I A , IppA and IppB , and particularly flagellin (fliC / fljR) and / or pagP , and / or msbB / pagP . For example, the immunostimulatory bacteria can include mutations in the genome, such as deletions or disruptions that reduce toxicity or infectivity of non-immune cells in a host. For example, the immunostimulatory bacteria can be pagP . As another example, the immunostimulatory bacteria can be flagellin (fliC / fljB ), and can also be pagP . The bacteria can be modified so that they accumulate and express the therapeutic product(s) in tumor-resident immune cells and in the tumor microenvironment (TME), thereby delivering an immunotherapeutic anti -turn or product into the environment in which it has beneficial activity, and avoiding adverse or toxic side effects from expression in other cells / environments. The nucleic acids encoding the

[0046] immunostimulatory protein(s) / therapeutic product(s) can be operatively linked for expression to nucleic acids encoding a secretory signal, whereby, upon expression, in a host, the immunostimulatory protein / therapeutic product is secreted into the tumor microenvironment.

[0047] As discussed above, the genome of the immunostimulatory bacteria also is modified so that the bacteria preferentially infect immune cells, such as tumor- resident immune cells, and / or the genome is modified so that the bacteria induce less cell death in tumor-resident immune cells (decreased pyroptosis) than the unmodified bacteria. As a result, the immunostimulatory bacteria accumulate, or accumulate to a greater extent than those without the modifications, in tumors or in the tumor microenvironment or in tumor-resident immune cells, to thereby deliver the immunostimulatory protein(s) and constitutively active variants thereof, and other therapeutic products, to the cell to stimulate or induce expression of type I interferon. The bacteria can be one or more of flagellin (fliC / fljBj, pagP , and msbB , and can include other such modifications as described herein.

[0048] The immunostimulatory bacteria can also be aspartate-semialdehyde dehydrogenase (asct), such as by virtue of disruption or deletion of all or a portion of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (asd), whereby endogenous asd is not expressed. These immunostimulatory bacteria can be modified to encode aspartate-semialdehyde dehydrogenase ( asd) on the plasmid under control of a bacterial promoter so that the bacteria can be produced in vitro.

[0049] The immunostimulatory bacteria can be rendered auxotrophic for particular nutrients, that are rich or that accumulate in the tumor microenvironment, such as adenosine and adenine. Also, they can be modified to be auxotrophic for such nutrients to reduce or eliminate their ability to replicate. The inactivated / deleted bacterial genome genes can be complemented by providing them on a plasmid under the control of promoters recognized by the host.

[0050] Additionally, the genome of the immunostimulatory bacterium is modified so that it preferentially infects tumor-resident immune cells. This is achieved by deleting or disrupting bacterial genes that play a role in invasiveness or infectivity of the bacteria, and / or that play a role in inducing cell death. The bacteria are modified to preferentially infect tumor-resident immune cells, and / or to induce less cell death in such cells, than unmodified bacteria, or than in other cells that the bacteria can infect.

[0051] The immunostimulatory bacteria provided herein can include a modification of the bacterial genome, whereby the bacterium induces less cell death in tumor- resident immune cells; and / or a modification of the bacterial genome, whereby the bacterium accumulates more effectively in tumors, the TME, or tumor-resident immune cells. These immunostimulatory bacteria can be further modified so that the bacteria preferentially infect tumor-resident immune cells, and / or the genome of the immunostimulatory bacterium can be modified so that it induces less cell death in tumor-resident immune cells (decreases pyroptosis), whereby the immunostimulatory bacterium accumulates in tumors or in the tumor microenvironment or in tumor- resident immune cells, to thereby deliver a constitutively active immunostimulatory protein, or other therapeutic product(s), to the cell to stimulate or induce expression of type I IFN.

[0052] The immunostimulatory bacteria can include deletions or modifications of one or more genes or operons involved in SPI-1 -dependent invasion (and / or SPI-2), whereby the immunostimulatory bacteria do not invade or infect epithelial cells. Exemplary of genes that can be deleted or inactivated are one or more of awA , hi I A , hill), invA, invB, invC, invE , invF, invG, invH , invl , invJ , iacP, iagB, spaO, spaP , spaQ, spaR, spaS, orgA, orgB , orgC, prgPI , prgl , prgj , prgK , sic A , sicP, sipA, sipB, sipC, si pi), sirC , sopB, sopD, sopE , sopE2, sprB , and sptP. Elimination of the ability to infect epithelial cells also can be achieved by engineering the immunostimulatory bacteria herein to contain knockouts or deletions of genes encoding proteins involved in SPI-1 -independent invasion, such as one or more of the genes selected from among rck , pagN, hlyE , pefl, srgD , srgA , srgB , and .sr^f \ Similarly, the immunostimulatory bacteria can include deletions in genes and / or operons in SPI-2, for example, to engineer the bacteria to escape the Salmonella- containing vacuole (SCV). These genes include, for example, sifA , sse.J, sseL , sopD2,pipB2 , sseF, sseG, spvB, and steA.

[0053] For example, the immunostimulatory bacteria can be modified to have reduced pathogenicity, whereby infection of epithelial and / or other non-immune cells is reduced, relative to the bacterium without the modification. These include

[0054] modification of the type 3 secretion system (T3SS) or type 4 secretion system (T4SS), such as modification of the SPI-1 pathway or T3SS system of Salmonella as described and exemplified herein. The bacteria further can be modified to induce less cell death, such as by deletion or disruption of nucleic acids encoding PagP (lipid A

[0055] palmitoyltransferase), which reduces virulence of the bacterium.

[0056] The genome of the immunostimulatory bacteria provided herein can be modified to increase or promote infection of immune cells, particularly immune cells in the tumor microenvironment, such as phagocytic cells. This includes reducing infection of non-immune cells, such as epithelial cells, or increasing infection of immune cells. The bacteria also can be modified to decrease pyroptosis in immune cells. Numerous modifications of the bacterial genome can do one or both of increasing infection of immune cells and decreasing pyroptosis. The

[0057] immunostimulatory bacteria provided herein include such modifications, for example, deletions and / or disruptions of genes involved in the SPI-1 T3SS pathway, such as disruption or deletion of hi l A , and / or disruption / deletion of genes encoding flagellin, rod protein (PrgJ), needle protein (Prgl) and QseC.

[0058] The therapeutic products encoded on the plasmids for expression in a eukaryotic, such as a human, host, are under control of eukaryotic regulatory sequences, including eukaryotic promoters, such as promoters recognized by RNA polymerase II or III. These include viral and mammalian RNA polymerase II promoters.

[0059] Exemplary viral promoters, include, but are not limited to, a cytomegalovirus (CMV) promoter, an SV40 promoter, an Epstein Barr virus (EBV) promoter, a herpes virus promoter, a respiratory syncytial virus (RSV) promoter, and an adenovirus promoter. Other RNA polymerase II promoters include, but are not limited to, an elongation factor- 1 (EF-1) alpha promoter, or a UbC promoter (lentivirus), or a PGK (3-phosphoglycerate kinase) promoter, a synthetic MND promoter, and a synthetic promoter such as a CAGG (or CAG) promoter. The synthetic CAG promoter contains the cytomegalovirus (CMV) early enhancer element (C); the promoter, the first exon and the first intron of chicken beta-actin gene (A); and the splice acceptor of the rabbit beta-globin gene (G). MND is a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer (murine leukemia virus-derived MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted; see, e.g., Li et al. (2010) J. Neurosci. Methods 189:56-64). Other strong regulatable or constitutive promoters can be used. Exemplary of the promoters are the EF-1 alpha promoter, CMV, SV40, PGK, EIF4A1, CAG, and CD68 promoters. The regulatory sequences also include terminators, enhancers, secretory and other trafficking signals.

[0060] The plasmids included in the immunostimulatory bacteria can be present in low copy number or medium copy number, such as by selection of an origin of replication that results in medium-to-low copy number, such as a low copy number origin of replication. It is shown herein that the anti-tumor activity and other properties of the bacteria are improved when the plasmid is present in low to medium copy number, where medium copy number is less than 150 or less than about 150 and more than 20 or about 20 or is between 20 or 25 and 150, and low copy number is less than 25 or less than 20 or less than about 25 or less than about 20 copies.

[0061] The immunostimulatory bacteria provided herein include any of the strains and bacteria described in U.S. Application Serial No. 16 / 033,187, further modified to express an immunostimulatory protein and / or to preferentially infect and / or to be less toxic in immune cells in the tumor microenvironment, or in tumor-resident immune cells, as described and exemplified herein. Encoded Therapeutic Proteins / Products

[0062] The immunostimulatory bacteria encode a therapeutic protein or product, on a plasmid in the bacterium, under control of a eukaryotic promoter, that, when expressed in a mammalian subject, confers or contributes to anti -turn or immunity in the tumor microenvironment.

[0063] Products encoded by the immunostimulatory bacteria include proteins that are part of a cytosolic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), and variants thereof. These include variant proteins with increased activity and variant proteins that result in constitutive expression of type I interferons. These also include proteins that naturally, or by mutation, have decreased signaling activity in pathways that lead to undesirable immune responses, but that have type I interferon stimulating activity and / or interferon-b stimulating activity comparable to or greater than the native human proteins. In particular, the immunostimulatory bacteria encode gain-of-function (GOF) variants of an immunostimulatory protein that, in unmodified form, is part of a cytosolic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN). Exemplary are gain-of function,

[0064] constitutively active variants of an immunostimulatory protein that, in humans, promotes or causes interferonopathies, where the genome of the immunostimulatory bacterium is modified so that the bacterium preferentially infects tumor-resident immune cells, and / or the genome of the immunostimulatory bacterium is modified so that it induces less cell death in tumor-resident immune cells (decreases pyroptosis), whereby the immunostimulatory bacterium accumulates in tumors or in the tumor microenvironment or in tumor-resident immune cells, to thereby deliver the constitutively active immunostimulatory protein to the cell to stimulate or induce expression of type I IFN. The variant can include a mutation that eliminates a phosphorylation site in the immunostimulatory protein, to thereby reduce nuclear factor kappa-light-chain-enhancer of activated B cell (NF-KB) signaling. These include, for example, STING, RIG-I, MDA-5, IRF-3, IRF-5, IRF-7, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, and SNRNP200, and variants thereof, such as those expressed in interferonopathies and conservative variations thereof that have constitutive activity or increased activity. In some embodiments, these include proteins that induce type I IFN, such as STING, RIG-I, IRF-3, IRF-7, or MDA5, and variants thereof that have increased activity or constitutive activity, where the immunostimulatory protein is STING, RIG-I, IRF-3, IRF-7, or MDA5.

[0065] Hence, provided herein are immunostimulatory bacteria comprising a plasmid that contains heterologous nucleic acid encoding a gain-of-function variant of an immunostimulatory protein that, in unmodified form, is part of a cytosolic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN). These gain-of- function proteins are encoded on a plasmid under control of eukaryotic regulatory signals, including promoters, and optionally other regulatory signals, such as enhancers, polyA and transcription terminators. The nucleic acids encoding the proteins / products on the plasmid can be multiplexed, whereby a plurality of products are encoded. Strategies for multigene co-expression include use of multiple promoters in a single vector, fusion proteins, proteolytic cleavage sites between genes, internal ribosome entry sites (IRES), and“self-cleaving” (ribosome skipping) 2A peptides. 2A peptides are 18-22 amino-acid (aa)-long viral oligopeptides that mediate“cleavage” of polypeptides during translation in eukaryotic cells. Thus, provided are plasmids that encode the therapeutic products on the plasmid under control of a single promoter by including 2A self-cleaving peptides between the coding portions, such as T2A, P2A, F2A, and E2A.

[0066] The unmodified forms of the immunostimulatory proteins are proteins in a signaling pathway that senses cytosolic DNA / RNA. They include those proteins that are modified with amino acid replacement s) or deletions that increase activity and / or render the activity constitutive. Provided are immunostimulatory bacteria that contain a plasmid encoding a gain-of-function, constitutively active variant of an

[0067] immunostimulatory protein. These gain-of-function proteins, include proteins in the signaling pathway that leads to expression of type I interferon, including proteins that, in humans, promote or cause interferonopathies, and gain-of-function mutants that are modified, having been selected, to result in constitutive expression of type I interferon. The immunostimulatory protein in its unmodified form is one that senses or interacts directly or indirectly as part of a signaling pathway with cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides, to induce expression of type I interferon, and the variant protein induces expression of type I interferon in the absence of the sensing or interacting with the cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides (CDNs). Included are gain-of-function variants that do not require cytosolic nucleic acid, nucleotides, dinucleotides, or cyclic dinucleotides to result in expression of a type I interferon. Exemplary of such proteins are STING, RIG-I, MDA-5, IRF-3, IRF-5, IRF-7, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, and SNRNP200.

[0068] In these immunostimulatory bacteria, the encoded variant gain-of-function protein can be one that eliminates a phosphorylation site in the immunostimulatory protein to thereby reduce nuclear factor kappa-light-chain-enhancer of activated B cell (NF-KB) signaling. Alternatively, the bacteria can include one or more replacements of the amino acid serine (S) or threonine (T) at a phosphorylation site with aspartic acid (D), which is phosphomimetic, and results in increased or constitutive activity. Exemplary of the proteins in signaling pathways that result in type I interferon expression are STING, RIG-I, IRF-3, IRF-7 and MDA5. Described herein are exemplary mutations that result in gain-of-function activity for each of these proteins. Mutations include those in which the encoded immunostimulatory protein is a variant STING, RIG-I, IRF-3, IRF-7 or MDA5, in which one or more serine (S) or threonine residue(s) that is / are phosphorylated as a consequence of viral infection, is / are replaced with an aspartic acid (D), whereby the resulting variant is a phosphomimetic that constitutively induces type I interferon. For example, provided are

[0069] immunostimulatory bacteria in which the immunostimulatory protein is IRF-3 that has one or more replacement s) at residues at positions 385, 386, 396, 398, 402, 404 and 405, and the residues are replaced with aspartic acid residues; this includes IRF-3 that has the replacement S396D with reference to SEQ ID NO:312, and IRF-3 that comprises the mutations S396D / S398D / S402D / T404D / S405D with reference to SEQ ID NO:312. Other examples are immunostimulatory bacteria wherein the

[0070] immunostimulatory protein is selected from among STING, MDA5, IRF-7 and RIG-I, in which the mutations are selected as follows: a) in STING, with reference to human STING of SEQ ID NOs: 305-309, one or more selected from among: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A,

[0071] S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A, and S324A / S326A; b) in MDA5, with reference to SEQ ID NO:310, one or more of: T331I, T331R, A489T, R822Q, G821 S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F, and A452T; c) in RIG-I, with reference to SEQ ID NO:311, one or both of E373 A and C268F; and d) in IRF-7, with reference to SEQ ID NO:313, one or more of: S477D / S479D, S475D / S477D / S479D,

[0072] S475D / S476D / S477D / S479D / S483D / S487D and D247-467. Any of these

[0073] replacements can be replaced with a conservative mutations in accord with the Table of Exemplary Conservative Amino Acid Substitutions below.

[0074] Also provided are delivery vehicles, such as exosomes, liposomes, oncolytic viruses, nanoparticles, the immunostimulatory bacteria, and other such vehicles, that contain nucleic acids encoding the gain-of-function proteins and other therapeutic products, as described above and elsewhere herein. For example, provided are delivery vehicles that contain nucleic acids, generally DNA encoding a gain-of- function immunostimulatory protein that is part of a signaling pathway that results in expression of type I interferon. The gain-of-function variants can render expression of type I interferon constitutive. For example, these variants include any discussed herein, such as a modified STING, where: the modifications in STING render its activity constitutive so that it does not require cGAMP (or other ligands / CDNs) for activity; modified STING is encoded by a modified TMEM173 gene; the

[0075] modifications comprise insertions, deletions or replacements of amino acid(s); and the modified STING has enhanced immunostimulatory activity compared to the unmodified STING. These amino acid replacement(s) in STING, with reference to human STING of SEQ ID NOs: 305-309, include one or more selected from among: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A, and S324A / S326A. The immunostimulatory bacteria provided herein also can contain a sequence of nucleotides encoding an immunostimulatory protein that, when expressed in a mammalian subject, confers or contributes to anti -turn or immunity in the tumor microenvironment; the immunostimulatory protein is encoded on a plasmid in the bacterium under control of a eukaryotic promoter. Exemplary promoters include, but are not limited to, an elongation factor- 1 (EF1) alpha promoter, or a UbC promoter, or a PGK promoter, or a CAGG or CAG promoter.

[0076] The immunostimulatory bacterial also can encode an inhibitory RNA (RNAi) that, when expressed in a mammalian subject, confers or contributes to anti -turn or immunity. The RNAi is encoded on a plasmid in the bacterium under control of a eukaryotic promoter. The genome of the immunostimulatory bacterium is modified so that it induces less cell death in tumor-resident immune cells and / or so that it accumulates in tumor-resident immune cells and in the tumor

[0077] microenvironment / tumors.

[0078] The immunostimulatory bacteria provided herein also can encode other immunostimulatory proteins. The immunostimulatory protein can be a cytokine, such as a chemokine. Exemplary of immunostimulatory proteins are IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-15, IL-15 / IL-15R alpha chain complex, IL-36 gamma, IL- 18, CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, proteins that are involved in or that effect or potentiate the recruitment / persistence of T cells, CD40, CD40 Ligand (CD40L), 0X40, 0X40 Ligand (OX40L), 4-1BB, 4-1BB Ligand (4-1BBL), members of the B7-CD28 family, and members of the tumor necrosis factor receptor (TNFR) superfamily. In some embodiments, these include, for example, IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-15, IL-23, IL-36 gamma, IL-2 that has attenuated binding to IL-2Ra, IL-15 / IL-15R alpha chain complex, IL-18, IL-2 modified so that it does not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-a, interferon-b, CCL3, CCL4, CCL5, proteins that are involved in or that effect or potentiate

[0079] recruitment / persistence of T cells, CD40, CD40 Ligand, 0X40, 0X40 Ligand, 4-1BB, 4- IBB Ligand, members of the B7-CD28 family, TGF-beta polypeptide antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily.

[0080] The immunostimulatory bacteria can optionally include a sequence of nucleotides encoding inhibitory RNA (RNAi) that inhibits, suppresses or disrupts expression of an immune checkpoint. The RNAi can be encoded on a plasmid in the bacterium. The nucleotides encoding the immunostimulatory protein, and optionally an RNAi, can be on a plasmid present in low to medium copy number.

[0081] The immunostimulatory bacteria also can encode therapeutic products, such as RNAi or a CRISPR cassette that inhibits, suppresses or disrupts expression of an immune checkpoint or other target whose inhibition, suppression or disruption increases the anti -turn or immune response in a subject; the RNAi or CRISPR cassette is encoded on a plasmid in the bacterium. Other therapeutic products include, for example, antibodies that bind to immune checkpoints to inhibit their activities, such as, for example, anti-PD-1, anti-PD-Ll and anti-CTLA-4 antibodies.

[0082] RNAi includes all forms of double-stranded RNA that can be used to silence the expression of targeted nucleic acids. RNAi includes shRNA, siRNA and microRNA (miRNA). Any of these forms can be interchanged in the embodiments disclosed and described herein. In general, the RNAi is encoded on a plasmid in the bacterium. The plasmids can include other heterologous nucleic acids that encode products of interest that modulate or add activities or products to the bacterium, or other such products that can modulate the immune system of a subject to be treated with the bacterium. Bacterial genes also can be added, deleted or disrupted. These genes can encode products for growth and replication of the bacteria, or products that also modulate the immune response of the host to the bacteria.

[0083] Bacterial species include, but are not limited to, for example, strains of Salmonella , Shigella , Listeria , E. coli , and Bifidobacteriae . For example, species include Shigella sonnei , Shigella flexneri , Shigella dysenteriae , Listeria

[0084] monocytogenes , Salmonella typhi , Salmonella typhimurium , Salmonella gallinarum , and Salmonella enteritidis.

[0085] Species include, for example, strains of Salmonella , Shigella , E. coli ,

[0086] Bifidobacteriae , 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 strain thereof or a modified strain thereof of any of the preceding list of bacterial strains. Other suitable bacterial species include Rickettsia , Klebsiella , Bordetella , Neisseria , Aeromonas, Franciesella , Coryne bacterium, Citrobacter , Chlamydia , Haemophilus , Brucella , Mycobacterium , Mycoplasma , Legionella , Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix. For example, Rickettsia Rikettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida,

[0087] Franciesella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus sornnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix

[0088] rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, and Agrobacterium tumerfacium.

[0089] Salmonella is exemplified herein, and particularly, Salmonella typhimurium strains, such as the strain designated YS1646 (ATCC #202165) or VNP20009, and the wild-type strain deposited as ATCC #14028, or a strain having all of the identifying characteristics of ATCC #14028. Other strains include, for example, RE88, SL7207, c 8429, c 8431, and c 8468. Exemplary Salmonella strains provided herein are immunostimulatory bacterium strains AST-104, AST-105, AST-106, AST-108, AST- 110, AST-112, AST-113, AST-115, AST-117, AST-118, AST-119, AST-120, AST-

[0090] 121, AST-122, and AST-123. These strains can be further modified to encode immunostimulatory proteins that are gain-of-function variants of proteins in signaling pathways that lead to expression of type I interferon or other immune modulatory proteins. The immunostimulatory bacteria also can encode immunostimulatory proteins that increase the immune response in the tumor microenvironment, such as cytokines. The immunostimulatory bacteria also can be modified to preferentially infect immune cells in the tumor microenvironment or to infect tumor-resident immune cells, and / or to induce less cell death in such immune cells, as described herein. Sequences thereof and descriptions are provided in the detailed description, examples and sequence listing. The immunostimulatory bacteria can be derived from attenuated strains of bacteria, or they become attenuated by virtue of the modifications described herein, such as deletion of asd, whereby replication is limited in vivo.

[0091] It is understood that instances in which bacterial genes are modified and referenced herein, they are referenced with respect to their designation (name) in Salmonella species, which is exemplary of bacteria from which immunostimulatory bacteria can be produced. The skilled person recognizes that other species have corresponding proteins, but that their designation or name can be different from the name in Salmonella. The generic disclosure herein, however, can be applied to other bacterial species. For example, as shown herein, deletion or inactivation of flagellin (fliC / fljB ) in Salmonella and / or pagP results in increased colonization of tumors.

[0092] Similar genes for flagella or similar functions for infection can be modified in other bacterial species to achieve increased tumor colonization. Similarly,

[0093] inactivation / deletion of bacterial products, such as the products of pagP and / or msbB , as described herein, can reduce complement activation and / or other inflammatory responses, thereby increasing targeting to tumors, tumor-resident immune cells and the tumor microenvironment. Corresponding genes in other species that are involved in activating the complement pathway or other inflammatory pathway, can be deleted, as exemplified herein for Salmonella.

[0094] The immunostimulatory bacteria provided herein encode inhibitors of various genes that contribute to reduced anti-tumor immune responses and / or express genes and / or gene products and / or products that stimulate the immune system, and thereby are immunostimulatory.

[0095] The immunostimulatory bacteria provided herein have properties that render them immunostimulatory. Adenosine auxotrophy also is immunostimulatory. They also they encode, on the plasmid, therapeutic payloads, such as gain-of- function / constitutively active STING mutants, and other immunostimulatory proteins. The effects of this combination are enhanced by the strains provided herein that are auxotrophic for adenosine, which provides preferential accumulation in, or recruitment into, adenosine-rich immunosuppressive tumor microenvironments (TMEs). Reducing adenosine in such TMEs further enhances the immunostimulatory effects. Such combinations of traits in any of the bacterial strains known, or that can be engineered for therapeutic administration, provide similar immunostimulatory effects.

[0096] Engineered immunostimulatory bacteria, such as the S. typhimurium immunostimulatory bacteria provided herein, contain multiple synergistic modalities to induce immune re-activation of cold tumors to promote tumor antigen-specific immune responses, while inhibiting immune checkpoint pathways that the tumor utilizes to subvert and evade durable anti-tumor immunity. Included in embodiments is adenosine auxotrophy and enhanced vascular disruption. This improvement in tumor targeting through adenosine auxotrophy and enhanced vascular disruption increases potency, while localizing the inflammation to limit systemic cytokine exposure and the autoimmune toxicities observed with other immunotherapy modalities.

[0097] The heterologous proteins, such as the immunostimulatory proteins and gain- of-function immunostimulatory proteins, and RNAs are expressed on plasmids under the control of promoters that are recognized by the eukaryotic host cell transcription machinery, such as RNA polymerase II (RNAP II) and RNA polymerase III (RNAP III) promoters. RNAP III promoters generally are constitutively expressed in a eukaryotic host; RNAP II promoters can be regulated. The therapeutic

[0098] products / immunostimulatory proteins are provided on plasmids stably expressed by the bacteria. Exemplary of such bacteria are Salmonella strains, generally attenuated strains, either attenuated by passage or other methods, or by virtue of modifications described herein, such as adenosine auxotrophy. Exemplary of Salmonella strains are modified S. typhimurium strains that have a defective asd gene. These bacteria can be modified to include carrying a functional asd gene on the introduced plasmid; this maintains selection for the plasmid so that an antibiotic-based plasmid

[0099] maintenance / selection system is not needed. The asd defective strains that do not contain a functional asd gene on a plasmid are autolytic in the host.

[0100] The promoters can be selected for the environment of the tumor cell, such as a promoter expressed in a tumor microenvironment (TME), a promoter expressed in hypoxic conditions, or a promoter expressed in conditions where the pH is less than 7.

[0101] The plasmids in any of the bacteria described and enumerated above encode therapeutic products. Plasmids can be present in many copies or fewer. This can be controlled by selection of elements, such as the origin of replication. Low and high and medium copy number plasmids and origins of replication are well known to those of skill in the art and can be selected. In embodiments of the immunostimulatory bacteria here, the plasmid can be present in low to medium copy number, such as about 150 or 150 and fewer copies, to low copy number which is less than about 25 or about 20 or 25 copies. Exemplary origins of replication are those derived from pBR322, pi 5 A, pSClOl, pMBl, colEl, colE2, pPSlO, R6K, Rl, RK2, and pUC.

[0102] The plasmids encode therapeutic polypeptides, such as the polypeptides that induce type I interferons, such as those expressed in interferonopathies, and / or any therapeutic proteins described herein, and / or known to those of skill in the art for use in cancer therapies. The plasmids also can include sequences of nucleic acids encoding listeriolysin O (LLO) protein lacking the signal sequence (cytoLLO), a CpG motif, a DNA nuclear targeting sequence (DTS), and a retinoic acid-inducible gene-I (RIG-I) binding element. The immunostimulatory bacterium that comprises nucleic acids can include a CpG motif recognized by toll-like receptor 9 (TLR9). The CpG motif can be encoded on the plasmid. The CpG motif can be included in, or is part of, a bacterial gene that is encoded on the plasmid. For example, the gene that comprises CpGs can be asd , encoded on the plasmid. Immunostimulatory bacteria provided herein can include one or more of a CpG motif, an asd gene selectable marker for plasmid maintenance and a DNA nuclear targeting sequence.

[0103] The immunostimulatory bacteria can be flagellin deficient, such as by deletion of or disruption in a gene(s) encoding the flagella. For example, provided are immunostimulatory bacteria that contain deletions in the genes encoding one or both of flagellin subunits fliC and fljB, whereby the bacterium is flagella deficient, and wherein the wild-type bacterium expresses flagella. The immunostimulatory bacteria also can have a deletion or modification in the gene encoding endonuclease I (endA), whereby endA activity is inhibited or eliminated.

[0104] The immunostimulatory bacteria provided herein can be aspartate- semialdehyde dehydrogenase (asd), which permits growth in DAP supplemented medium, but limits replication in vivo when administered to subjects for treatment. Such bacteria will be self-limiting, which can be advantageous for treatment. The bacterium can be asc by virtue of disruption or deletion of all or a portion of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (asd), whereby the endogenous asd is not expressed. In other embodiments, the gene encoding aspartate- semialdehyde dehydrogenase can be included on the plasmid for expression in vivo.

[0105] Any of the immunostimulatory bacteria provided herein can include nucleic acid, generally on the plasmid, that includes a CpG motif or a CpG island, wherein the CpG motif is recognized by toll-like receptor 9 (TLR9). Nucleic acid encoding CpG motifs or islands are plentiful in prokaryotes, and, thus, the CpG motif can be included in, or can be a part of, a bacterial gene that is encoded on the plasmid. For example, the bacterial gene asd contains immunostimulatory CpGs.

[0106] The immunostimulatory bacteria provided herein can be auxotrophic for adenosine, or adenosine and adenine. Any of the bacteria herein can be rendered autotrophic for adenosine, which advantageously can increase the anti-tumor activity, since adenosine accumulates in many tumors, and is immunosuppressive.

[0107] The immunostimulatory bacteria provided herein can be flagellin deficient, where the wild-type bacterium comprises flagella. They can be rendered flagellin deficient by disrupting or deleting all or a part of the gene or genes that encode flagella. For example, provided are immunostimulatory bacteria that have deletions in the genes encoding one or both of flagellin subunits FliC and FljB, whereby the bacteria is flagella deficient.

[0108] The immunostimulatory bacteria provided herein can include a nucleic acid encoding cytoLLO, which is a listeriolysin O (LLO) protein lacking the periplasmic secretion signal sequence so that it accumulates in the cytoplasm. This mutation is advantageously combined with asd bacteria. LLO is a cholesterol-dependent pore forming hemolysin from Listeria monocytogenes that mediates phagosomal escape of bacteria. When the autolytic strain is introduced into tumor-bearing hosts, such as humans, the bacteria are taken up by phagocytic immune cells and enter the vacuole. In this environment, the lack of DAP prevents bacterial replication, and results in autolysis of the bacteria in the vacuole. Lysis then releases the plasmid and the accumulated LLO forms pores in the cholesterol-containing vacuole membrane and allows for delivery of the plasmid into the cytosol of the host cell. Here, the therapeutic products can be expressed using the host cell machinery, and released into the tumor microenvironment to effect anti-tumor therapy. The immunostimulatory bacteria can include a DNA nuclear targeting sequence (DTS), such as an SV40 DTS, encoded on the plasmid.

[0109] The immunostimulatory bacteria can have a deletion or modification in the gene encoding endonuclease- 1 (endA), whereby end A activity is inhibited or eliminated. Exemplary of these are immunostimulatory bacteria that contain one or more of a CpG motif, an asd gene selectable marker for plasmid maintenance and a DNA nuclear targeting sequence.

[0110] The immunostimulatory bacteria can contain nucleic acids on the plasmid encoding two or more different RNA molecules that inhibit, suppress or disrupt expression of an immune checkpoint or an RNA molecule that encodes an inhibitor of a metabolite that is immunosuppressive or is in an immunosuppressive pathway.

[0111] The nucleic acids encoding the RNAi, such as shRNA or miRNA or siRNA can include a transcriptional terminator following the RNA-encoding nucleic acid. In all embodiments, the RNAi encoded on the plasmid in the immunostimulatory bacteria can be short hairpin RNAs (shRNAs) or micro-RNAs (miRNAs).

[0112] The plasmids in any of the immunostimulatory bacteria also can encode a sequence of nucleotides that is an agonist of retinoic acid-inducible gene I (RIG-I) or a RIG-I binding element.

[0113] The immunostimulatory bacteria can include one or more of deletions in genes, such as one or more of purl (purM ), mshB purl) flagellin (flidfljB ), pagP , adrA , csgD and hilA . The immunostimulatory bacteria can be msbB . For example, the immunostimulatory bacteria can contain one or more of a purl deletion, an msbB deletion, an asd deletion, and adrA deletion, and optionally a csgD deletion. Exemplary of bacterial gene deletions / modifications are any of the following:

[0114] one or more of a mutation in a gene that alters the biosynthesis of

[0115] lipopolysaccharide selected from among one or more of rfaL, rfaG , rfaH rfaD , rfaP , rFb , rfa, msbB , htrB,firA,pagL,pagP , IpxR, arnJ , ep / A , and IpxT ; and / or

[0116] one or more of a mutation that introduces a suicide gene and is selected from one or more of sacB , nuk, hok , gef kil or phi A ; and / or

[0117] one or more of a mutation that introduces a bacterial lysis gene and is selected from one or both of hly and cly and / or a mutation in one or more virulence factor(s) selected from among IsyA,pag, prg, iscA, virG,plc and act, and / or

[0118] one or more mutations that modify the stress response selected from among recA , htrA , htpR , hsp and groEL; and / or

[0119] a mutation in min that disrupts the cell cycle; and / or

[0120] one or more mutations that disrupt or inactivate regulatory functions selected from among cya, crp , phoP / phoQ , and ompR.

[0121] The immunostimulatory bacterium can be a strain of Salmonella , Shigella , E. coli , Bifidohacteriae , Rickettsia , Vibrio , Listeria , Klebsiella , Bordetella , Neisseria , Aeromonas, Francisella , Cholera , Corynebacterium , Citrobacter , Chlamydia ,

[0122] Haemophilus , Brucella , Mycobacterium , Mycoplasma , Legionella , Rhodococcus, Pseudomonas , Helicobacter , Bacillus , or Erysipelothrix , or an attenuated strain thereof or modified strain thereof of any of the preceding list of bacterial strains.

[0123] Exemplary of the immunostimulatory bacteria are those where the plasmid contains one or more of a sequence of nucleic acids encoding a listeriolysin O (LLO) protein lacking the signal sequence (cytoLLO), a CpG motif, a DNA nuclear targeting sequence (DTS), and a retinoic acid-inducible gene-I (RIG-I) binding element.

[0124] Where the plasmid contains two or more therapeutic products under control of separate promoters each is separated by at least about 75 nucleotides, or 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 1600 or 1600 nucleotides (or base pairs), or between 75-1500 or 1600 nucleotides (or base pairs).

[0125] Other exemplary immunostimulatory bacteria include those that are auxotrophic for adenosine, and comprise: one or more of a deletion in the gene(s) encoding the flagella; a deletion in endA ; a plasmid that encodes CytoLLO; a nuclear localization sequence; and an asd plasmid complementation system; and encode a therapeutic product, including a gain-of-function variants of an immunostimulatory protein that, in unmodified form, is part of a cytosolic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), such as any described herein.

[0126] Such immunostimulatory bacteria include strains of Salmonella , such as a wild type Salmonella typhimurium strain, such as the strain deposited under ATCC accession no. 14028, or a strain having all of the identifying characteristics of the strain deposited under ATCC accession #14028. Other strains include, for example, an attenuated Salmonella typhimurium strain selected from among strains designated as AST-100, VNP20009, or strains YS1646 (ATCC #202165), RE88, SL7207, c 8429, c 8431, and c 8468.

[0127] The immunostimulatory bacteria can contain one or more of a purl deletion, an msbB deletion, an asd deletion, and an adrA deletion, in addition to the modifications that increase accumulation in tumor cells and / or reduce cell death, and can encode an immunostimulatory protein as described herein. The

[0128] immunostimulatory bacteria also can include:

[0129] one or more of a mutation in a gene that alters the biosynthesis of

[0130] lipopolysaccharide selected from among one or more of rfaL, rfaG , rfaH rfaD , rfaP , rFb , rfa, msbB , htrB,firA,pagL,pagP , IpxR, arnL , ep / A , and IpxT ; and / or

[0131] one or more of a mutation that introduces a suicide gene and is selected from among one or more of sacB , nuk, hok , gef kil and phi A ; and / or

[0132] one or more of a mutation that introduces a bacterial lysis gene and is selected from among one or both of hly and cly and / or

[0133] a mutation in one or more virulence factor(s) selected from among IsyA,pag, prg, iscA, virG,plc and acl and / or

[0134] one or more mutations that modify the stress response selected from among recA , htrA , htpR , hsp and groEL; and / or

[0135] a mutation in min that disrupts the cell cycle; and / or

[0136] one or more mutations that disrupt or inactivate regulatory functions selected from among cya, crp , phoP / phoQ and ompR.

[0137] The strains can be one or more of msbB , asd , hi l A and / or flagellin (fliC / fljB

[0138] ), and / or pagP . The therapeutic product, such as gain-of-function variants of an immunostimulatory protein that, in unmodified form, is part of a cytosolic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), RNAi, and immunostimulatory proteins, such as chemokines / cytokines, are expressed under control of a promoter recognized by the host, such as an RNAP III promoter or an RNAP II promoter, as described herein. The immunostimulatory bacterium can be a strain of Salmonella , Shigella , E. coli , Bifidobacteriae , Rickettsia , Vibrio , Listeria , Klebsiella , Bordetella , Neisseria , Aeromonas, Francisella , Cholera ,

[0139] Coryne bacterium, Citrobacter , Chlamydia , Haemophilus , Brucella , Mycobacterium , Mycoplasma , Legionella , Rhodococcus, Pseudomonas , Helicobacter , Bacillus , or Erysipelothrix , or an attenuated strain thereof or a modified strain thereof of any of the preceding list of bacterial strains. Generally, the strain is one that is attenuated in the host, either as an attenuated strain or by virtue of the modifications that alter its properties, including cells it can infect and ability to replication in certain cells or all cells. Salmonella strains, such as A typhimurium , are exemplary of the bacteria.

[0140] Exemplary strains include Salmonella typhimurium strains derived from strains designated as AST-100, VNP20009, or strains YS1646 (ATCC #202165), RE88, SL7207, c 8429, c 8431, c 8468, and the wild-type strain ATCC #14028.

[0141] Compositions containing the immunostimulatory bacteria are provided. Such compositions contain the bacteria and a pharmaceutically acceptable excipient or vehicle. The immunostimulatory bacteria include any described herein or in patents / applications incorporated herein or known to those of skill in the art. Such bacteria are modified to encode a variant of an immunostimulatory protein that is part of a signaling pathway resulting in expression of a type I interferon. The protein, such as a STING protein, is modified so that it has increased activity and / or leads to constitutive expression of the type I interferon, such as interferon-a or interferon-b. The bacteria can also encode an immunostimulatory protein that increases anti-tumor activity in the tumor microenvironment or in the tumor, such as a cytokine. The genomes of the bacteria can be modified to have increased infectivity of immune cells, and or reduced infectivity of non-immune cells, and / or reduced ability to induce cell death of immune cells. Hence, the bacteria are modified as described herein to accumulate in tumors or the tumor microenvironment or tumor-resident immune cells, and / or to deliver immunostimulatory proteins that promote anti-tumor activity. The immunostimulatory bacteria can additionally contain a plasmid encoding RNAi, such as miRNA or shRNA, or a CRISPR cassette, that target an immune checkpoint, or otherwise enhance the anti-tumor activity of the bacteria.

[0142] A single dose is therapeutically effective for treating a disease or disorder in which immune stimulation effects treatment. Exemplary of such stimulation is an immune response, that includes, but is not limited to, one or both of a specific immune response and non-specific immune response, specific and non-specific immune responses, an innate response, a primary immune response, adaptive immunity, a secondary immune response, a memory immune response, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression.

[0143] Provided are immunostimulatory bacteria that are cGAS agonists. Exemplary of such bacteria are Salmonella species, such as S. typhimurium , that is one or both of a cGAS agonist and Stimulator of Interferon Genes (STING) agonist. These can be administered, for example, in uses and methods, such as radiotherapy and

[0144] chemotherapy, in which cytosolic DNA is produced or accumulates. STING activates innate immunity in response to sensing nucleic acids in the cytosol. Downstream signaling is activated through binding of cyclic dinucleotides (CDNs), which are synthesized by bacteria or by host enzyme cGAS in response to binding to cytosolic dsDNA. Bacterial and host-produced CDNs have distinct phosphate bridge structures, which differentiates their capacity to activate STING. CDNs are synthesized by bacteria or by host enzyme cGAS in response to binding cytosolic dsDNA. IFN-b is the signature cytokine of activated STING.

[0145] Also provided are modified non-human STING proteins and STING protein chimeras, as well as delivery vehicles, including any described herein, including the bacteria, liposomes, exosomes, minicells, nanoparticles, vectors, such as oncolytic virus, pharmaceutical compositions containing the proteins and / or the delivery vehcles, cells encoding or containing these STING proteins and / or containing the delivery vehicles, and uses thereof and methods of treatment of cancers. The modified non-human STING proteins and STING protein chimeras, as well as the delivery vehicles, cells, immunostimulatory bacteria, uses, methods and pharmaceutical compositions, include, but are not limited to:

[0146] 1. Modified non-human STING proteins, where the non-human STING protein is one that has lower NF-KB activation than the human STING protein, and, optionally, higher type I interferon activation / signaling activity, compared to the wild type (WT) human STING protein. These non-human STING proteins are modified to include a mutation or mutations so that they have increased activity or act

[0147] constitutively, in the absence of cytosolic nucleic acid signaling. The mutations are typically amino acid mutations that occur in interferonopathies in humans, such as those described above for human STING. The corresponding mutations are introduced into the non-human species STING proteins, where corresponding amino acid residues are identified by alignment (see, e.g ., figures 1-13). Also, in some embodiments, the TRAF6 binding site in the C-terminal tail (CTT) of the STING protein is deleted, reducing NF-KB signaling activity.

[0148] 2. Modified STING proteins, particularly human STING proteins, that are chimeras, in which the CTT (C-terminal tail) region in the STING protein from one species, such as human, is replaced with the CTT from the STING protein of another, non-human species that has lower NF-KB signaling activity and / or higher type I IFN signaling activity than human STING. Also, the TRAF6 binding site is optionally deleted from the CTT in these chimeras.

[0149] 3. The modified STING proteins of 2 that also include the mutations of 1.

[0150] 4. Delivery vehicles, such as immunostimulatory bacteria, any provided herein or known to those of skill in the art, including exosomes, minicells, liposomes, nanoparticles, oncolytic viruses, and other viral vectors, that encode the modified STING proteins of any of 1-3.

[0151] 5. Delivery vehicles, such as immunostimulatory bacteria, any provided herein or known to those of skill in the art, including exosomes, minicells, liposomes, nanoparticles, oncolytic viruses, and other viral vectors, that encode unmodified

[0152] STING from non-human species whose STING protein has reduced NF-KB signaling activity compared to that of human STING, and optionally increased type I interferon stimulating / signaling activity.

[0153] 6. Cells (non-zygotes, if human), such as cells used for cell therapy, such as T-cells and stem cells, and cells used to produce the proteins of any of 1-3.

[0154] 7. Pharmaceutical compositions that contain the STING proteins of 1-3 or the delivery vehicles of 4 and 5, or the cells of 6.

[0155] 8. Uses and methods of treatment of cancer by administering any of 1-7, as described herein for the immunostimulatory bacteria.

[0156] 9. Also provided are immunostimulatory bacteria that encode non-human

[0157] STING proteins, particularly any that have lower NF-KB activity (signaling activity) and similar or greater type I interferon stimulating activity or interferon-b stimulating activity compared to human STING.

[0158] Assays and methods to assess NF-KB activity (signaling activity) and type I interferon stimulating activity or interferon-b stimulating activity of STING are described herein, and also are known to those of skill in the art. Methods include those described, for example, in de Oliveira Mann et al. (2019) Cell Reports 27 1165- 1175, which describes, inter alia , the interferon-b and NF-KB signaling activity of STING proteins from various species, including human, thereby identifying STING proteins from various species that have lower NF-KB activity than human STING, and those that also have comparable or higher interferon-b activity than human STING de Oliveira Mann et al. (2019) provides species alignments and identifies domains of STING in each species, including the CTT domain (see, also, the Supplemental Information for de Oliveira Mann et al. (2019)).

[0159] The non-human STING proteins can be, but are not limited to, STING proteins from the following species: Tasmanian devil Sarcophilus harrisii ; SEQ ID NO:331), marmoset (i Callithrix jacchus, SEQ ID NO:341), cattle ( Bos taurus ; SEQ ID NO:342), cat (Felis catus ; SEQ ID NO:338), ostrich (, Struthio camelus australis ; SEQ ID NO:343), crested ibis (Nipponia nippon ; SEQ ID NO:344), coelacanth ( Latimeria chalumnae^ SEQ ID NOs:345-346), boar ( Sus scrofa SEQ ID NO:347), bat

[0160] ( Rousettus aegyptiacus SEQ ID NO:348), manatee ( Trichechus manatus latirostris ;

[0161] SEQ ID NO:349), ghost shark ( Callorhinchus milir, SEQ ID NO:350), mouse (Mus musculus ; SEQ ID NO:351), and zebrafish ( Danio rerio; SEQ ID NO:330). These vertebrate STING proteins readily activate immune signaling in human cells, indicating that the molecular mechanism of STING signaling is shared in vertebrates (see, de Oliveira Mann et al. (2019) Cell Reports 27: 1165-1175).

[0162] Pharmaceutical compositions containing any of the immunostimulatory bacteria and other delivery vehicles also are provided. As are uses thereof for treatment of cancers, and methods of treatment of cancer. Methods and uses include treating a subject who has cancer, comprising administering an immunostimulatory bacterium or the pharmaceutical composition to a subject, such as a human. A method of treating a subject who has cancer, comprising administering an immunostimulatory bacterium, is provided. Methods and uses include combination therapy in which a second anti-cancer agent or treatment is administered. The second anti-cancer agent can be a

[0163] chemotherapeutic agent that results in cytosolic DNA, or radiotherapy, or an immune checkpoint inhibitor, such as an anti-PD-1, or anti-PD-Ll or anti-CTLA-4 antibody, or CAR-T cells or other therapeutic cells, such as stem cells, TIL cells and modified cells for cancer therapy.

[0164] 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 into the lung, or intratumoral, intravenously,

[0165] intramuscularly, or subcutaneously. Administration can be by any suitable route, including systemic or local or topical, such as parenteral, including, for example, oral or rectal or by aerosol into the lung, intratumoral, intravenously, intramuscularly, or subcutaneously.

[0166] Cancers include solid tumors and hematologic malignancies, such as, but not limited to, lymphoma, leukemia, gastric cancer, and cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix, and liver.

[0167] The immunostimulatory bacteria can be formulated into compositions for administration, such as suspensions. They can be dried and stored as powders.

[0168] Combinations of the immunostimulatory bacteria with other anti-cancer agents also are provided.

[0169] Combination therapies for treatment of cancers and malignancies are provided. The immunostimulatory bacteria can be administered before, after, intermittently with, or concurrently with, other cancer therapies, including radiotherapy,

[0170] chemotherapies, particularly genotoxic chemotherapies that result in cytosolic DNA, and immunotherapies, such as checkpoint inhibitor antibodies, including anti-PD-1 antibodies, anti-PD-Ll antibodies, and anti-CTLA-4 antibodies, and other such immunotherapies.

[0171] Also provided are isolated cells that contain the immunostimulatory bacteria or that contain any of the other delivery vehicles, such as exosomes, liposomes and other such vehicles, that contain nucleic acids encoding the gain-of-function variant proteins and other therapeutic products as described herein. Cells include, but are not limited to, immune cells, stem cells, tumor cells, primary cell lines, and other cells used in cell therapy. Exemplary cells include, for example, hematopoietic cells, such as T-cells, and hematopoietic stem cells. The hematopoietic cell can be a chimeric antigen myeloid cell, such as a macrophage. The delivery vehicles and

[0172] immunostimulatory bacteria can be introduced into the cells ex vivo. Thus, for example, provided are isolated cells that contain immunostimulatory bacteria, where: the immunostimulatory bacterium is modified so that it preferentially infects tumor- resident immune cells, and / or the genome of the immunostimulatory bacterium is modified so that it induces less cell death in tumor-resident immune cells; and the cell is an immune cell, a stem cell, a cell from a primary cell line, or a tumor cell. The cells are used in methods of cell therapy, such as for the treatment of cancers. The cells can be allogeneic or autologous to the subject treated.

[0173] Also provided are methods for increasing tumor / tumor microenvironment colonization by an immunostimulatory bacterium. The methods include, for example, modifying the genome of a bacterium to render the bacterium flagellin (fliC / fljB ) and / or pagP .

[0174] The terms and expressions that are employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and

[0175] expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are contemplated. BRIEF DESCRIPTION OF THE DRAWINGS

[0176] Figure 1 depicts the alignment of wild-type human and Tasmanian devil STING proteins.

[0177] Figure 2 depicts the alignment of wild-type human and marmoset STING proteins.

[0178] Figure 3 depicts the alignment of wild-type human and cattle STING proteins.

[0179] Figure 4 depicts the alignment of wild-type human and cat STING proteins.

[0180] Figure 5 depicts the alignment of wild-type human and ostrich STING proteins.

[0181] Figure 6 depicts the alignment of wild-type human and crested ibis STING proteins. Figure 7 depicts the alignment of wild-type human and coelacanth (SEQ ID NO:345) STING proteins.

[0182] Figure 8 depicts the alignment of wild-type human and zebrafish STING proteins.

[0183] Figure 9 depicts the alignment of wild-type human and boar STING proteins. Figure 10 depicts the alignment of wild-type human and bat STING proteins. Figure 11 depicts the alignment of wild-type human and manatee STING proteins.

[0184] Figure 12 depicts the alignment of wild-type human and ghost shark STING proteins.

[0185] Figure 13 depicts the alignment of wild-type human and mouse STING proteins.

[0186] DETAILED DESCRIPTION OUTLINE

[0187] A. DEFINITIONS

[0188] B. OVERVIEW OF THE IMMUNOSTIMULATORY BACTERIA

[0189] C. CANCER IMMUNOTHERAPEUTICS

[0190] 1. Immunotherapies

[0191] 2. Adoptive Immunotherapies

[0192] 3. Cancer Vaccines and Oncolytic Viruses

[0193] D. BACTERIAL CANCER IMMUNOTHERAPY

[0194] 1. Bacterial Therapies

[0195] 2. Comparison of the Immune Responses to Bacteria and Viruses

[0196] 3. Salmonella Therapy

[0197] a. Tumor-tropic Bacteria

[0198] b. Salmonella enterica serovar typhimurium

[0199] c. Bacterial Attenuation

[0200] i. msbBr Mutants

[0201] ii. purl Mutants

[0202] iii. Combinations of Attenuating Mutations iv. VNP20009 and Other Attenuated S.

[0203] typhimurium strains

[0204] v. S. typhimurium Engineered To Deliver Macromolecules

[0205] 4. Enhancements of Immunostimulatory Bacteria to Increase Therapeutic Index and Expression in Tumor-Resident Immune Cells

[0206] a. asd Gene Deletion

[0207] b. Adenosine Auxotrophy

[0208] c. Flagellin Deficient Strains

[0209] d. Deletion of Genes in the LPS Biosynthetic Pathway e. Deletions in Genes Required for Biofilm Formation f. Salmonella Engineered to Escape the Salmonella Containing Vacuole (SCV)

[0210] g. Deletions of SPI-1 and SPI-2 Genes and / or Other Genes to Eliminate the Ability of the Bacteria to Infect Epithelial Cells, Including Deletion of Flagella i. Salmonella Pathogenicity Island 1 (SPI-1)

[0211] SPI-l-Dependent Host Cell Invasion SPI-l-Independent Host Cell Invasion ii. Salmonella Pathogenicity Island 2 (SPI-2) h. Endonuclease (endA) Mutations to Increase Plasmid Delivery

[0212] i. RIG-I Binding Sequences

[0213] j. DNase II Inhibition

[0214] k. RNase H2 Inhibition

[0215] l. Stabilin-l / CLEVER-1 Inhibition

[0216] m. CpG Motifs and CpG Islands

[0217] 5. Modifications that Increase Uptake of Gram-Negative Bacteria, such as Salmonella , by Immune Cells, and Reduce Immune Cell Death

[0218] a. Bacterial Uptake by Immune cells b. Macrophage Pyroptosis

[0219] i. Flagellin

[0220] ii. SPI-1 Proteins

[0221] Rod Protein (PrgJ)

[0222] Needle protein (Prgl)

[0223] iii. QseC

[0224] 6. Bacterial Culture Conditions

[0225] 7. Increased Tumor Colonization

[0226] E. NON-HUMAN STING PROTEINS AND GAIN-OF-FUNCTION MUTATIONS IN PROTEINS THAT STIMULATE THE IMMUNE RESPONSE IN THE TUMOR MICROENVIRONMENT

[0227] 1. Type I Interferons and Pathways

[0228] 2. Type I Interferonopathies and Gain-of-Function Mutants

[0229] 3. STING-Mediated Immune Activation

[0230] 4. TMEM173 Alleles

[0231] 5. Constitutive STING Expression and Gain-of-Function Mutations

[0232] 6 Non-human STING Proteins, and Variants Thereof with Increased or Constitutive Activity, and STING Chimeras, and Variants Thereof with Increased or Constitutive Activity

[0233] 7. Other Gene Products that Act as Cytosolic DNA / RNA Sensors and Constitutive Variants

[0234] a. Retinoic Acid-Inducible Gene I (RIG-I)-Like

[0235] Receptors (RLRs)

[0236] b. MDA5 / IFIH1

[0237] c. RIG-I

[0238] d. IRF-3 and IRF-7

[0239] 8. Other Type I IFN Regulatory Proteins

[0240] 9. Other Therapeutic Products F. IMMUNOSTIMULATORY BACTERIA ENCODING THE PROTEINS AND CONSTRUCTION OF EXEMPLARY PLASMIDS AND DELIVERY VEHICLES

[0241] 1. Origin of Replication and Plasmid Copy Number

[0242] 2. Plasmid Maintenance / Selection Components

[0243] 3. RNA Polymerase Promoters

[0244] 4. DNA Nuclear Targeting Sequences

[0245] 5. CRISPR

[0246] G. OTHER DELIVERY VEHICLES ENCODING THE NON

[0247] HUMAN STING PROTEINS AND GAIN-OF-FUNCTION MODIFIED PROTEINS THAT CONSTITUTIVELY INDUCE TYPE I INTERFERON AND OTHER THERAPEUTIC PRODUCTS

[0248] 1. Exosomes, Extracellular Vesicles, And Other Vesicular Delivery Vehicles

[0249] 2. Oncolytic Viruses

[0250] a. Adenovirus

[0251] b. Herpes Simplex Virus

[0252] c. Poxvirus

[0253] d. Measles Virus

[0254] e. Reovirus

[0255] f. Vesicular Stomatitis Virus (VSV)

[0256] g. Newcastle Disease Virus

[0257] h. Parvovirus

[0258] i. Coxsackie Virus

[0259] j. Seneca Valley Virus

[0260] H. PHARMACEUTICAL PRODUCTION, COMPOSITIONS, AND FORMULATIONS

[0261] 1. Manufacturing

[0262] a. Cell Bank Manufacturing

[0263] b. Drug Substance Manufacturing

[0264] c. Drug Product Manufacturing 2. Compositions

[0265] 3. Formulations

[0266] a. Liquids, Injectables, Emulsions b. Dried Thermostable Formulations

[0267] 4. Compositions for Other Routes of Administration

[0268] 5. Dosages and Administration

[0269] 6. Packaging and Articles of Manufacture

[0270] I. METHODS OF TREATMENT AND USES

[0271] 1. Tumors

[0272] 2. Administration

[0273] 3. Monitoring

[0274] J. EXAMPLES

[0275] A. DEFINITIONS

[0276] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0277] As used herein, therapeutic bacteria are bacteria that effect therapy, such as cancer or anti -tumor therapy, when administered to a subject, such as a human.

[0278] As used herein, immunostimulatory bacteria are therapeutic bacteria that, when introduced into a subject, accumulate in immunoprivileged tissues and cells, such as tumors, and replicate and / or express products that are immunostimulatory or that result in immunostimulation. For example, the immunostimulatory bacteria are attenuated in the host by virtue of reduced toxicity or pathogenicity and / or by virtue of encoded products that reduce toxicity or pathogenicity, as the immunostimulatory bacteria cannot replicate and / or express products (or have reduced replication / product expression), except primarily in immunoprivileged environments. Immunostimulatory bacteria provided herein are modified to encode a product or products or exhibit a trait or property that renders them immunostimulatory. Such products, properties and traits include, but are not limited to, for example, at least one of: an immunostimulatory protein, such as a cytokine or co-stimulatory molecule; a DNA / RNA sensor or gain- of-function variant thereof ( e.g ., STING, MDA5, RIG-I); RNAi, such as siRNA (shRNA and microRNA), CRISPR, that targets, disrupts or inhibits a checkpoint gene such as TREX1 and / or PD-L1; or an inhibitor of an immune checkpoint such as an anti-immune checkpoint antibody. Immunostimulatory bacteria also can include a modification that renders the bacterium auxotrophic for a metabolite that is immunosuppressive or that is in an immunosuppressive pathway, such as adenosine.

[0279] As used herein, the strain designations VNP20009 (see, e.g., International PCT Application Publication No. WO 99 / 13053, see, also U.S. Patent 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 No. 202165. VNP20009 is a modified attenuated strain of Salmonella typhimurium, which contains deletions in msbB and purl, and was generated from wild type strain ATCC 14028.

[0280] As used herein, the strain designations YS1456 and 8.7 are used

[0281] interchangeably and each refer to the strain deposited with the American Type Culture Collection and assigned Accession No. 202164 (see, U.S. Patent No. 6,863,894).

[0282] As used herein, an interferonopathy refers to a disorder associated with an upregulation of interferon by virtue of a mutation in a gene product involved in a pathway that regulates or induces expression of interferon. The activity of the products normally is regulated by a mediator, such as cytosolic DNA or RNA or nucleotides; when mutated, the activity is constitutive. Type I interferonopathies include a spectrum of conditions, including the severe forms of Aicardi-Goutieres Syndrome (AGS) and the milder Familial Chilblain Lupus (FCL). Nucleic acid molecules encoding mutated products with these properties can be produced in vitro, such as by selecting for mutations that result in a gain-of-function in the product, compared to the product of an allele that has normal activity, or has further gain-of- function compared to the disease-associated gain-of-function mutants described herein.

[0283] As used herein, a gain-of-function mutation is one that increases the activity of a protein compared to the same protein that does not have the mutation. For example, if the protein is a receptor, it will have increased affinity for a ligand; if it is an enzyme, it will have increased activity, including constitutive activity.

[0284] As used herein, an origin of replication is a sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. For small DNA, including bacterial plasmids and small viruses, a single origin is sufficient.

[0285] The origin of replication determines the vector copy number, which depends upon the selected origin of replication. For example, if the expression vector is derived from the low-copy-number plasmid pBR322, it is between about 25-50 copies / cell, and if derived from the high-copy -number plasmid pUC, it can be 150- 200 copies / cell.

[0286] As used herein, medium copy number of a plasmid in cells is about or is 150 or less than 150, low copy number is 15-30, such as 20 or less than 20. Low to medium copy number is less than 150. High copy number is greater than 150 copies / cell.

[0287] As used herein, 2A peptides are 18-22 amino-acid (aa)-long viral

[0288] oligopeptides that mediate cleavage of polypeptides during translation in eukaryotic cells. The designation“2A” refers to a specific region of the viral genome and different viral 2As have generally been named after the virus they were derived from. Exemplary of these are F2A (foot-and-mouth disease virus 2A), E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (Thosea asigna virus 2A). (See, e.g., Liu et al. (2017) Scientific Reports 7:2193, Fig. 1, for encoding sequences; see, also, SEQ ID NOs:367-370).

[0289] As used herein, a CpG motif is a pattern of bases that include an unmethylated central CpG ("p" refers to the phosphodiester link between consecutive C and G nucleotides) surrounded by at least one base flanking (on the 3' and the 5' side of) the central CpG. A CpG oligodeoxynucleotide is an oligodeoxynucleotide that is at least about ten nucleotides in length and includes an unmethylated CpG. At least the C of the 5' CG 3' is unmethylated. As used herein, a RIG-I binding sequence refers to a 5’ triphosphate (5’ppp) structure directly, or that which is synthesized by RNA pol III from a poly(dA-dT) sequence, which by virtue of interaction with RIG-I can activate type I IFN via the RIG-I pathway. The RNA includes at least four A ribonucleotides (A-A-A-A); it can contain 4, 5, 6, 7, 8, 9, 10 or more. The RIG-I binding sequence is introduced into a plasmid in the bacterium for transcription into the polyA.

[0290] As used herein,“cytokines” are a broad and loose category of small proteins (-5-20 kDa) that are important in cell signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are cell signaling molecules that aid cell to cell communication in immune responses, and stimulate the movement of cells towards sites of inflammation, infection and trauma.

[0291] As used herein,“chemokines” refer to chemoattractant (chemotactic) cytokines that bind to chemokine receptors and include proteins isolated from natural sources as well as those made synthetically, as by recombinant means or by chemical synthesis. Exemplary chemokines include, but are not limited to, IL-8, IL-10, GCP-2, GRO-a, GRO-b, GRO-g, ENA-78, PBP, CTAP III, NAP-2, LAPF-4, MIG (CXCL9), CXCL10, CXCL11, PF4, IP-10, SDF-la, SDF-Ib, SDF-2, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-la (CCL3), MPMb (CCL4), MPMg, MIP-2, MIP-2a, MIP-3a, MIR-3b, MPM, MIP-5, MDC, HCC-1, ALP, lungkine, Tim-1, eotaxin-1, eotaxin-2, 1- 309, SCYA17, TRAC, RANTES (CCL5), DC-CK-1, lymphotactin, and fractalkine, and others known to those of skill in the art. Chemokines are involved in the migration of immune cells to sites of inflammation, as well as in the maturation of immune cells and in the generation of adaptive immune responses.

[0292] As used herein, an“immunostimulatory protein” is a protein that exhibits or promotes an anti-tumor immune response in the tumor microenvironment. Exemplary of such proteins are cytokines, chemokines, and co-stimulatory molecules, such as, but not limited to, GM-CSF, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-36 gamma, IFNa, PTNίb, IL-12p70 (IL-12p40 + IL-12p35), IL-15 / IL-15R alpha chain complex, CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, molecules involved in the potential recruitment / persistence of T cells, CD40, CD40 ligand (CD40L), 0X40, 0X40 ligand (OX40L), 4- IBB, 4- IBB ligand (4-1BBL), members of the B7-CD28 family and members of the TNFR superfamily. As used herein, a cytosolic DNA / RNA sensor pathway is one that is initiated by the presence of DNA, RNA, nucleotides, dinucleotides, cyclic nucleotides and / or cyclic dinucleotides or other nucleic acid molecules, that leads to production of type I interferon. The nucleic acid molecules in the cytosol occur from viral or bacterial or radiation or other such exposure, leading to activation of an immune response in a host.

[0293] As used herein, an immunostimulatory protein that induces an innate immune response, such as induction of type I interferon, is a protein that is part of a cytosolic DNA / RNA sensor pathway that leads to expression of the immune response mediator, such as type I interferon. For example, as described herein and known to those of skill in the art, cytosolic DNA is sensed by cGAS, leading to the production of cGAMP and subsequent STING (Stimulator of Interferon Genes) / TBK1 (TANK-binding kinase 1) / IRF3 (interferon regulatory factor) signaling, and type I IFN production. Bacterial cyclic dinucleotides (CDNs, such as bacterial cyclic di-AMP) also activate STING. Hence, STING is an immunostimulatory protein that induces type I interferon. 5’- triphosphate RNA and double stranded RNA are sensed by RIG-I and either MDA-5 alone or MDA-5 / LGP2. This leads to polymerization of mitochondrial MAVS (mitochondrial anti-signaling protein), and also activates TBK1 and IRF3. The proteins in such pathways are immunostimulatory proteins that lead to expression of innate immune response mediators, such as type I interferon. The immunostimulatory proteins in the DNA / RNA sensor pathways can be modified so that they have increased activity or act constitutively, in the absence of cytosolic nucleic acid, to lead to the immune response, such as expression of type I interferon.

[0294] As used herein, the’’carboxy-terminal tail” or“C-terminal tail” (CTT) of the innate immune protein STING refers to the C-terminal portion of a STING protein that, in a wild-type STING protein, is tethered to the cGAMP -binding domain by a flexible linker region. The CTT includes an IRF3 binding site, a TBK1 binding site, and a TRAF6 binding site. STING promotes the induction of interferon beta (IFN-b) production via the phosphorylation of the STING protein C-terminal tail (CTT) by TANK-binding kinase 1 (TBK1). The interaction between STING and TBK1 is mediated by an evolutionarily conserved stretch of eight amino-acid residues in the carboxy-terminal tail (CTT) of STING. TRAF6 catalyzes the formation of K63 -linked ubiquitin chains on STING, leading to the activation of the transcription factor NF-KB and the induction of an alternative STING-dependent gene expression program.

[0295] Deletion of the TRAF6 binding site in the CTT can reduce activation of NF-KB signaling. Substitution of the human CTT (or portions thereof) with the CTT (or corresponding portion thereof) from STING of species with low NF-KB activation can decrease NF-KB activation by human STING. The STING CTT is an unstructured stretch of ~40 amino acids that contains sequence motifs required for STING phosphorylation and recruitment of IRF3 (see, de Oliveira Mann et al. (2019) Cell Reports 27 1165-1175). Human STING residue S366 has been identified as a primary TBK1 phosphorylation site that is part of an LxIS motif shared among innate immune adaptor proteins that activate interferon signaling (see, de Oliveira Mann et al. (2019) Cell Reports 27 1165-1175). The human STING CTT contains a second PxPLR motif that includes the residue L374, which is required for TBK1 binding; the LxIS and PxPLR sequences are conserved among vertebrate STING alleles (see, de Oliveira Mann et al. (2019) Cell Reports 27: 1165-1175). Exemplary STING CTT sequences, and the IRF3, TBK1 and TRAF6 binding sites are set forth in the following table:

[0296]

[0297] As used herein, a bacterium that is modified so that it“induces less cell death in tumor-resident immune cells” is one that is less toxic than the bacterium without the modification, or one that has reduced virulence compared to the bacterium without the modification. Exemplary of such modifications are those that eliminate pyroptosis and that alter LPS profiles on the bacterium. These modifications include disruption of or deletion of flagellin genes, one or more components of the SPI-1 pathway, such as hi I A , rod protein, needle protein, QseC and pagP.

[0298] As used herein, a bacterium that is“modified so that it preferentially infects tumor-resident immune cells” has a modification in its genome that reduces its ability to infect cells other than immune cells. Exemplary of such modifications are modifications that disrupt the type 3 secretion system or type 4 secretion system or other genes or systems that affect the ability of a bacterium to invade a non-immune cell. For example, disruption / deletion of an SPI-1 component, which is needed for infection of cells, such as epithelial cells, but does not affect infection of immune cells, such as phagocytic cells, by Salmonella.

[0299] As used herein, a“modification” is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids or nucleotides, respectively. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.

[0300] As used herein, a modification to a bacterial genome or to a plasmid or gene includes deletions, replacements and insertions of nucleic acid.

[0301] As used herein, RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules to inhibit translation and thereby expression of a targeted gene. As used herein, RNA molecules that act via RNAi are referred to as inhibitory by virtue of their silencing of expression of a targeted gene. Silencing expression means that expression of the targeted gene is reduced or suppressed or inhibited.

[0302] As used herein, gene silencing via RNAi is said to inhibit, suppress, disrupt or silence expression of a targeted gene. A targeted gene contains sequences of nucleotides that correspond to the sequences in the inhibitory RNA, whereby the inhibitory RNA silences expression of mRNA. Small interfering RNAs (siRNAs) are small pieces of double-stranded (ds) RNA, usually about 21 nucleotides long, with 3' overhangs (2 nucleotides) at each end that can be used to "interfere" with the translation of proteins by binding to and promoting the degradation of messenger RNA (mRNA) at specific sequences. In doing so, siRNAs prevent the production of specific proteins based on the nucleotide sequences of their corresponding mRNAs. The process is called RNA interference (RNAi), and also is referred to as siRNA silencing or siRNA knockdown. A short-hairpin RNA or small-hairpin RNA

[0303] (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors.

[0304] As used herein, inhibiting, suppressing, disrupting or silencing a targeted gene refers to processes that alter expression, such as translation, of the targeted gene, whereby activity or expression of the product encoded by the targeted gene is reduced. Reduction, includes a complete knock-out or a partial knockout, whereby, with reference to the immunostimulatory bacteria provided herein and administration herein, treatment is effected.

[0305] As used herein, a tumor microenvironment (TME) is the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM). Conditions that exist include, but are not limited to, increased vascularization, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure and altered metabolites or metabolism, such as higher levels of adenosine, indicative of a tumor. As used herein, human type I interferons (IFNs) are a subgroup of interferon proteins that regulate the activity of the immune system. All type I IFNs bind to a specific cell surface receptor complex, such as the IFN-a receptor. Type I interferons include IFN-a and IFN-b, among others. IFN-b proteins are produced by fibroblasts, and have antiviral activity that is involved mainly in innate immune response. Two types of IFN-b are IFN-bI (IFNBl) and IRN-b3 (IFNB3).

[0306] As used herein, recitation that a nucleic acid or encoded RNA targets a gene means that it inhibits or suppresses or silences expression of the gene by any mechanism. Generally, such nucleic acid includes at least a portion complementary to the targeted gene, where the portion is sufficient to form a hybrid with the complementary portion.

[0307] As used herein,“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 a native or wild-type sequence.

[0308] 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 compared to a wild-type sequence, contains one or more additional nucleotides within the linear length of the sequence.

[0309] As used herein,“additions” to nucleic acid and amino acid sequences describe addition of nucleotides or amino acids onto either termini compared to another sequence.

[0310] As used herein,“substitution” or“replacement” refers to the replacing of one or more nucleotides or amino acids in a native, target, wild-type or other nucleic acid or polypeptide sequence with an alternative nucleotide or amino acid, without changing the length (as described in numbers of residues) of the molecule. Thus, one or more substitutions in a molecule does not change the number of amino acid residues or nucleotides of the molecule. Amino acid replacements compared to a particular polypeptide can be expressed in terms of the number of the amino acid residue along the length of the polypeptide sequence. As used herein,“at a position corresponding to,” or recitation that nucleotides or amino acid positions“correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence Listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:

[0311] 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., M Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAM J Applied Math 48: 1073).

[0312] As used herein, alignment of a sequence refers to the use of homology to align two or more sequences of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence. Related or variant polypeptides or nucleic acid molecules can be aligned by any method known to those of skill in the art. Such methods typically maximize matches, and include methods, such as using manual alignments and by using the numerous alignment programs available (e.g, BLASTP) and others known to those of skill in the art. By aligning the sequences of polypeptides or nucleic acids, one skilled in the art can identify analogous portions or positions, using conserved and identical amino acid residues as guides. Further, one skilled in the art also can employ conserved amino acid or nucleotide residues as guides to find corresponding amino acid or nucleotide residues between and among human and non-human sequences. Corresponding positions also can be based on structural alignments, for example by using computer simulated alignments of protein structure. In other instances, corresponding regions can be identified. One skilled in the art also can employ conserved amino acid residues as guides to find corresponding amino acid residues between and among human and non-human sequences.

[0313] As used herein, a“property” of a polypeptide, such as an antibody, refers to any property exhibited by a polypeptide, including, but not limited to, binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, thermal tolerance, and tolerance to pH

[0314] conditions. Changes in properties can alter an“activity” of the polypeptide. For example, a change in the binding specificity of the antibody polypeptide can alter the ability to bind an antigen, and / or various binding activities, such as affinity or avidity, or in vivo activities of the polypeptide.

[0315] As used herein, an“activity” or a“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, ability to interact with a biomolecule, for example, through antigen-binding, DNA binding, ligand binding, or dimerization, or enzymatic activity, for example, kinase activity or proteolytic activity. For an antibody (including antibody fragments), activities include, but are not limited to, the ability to specifically bind a particular antigen, affinity of antigen-binding ( e.g ., high or low affinity), avidity of antigen-binding (e.g, high or low avidity), on-rate, off-rate, effector functions, such as the ability to promote antigen neutralization or clearance, virus neutralization, and in vivo activities, such as the ability to prevent infection or invasion of a pathogen, or to promote clearance, or to penetrate a particular tissue or fluid or cell in 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 to measure on- or off-rate,

[0316] immunohistochemistry and immunofluorescence histology and microscopy, cell- based assays, flow cytometry and binding assays (e.g, panning assays).

[0317] As used herein,“bind,”“bound” or grammatical variations thereof refers to the participation of a molecule in any attractive interaction with another molecule, resulting in a stable association in which the two molecules are in close proximity to one another. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as chemical compounds including drugs.

[0318] As used herein,“antibody” refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantly produced, including any fragment thereof containing at least a portion of the variable heavy chain and light region of the immunoglobulin molecule that is sufficient to form an antigen binding site and, when assembled, to specifically bind an antigen. Hence, 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 that contains two heavy chains (which can be denoted H and H’) and two light chains (which can be denoted L and L’), where each heavy chain can be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen binding site ( e.g ., heavy chains include, but are not limited to, VH chains, VH-CH1 chains and VH-CH1-CH2- CH3 chains), and each light chain can be a full-length light chain or a portion thereof sufficient to form an antigen binding site (e.g., light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H’) pairs with one light chain (L and L’, respectively). Typically, antibodies minimally include all or at least a portion of the variable heavy (VH) chain and / or the variable light (VL) chain. The antibody also can include all or a portion of the constant region.

[0319] 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')2fragments, 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. Antibody also includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g, bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies. Antibodies provided herein include members of any immunoglobulin class (e.g, IgG, IgM, IgD, IgE, IgA and IgY), any subclass (e.g, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or sub-subclass (e.g, IgG2a and IgG2b). As used herein,“nucleic acid” refers to at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA), joined together, typically by phosphodiester linkages. Also included in the term“nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or

[0320] combinations thereof. Nucleic acids also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid). The term also includes, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double- stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deox ythymi dine. For RNA, the uracil base is uridine.

[0321] As used herein, an isolated nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding an antibody or antigen-binding fragments provided.

[0322] As used herein,“operably linked” with reference to nucleic acid sequences, regions, elements or domains 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, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide effects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide ( e.g ., a leader peptide) is operably linked to a nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. 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 either a fusion protein containing 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 nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0323] As used herein,“synthetic,” with reference to, for example, 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.

[0324] As used herein, the residues of naturally occurring a-amino acids are the residues of those 20 a-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.

[0325] As used herein,“polypeptide” refers to two or more amino acids covalently joined. The terms“polypeptide” and“protein” are used interchangeably herein.

[0326] As used herein, a“peptide” refers to a polypeptide that is from 2 to about or 40 amino acids in length.

[0327] As used herein, an“amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids contained in the antibodies provided include the twenty naturally-occurring amino acids (see Table below), non-natural amino acids, and amino acid analogs ( e.g ., amino acids wherein the a-carbon has a side chain). As used herein, the amino acids, which occur in the various amino acid sequences of polypeptides appearing herein, are identified according to their well-known, three- letter or one-letter abbreviations (see Table below). The nucleotides, which occur in the various nucleic acid molecules and fragments, are designated with the standard single-letter designations used routinely in the art.

[0328] As used herein,“amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The 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 property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J Biol. Chem ., 243:3557-59 (1968) and adopted at 37 C.F.R. §§ 1.821 - 1.822, abbreviations for amino acid residues are shown in the following Table:

[0329] Table of Correspondence

[0330]

[0331] All sequences of amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl- terminus. The phrase“amino acid residue” is defined to include the amino acids listed in the above Table of Correspondence, 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 NEE or to a carboxyl-terminal group such as COOH.

[0332] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in the art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide 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).

[0333] Such substitutions, such as in the gain-of-function mutations described and provided herein, can be made in accordance with the exemplary substitutions set forth in the following Table:

[0334] Exemplary conservative amino acid substitutions

[0335]

[0336] Other substitutions also are permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.

[0337] As used herein,“naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides.

[0338] As used herein, the term "non-natural amino acid" refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non- naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids and include, but are not limited to, the D-stereoi somers of amino acids. Exemplary non-natural amino acids are known to those of skill in the art, and include, but are not limited to, 2-

[0339] Aminoadipic acid (Aad), 3-Aminoadipic acid (bAad), P-alanine / p- Amino-propionic acid (Bala), 2-Aminobutyric acid (Abu), 4-Aminobutyric acid / piperidinic 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), allo-Hydroxylysine (Ahyl), 3-Hydroxyproline (3 Hyp), 4-Hydroxyproline (4Hyp), Isodesmosine (Ide), allo-Isoleucine (Aile), N- Methylglycine, sarcosine (MeGly), N-Methylisoleucine (Melle), 6-N-Methyllysine (MeLys), N-Methylvaline (MeVal), Norvaline (Nva), Norleucine (Me), and Ornithine (Orn).

[0340] As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.

[0341] As used herein, a DNA segment is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5’ to 3’ direction, encodes the sequence of amino acids of the specified polypeptide.

[0342] As used herein, the term polynucleotide means a single- or double-stranded polymer of deoxyribonucleotides 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 terms of nucleotides (abbreviated“nt”) or base pairs (abbreviated“bp”). The term nucleotides is used for single- and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide can differ slightly in length and that the ends thereof can be staggered; thus all nucleotides within a double-stranded polynucleotide molecule cannot be paired. Such unpaired ends will, in general, not exceed 20 nucleotides in length.

[0343] As used herein, production by recombinant methods refers means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.

[0344] As used herein,“heterologous nucleic acid” is nucleic acid that encodes products (i.e., RNA and / or proteins) that are not normally produced in vivo by the cell in which it is expressed, or nucleic acid that is in a locus in which it does not normally occur, or that mediates or encodes mediators that alter expression of endogenous nucleic acid, such as DNA, by affecting transcription, translation, or other regulatable biochemical processes. Heterologous nucleic acid, such as DNA, also is referred to as foreign nucleic acid. Any nucleic acid, such as DNA, that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed, is herein encompassed by heterologous nucleic acid; heterologous nucleic acid includes exogenously added nucleic acid that is also expressed endogenously. Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically or is introduced into a genomic locus in which it does not occur naturally, or its expression is under the control of regulatory sequences or a sequence that differs from the natural regulatory sequence or sequences.

[0345] Examples of heterologous nucleic acid herein include, but are not limited to, nucleic acid that encodes a protein in a DNA / RNA sensor pathway or a gain-of- function variant thereof, or an immunostimulatory protein, such as a cytokine, that confers or contributes to anti-tumor immunity in the tumor microenvironment. In the immunostimulatory bacteria, the heterologous nucleic acid generally is encoded on the introduced plasmid, but it can be introduced into the genome of the bacterium, such as a promoter that alters expression of a bacterial product. Heterologous nucleic acid, such as DNA, includes nucleic acid that can, in some manner, mediate expression of DNA that encodes a therapeutic product, or it can encode a product, such as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product.

[0346] As used herein, cell therapy involves the delivery of cells to a subject to treat a disease or condition. The cells, which can be allogeneic or autologous, are modified ex vivo , such as by infection of cells with immunostimulatory bacteria provided herein, so that they deliver or express products when introduced to a subject.

[0347] As used herein, genetic therapy involves the transfer of heterologous nucleic acid, such as DNA, into certain cells, such as target cells, of a mammal, particularly a human, with a disorder or condition for which such therapy is sought. The nucleic acid, such as DNA, is introduced into the selected target cells in a manner such that the heterologous nucleic acid, such as DNA, is expressed and a therapeutic product(s) encoded thereby is produced. Genetic therapy can also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced. The introduced nucleic acid can encode a therapeutic compound, such as a growth factor or inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor thereof, that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time. The heterologous nucleic acid, such as DNA, encoding the therapeutic product, can be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. Genetic therapy can also involve delivery of an inhibitor or repressor or other modulator of gene expression.

[0348] As used herein,“expression” refers to the process by which polypeptides are produced by transcription and translation of polynucleotides. The level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.

[0349] As used herein, a“host cell” is a cell that is used to receive, maintain, reproduce and / or amplify a vector. A host cell also can be used to express the polypeptide encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids.

[0350] 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 includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation. Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide, such as a modified anti-EGFR antibody. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well-known to those of skill in the art. A vector also includes“virus vectors” or“viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0351] As used herein, an“expression vector” includes vectors capable of expressing DNAthat is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. 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, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well- known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. 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.

[0352] As used herein,“sequence identity” refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference poly- peptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. The alignment can be local or global. Matches, mismatches and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. When using gap penalties, sequence identity can be determined with no penalty for end gaps ( e.g ., terminal gaps are not penalized). Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions / length of the total aligned sequence x 100.

[0353] As used herein, a“global alignment” is an alignment that aligns two sequences from beginning to end, aligning each letter in each sequence only once. An alignment is produced, regardless of whether or not there is similarity or identity between the sequences. For example, 50% sequence identity based on“global alignment” means that in an alignment of the full sequence of two compared sequences each of 100 nucleotides in length, 50% of the residues are the same. It is understood that global alignment also can be used in determining sequence identity even when the length of the aligned sequences is not the same. The differences in the terminal ends of the sequences will be taken into account in determining sequence identity, unless the“no penalty for end gaps” is selected. Generally, a global alignment is used on sequences that share significant similarity over most of their length. Exemplary algorithms for performing global alignment include the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48: 443). Exemplary programs for performing global alignment 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

[0354] deepc2. p si . i astate . edu / aat / align / ali gn . html . As used herein, a“local alignment” is an alignment that aligns two sequences, but only aligns those portions of the sequences that share similarity or identity.

[0355] Hence, a local alignment determines if sub-segments of one sequence are present in another sequence. If there is no similarity, no alignment will be returned. Local alignment algorithms include BLAST or Smith-Waterman algorithm (A civ. Appl.

[0356] Math. 2: 482 (1981)). For example, 50% sequence identity based on“local alignment” means that in an alignment of the full sequence of two compared sequences of any length, a region of similarity or identity of 100 nucleotides in length has 50% of the residues that are the same in the region of similarity or identity.

[0357] For purposes herein, sequence identity can be determined by standard alignment algorithm programs used with default gap penalties established by each supplier. Default parameters for the GAP program can include: (1) a unary

[0358] comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, as described by 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 reciting a percent identity, can be determined using known computer algorithms based on local or global alignment (see e.g,

[0359] wikipedia.org / wiki / Sequence_alignment_software, providing links to dozens of known and publicly available alignment databases and programs). Generally, for purposes herein sequence identity is determined using computer algorithms based on global alignment, such as the Needleman-Wunsch Global Sequence Alignment tool available from NCBI / BLAST

[0360] (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (William Pearson implementing the Huang and Miller algorithm (Adv. Appl. Math. (1991) 12:337-357)); and program from Xiaoqui Huang available at

[0361] deepc2.psi.iastate.edu / aat / align / align.html. Typically, the full-length sequence of each of the compared polypeptides or nucleotides is aligned across the full-length of each sequence in a global alignment. Local alignment also can be used when the sequences being compared are substantially the same length.

[0362] Therefore, as used herein, the term "identity" represents 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 percent identities from 90 to 100% relative to the reference polypeptide or polynucleotide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide or polynucleotide length of 100 amino acids or nucleotides are compared, no more than 10% (z.e., 10 out of 100) of amino acids or nucleotides in the test polypeptide or polynucleotide differ from those of the reference polypeptide. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g ., 10 / 100 amino acid difference (approximately 90% identity). Differences also can be due to deletions or truncations of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. Depending on the length of the compared sequences, at the level of homologies or identities above about 85-90%, the result can be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.

[0363] As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms.

[0364] As used herein, "treating" a subject with a disease or condition means that the subject’s symptoms are partially or totally alleviated, or remain static following treatment.

[0365] As used herein, treatment refers to any effects that ameliorate symptoms of a disease or disorder. Treatment encompasses prophylaxis, therapy and / or cure.

[0366] Treatment also encompasses any pharmaceutical use of any immunostimulatory bacterium or composition provided herein.

[0367] As used herein, prophylaxis refers to prevention of a potential disease and / or a prevention of worsening of symptoms or progression of a disease. As used herein,“prevention” or prophylaxis, and grammatically equivalent forms thereof, refers to methods in which the risk or probability of developing a disease or condition is reduced.

[0368] As used herein, a“pharmaceutically effective agent” includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, and conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.

[0369] As used herein, a“therapeutic effect” means an effect resulting from treatment of a subject that alters, typically improves or ameliorates, the symptoms of a disease or condition or that cures a disease or condition.

[0370] As used herein, a“therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.

[0371] As used herein,“therapeutic efficacy” refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, or composition containing a compound has been administered.

[0372] As used herein, a“prophylactically effective amount” or a“prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that when administered to a subject, will have the intended prophylactic effect, e.g ., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection. The full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0373] As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.

[0374] As used herein, an“anti-cancer agent” refers to any agent that is destructive or toxic to malignant cells and tissues. For example, anti-cancer agents include agents that kill cancer cells or otherwise inhibit or impair the growth of tumors or cancer cells. Exemplary anti-cancer agents are chemotherapeutic agents.

[0375] As used herein“therapeutic activity” refers to the in vivo activity of a therapeutic polypeptide. Generally, the therapeutic activity is the activity that is associated with treatment of a disease or condition.

[0376] As used herein, the term "subject" refers to an animal, including a mammal, such as a human being.

[0377] As used herein, a patient refers to a human subject.

[0378] As used herein, animal includes any animal, such as, but not limited to, primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; pigs and other animals. Non-human animals exclude humans as the contemplated animal. The polypeptides provided herein are from any source, animal, plant, prokaryotic and fungal. Most polypeptides are of animal origin, including mammalian origin.

[0379] As used herein, a“composition” refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

[0380] As used herein, a“combination” refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, a mixture thereof, such as a single mixture of the two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.

[0381] As used herein, combination therapy refers to administration of two or more different therapeutics. The different therapeutic agents can be provided and administered separately, sequentially, intermittently, or can be provided in a single composition.

[0382] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof, for a purpose including, but not limited to, activation,

[0383] administration, diagnosis, and assessment of a biological activity or property.

[0384] As used herein, a“unit dose form” refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.

[0385] As used herein, a“single dosage formulation” refers to a formulation for direct administration.

[0386] As used herein, a multi-dose formulation refers to a formulation that contains multiple doses of a therapeutic agent and that can be directly administered to provide several single doses of the therapeutic agent. The doses can be administered over the course of minutes, hours, weeks, days or months. Multi-dose formulations can allow dose adjustment, dose-pooling and / or dose-splitting. Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.

[0387] As used herein, an“article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass any of the compositions provided herein contained in articles of packaging.

[0388] As used herein, a“fluid” refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.

[0389] 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 substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification does not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound. As used herein, a “cellular extract” or“lysate” refers to a preparation or fraction which is made from a lysed or disrupted cell.

[0390] As used herein, a“control” refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest.

[0391] A control also can be an internal control.

[0392] 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 polypeptides with one or a plurality of immunoglobulin domains.

[0393] As used herein, the term "or" is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0394] As used herein, ranges and amounts can be expressed as“about” a particular value or range. About also includes the exact amount. Hence“about 5 amino acids” means“about 5 amino acids” and also“5 amino acids.”

[0395] As used herein,“optional” or“optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0396] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical

[0397] Nomenclature (see, Biochem. (1972) 11 (9): 1726- 1732).

[0398] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0399] B. OVERVIEW OF THE IMMUNOSTIMULATORY BACTERIA

[0400] Provided are modified bacteria, called immunostimulatory bacteria herein that accumulate and / or replicate in tumors and encode inhibitory RNAs, such as designed shRNAs and designed micro RNAs, that target genes whose inhibition, suppression or silencing effects tumor therapy, upon expression of the RNAs in the treated subject. Strains of bacteria for modification are any suitable for therapeutic use. The modified immunostimulatory bacteria provided herein are for use and for methods for treating cancer. The bacteria are modified for such uses and methods.

[0401] The immunostimulatory bacteria provided herein are modified by deletion or modification of bacterial genes to attenuate their inflammatory responses, and are modified to enhance anti-tumor immune responses in hosts treated with the bacteria. For example, the plasmids encoding therapeutic, such as anti-tumor, products in the host are included in the bacteria, and the bacteria can be auxotrophic for adenosine. Attenuation of the inflammatory response to the bacteria can be effected by deletion of the msbB gene, which decreases TNF-alpha in the host, and / or knocking out flagellin genes. The bacteria are modified to stimulate host anti-tumor activity, for example, by adding plasmids encoding immunostimulatory proteins, STING proteins, variant STING proteins, and proteins that target host immune checkpoints, and by adding nucleic acid with CpGs.

[0402] Bacterial strains can be attenuated strains or strains that are attenuated by standard methods or that by virtue of the modifications provided herein are attenuated in that their ability to colonize is limited primarily to immunoprivileged tissues and organs, particularly immune and tumor cells, including solid tumors. For purposes herein, the bacteria are not necessarily attenuated per se, but rather, contain modification(s), such as genomic modifications, that limit or alter the cells that are infected by the bacteria. Bacteria include, but are not limited to, for example, strains of Salmonella , Shigella , Listeria , E. coli , and Bifidobacteriae . For example, species include Shigella sonnei , Shigella flexneri , Shigella disenteriae , Listeria

[0403] 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

[0404] Erysipelothrix. For example, Rickettsia Rikettsiae , Rickettsia prowazekii , Rickettsia tsutsugamuchi , Rickettsia mooseri, Rickettsia sibirica , Bordetella bronchiseptica , Neisseria meningitidis , Neisseria gonorrhoeae , Aeromonas eucrenophila , Aeromonas salmonicida , Francisella tularensis , Corynebacterium pseudotuberculosis ,

[0405] Citrobacter freundii , Chlamydia pneumoniae , Haemophilus sornnus , Brucella abortus , Mycobacterium intr acellular e, Legionella pneumophila , Rhodococcus equi , Pseudomonas aeruginosa , Helicobacter mustelae , Vibrio cholerae , Bacillus subtilis , Erysipelothrix rhusiopathiae , Yersinia enterocolitica , Rochalimaea quintana , and

[0406] Agrobacterium tumerfacium.

[0407] The bacteria accumulate by virtue of one or more properties, including, diffusion, migration and chemotaxis to immunoprivileged tissues or organs or environments, environments that provide nutrients or other molecules for which they are auxotrophic and / or environments that contain replicating cells that provide environments for entry and replication of bacteria. The immunostimulatory bacteria provided herein and species that effect such therapy include species of Salmonella , Listeria , and E. coli. The bacteria contain plasmids that encode a therapeutic product or products expressed under control of a eukaryotic promoter, such as an RNA polymerase (RNAP) II or III promoter. Typically, RNAPIII (also referred to as

[0408] POLIII) promoters are constitutive, and RNAP II (also referred to as POLII) can be regulated. Where a plurality of products are encoded, expression of each can be under control of different promoters.

[0409] Among the bacteria provided herein, are bacteria that are modified so that they are auxotrophic for adenosine. This can be achieved by modification or deletion of genes involved in purine synthesis, metabolism, or transport. For example, disruption of the tsx gene in Salmonella species, such as Salmonella typhi , results in adenosine auxotrophy. Adenosine is immunosuppressive and accumulates to high concentrations in tumors; auxotrophy for adenosine improves the anti-tumor activity of the bacteria because the bacteria selectively replicate in tissues rich in adenosine.

[0410] Also provided are bacteria that are modified so that they have a defective asd gene. These bacteria for use in vivo are modified to include carrying a functional asd gene on the introduced plasmid; this maintains selection for the plasmid so that an antibiotic-based plasmid maintenance / selection system is not needed. Also provided is the use of asd defective strains that do not contain a functional asd gene on a plasmid and are thus engineered to be autolytic in the host. Also provided are bacteria that are modified so that they are incapable of producing flagella. This can be achieved by modifying the bacteria by means of deleting the genes that encode the flagellin subunits. The modified bacteria lacking flagellin are less inflammatory and therefore better tolerated and induce a more potent anti-tumor response.

[0411] Also provided are bacteria that are modified to produce listeriolysin O, which improves plasmid delivery in phagocytic cells.

[0412] Also provided are bacteria modified to carry a low copy, CpG-containing plasmid. The plasmid further can include other modifications.

[0413] The bacteria also can be modified to grow in a manner such that the bacteria, if a Salmonella species, expresses less of the toxic SPI-1 (<Salmonella pathogenicity island-1) genes. In Salmonella , genes responsible for virulence, invasion, survival, and extra intestinal spread are located in Salmonella pathogenicity islands (SPIs).

[0414] The bacteria can be further modified for other desirable traits, including for selection of plasmid maintenance, particularly for selection without antibiotics, for preparation of the strains. The immunostimulatory bacteria optionally can encode therapeutic polypeptides, including anti-tumor therapeutic polypeptides and agents.

[0415] Exemplary of the immunostimulatory bacteria provided herein are species of Salmonella. Exemplary of bacteria for modification as described herein are engineered strains of Salmonella typhimurium , such as strain YS1646 (ATCC Catalog # 202165; see, also, International PCT Application Publication No. WO 99 / 13053, also referred to as VNP20009) that is engineered with plasmids to complement an asd gene knockout and antibiotic-free plasmid maintenance.

[0416] Modified immunostimulatory bacterial strains that are rendered auxotrophic for adenosine are provided herein as are pharmaceutical compositions containing such strains formulated for administration to a subject, such as a human, for use in methods of treating tumors and cancers.

[0417] The engineered immunostimulatory bacteria provided herein contain multiple synergistic modalities to induce immune re-activation of cold tumors and to promote tumor antigen-specific immune responses, while inhibiting immune checkpoint pathways that the tumor utilizes to subvert and evade durable anti-tumor immunity. Improved tumor targeting through adenosine auxotrophy and enhanced vascular disruption have improved potency, while localizing the inflammation to limit systemic cytokine exposure and the autoimmune toxicities observed with other immunotherapy modalities. Exemplary of the bacteria so-modified are S. typhimurium strains, including such modifications of the strain YS1646, particularly asc strains, and of wild-type strains.

[0418] C. CANCER IMMUNOTHERAPEUTICS

[0419] The immunosuppressive milieu found within the tumor microenvironment (TME) is a driver of tumor initiation and progression. Cancers emerge after the immune system fails to control and contain tumors. Multiple tumor-specific mechanisms create tumor environments wherein the immune system is forced to tolerate tumors and their cells instead of eliminating them. The goal of cancer immunotherapy is to rescue the immune system’s natural ability to eliminate tumors.

[0420] 1. Immunotherapies

[0421] Several clinical cancer immunotherapies have sought to perturb the balance of immune suppression towards anti -tumor immunity. Strategies to stimulate immunity through directly administering cytokines such as IL-2 and IFN-a have seen modest clinical responses in a minority of patients, while inducing serious systemic inflammation-related toxicities (Sharma et al. (2011 ) Nat. Rev. Cancer 11 :805-812). The immune system has evolved several checks and balances to limit autoimmunity, such as 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), which is expressed on both antigen presenting cells (APCs) and tumor cells. The binding of PD-L1 to PD-1 interferes with CD8+T cell signaling pathways, impairing the proliferation and effector function of CD8+T cells, 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 a (SIRPa), V-domain Ig suppressor of T cell activation (VISTA),

[0422] 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), T cell immunoglobulin and mucin-domain containing-3 (TIM-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,

[0423] 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).

[0424] Antibodies designed to block immune checkpoints, such as anti -PD- 1 (for example, pembrolizumab, nivolumab) and anti-PD-Ll (for example, atezolizumab, avelumab, durvalumab), have had durable success in preventing T cell anergy and breaking immune tolerance. Only a fraction of treated patients demonstrate clinical benefit, and those that do often present with autoimmune-related toxi cities (see, e.g ., Ribas (2015) A. Engl. . / . Med. 373: 1490-1492; Topalian et al. (2012) A. Engl. J. Med. 3(5(5:3443-3447). This is further evidence for the need for therapies, provided herein, that are more effective and less toxic.

[0425] Another checkpoint blockade strategy inhibits the induction of CTLA-4 on T cells, which binds to and inhibits co-stimulatory receptors on APCs, such as CD80 or CD86, out-competing the co-stimulatory cluster differentiation 28 (CD28), which binds the same receptors, but with a lower affinity. This blocks the stimulatory signal from CD28, while the inhibitory signal from CTLA-4 is transmitted, preventing T cell activation (see, Phan et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:8372-8377). Anti- CTLA-4 therapy (for example, ipilimumab) has had clinical success and durability in some patients, whilst exhibiting an even greater incidence of severe immune-related adverse events (see, e.g. , Hodi et al. (2010) A Engl. J. Med. 363:711-723;

[0426] Schadendorf et al. (2015) J. Clin. Oncol. 33: 1889-1894). It also has been shown that tumors develop resistance to anti-immune checkpoint antibodies, highlighting the need for more durable anticancer therapies, such as those provided herein.

[0427] 2. Adoptive Immunotherapies

[0428] In seeking to reactivate a cold tumor to become more immunogenic, a class of immunotherapies known as adoptive cell therapy (ACT) encompasses a variety of strategies to harness 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 more immune-stimulatory properties. These therapies have not been successful because they fail to break immune tolerance to cancer (see, e.g., Rosenberg et al. (2004) Nat. Med. 12: 1279). A method using whole irradiated tumor cells containing endogenous tumor antigens and granulocyte macrophage colony-stimulating factor (GM-CSF) to stimulate DC recruitment, known as GVAX, similarly failed in the clinic due to the lack of ability to break tumor tolerance (Copier et al. (2010) Curr. Opin. Mol. Ther. 12:647-653). A separate autologous cell-based therapy, Sipuleucel-T (Provenge), was FDA approved in 2010 for castration-resistant prostate cancer. It utilizes APCs retrieved from the patient and re-armed to express prostatic acid phosphatase (PAP) antigen to stimulate a T cell response, then re-introduced following lymphablation. Unfortunately, its broader adoption is limited by low observed objective response rates and high costs, and its use is limited only to the early stages of prostate cancer (Anassi et al. (2011) P T. 36(4): 197-202). Similarly, autologous T cell therapies (ATCs) harvest a patient’s own T cells and reactivate them ex vivo to overcome tumor tolerance, then reintroduce them to the patient following lymphablation. ATCs have had limited clinical success, and only in melanoma, while generating serious safety and feasibility issues that limit their utility (Yee et al. (2013) Clin. Cancer Res. 19: 1-3).

[0429] Chimeric antigen receptor T cell (CAR-T) therapies are T cells harvested from patients that have been re-engineered to express a fusion protein between the T cell receptor and an antibody Ig variable extracellular domain. This confers upon them the antigen-recognition properties of antibodies with the cytolytic properties of activated T cells (Sadelain (2015) Clin. Invest. 125:3392-400). Success has been limited to B cell and hematopoietic malignancies, at the cost of deadly immune- related adverse events (Jackson et al. (2016) Nat. Rev. Clin. Oncol. 73:370-383). Tumors can also mutate to escape recognition by a target antigen, including CD 19 (Ruella et al., (2016) Comput Struct Biotechnol . / . 14: 357-362) and EGFRvIII (ORourke et al. (2017) Sci Transl Med. Jul 19;9:399), thereby fostering immune escape. While CAR-T therapies are approved in the context of hematological malignancies, they face a significant hurdle for feasibility to treat solid tumors:

[0430] overcoming the highly immunosuppressive nature of the solid tumor

[0431] microenvironment. A number of additional modifications to existing CAR-T therapies are needed to potentially provide feasibility against solid tumors (Kakarla, et al.

[0432] (2014) Cancer J. Mar-Apr; 20(2): 151—155). 3. Cancer Vaccines and Oncolytic Viruses

[0433] Cold tumors lack T cell and dendritic cell (DC) infiltration, and are non-T- cell-inflamed (Sharma et al. (2017) Cell 9;168(4):707-723). In seeking to reactivate a cold tumor to become more immunogenic, another class of immunotherapies harness microorganisms that can accumulate in tumors, either naturally or by virtue of engineering. These include viruses designed to stimulate the immune system to express tumor antigens, thereby activating and reprogramming the immune system to reject the tumor. Virally-based cancer vaccines have failed clinically for a number of factors, including pre-existing or acquired immunity to the viral vector itself, as well as a lack of sufficient immunogenicity to the expressed tumor antigens (Larocca et al. (2011) Cancer J 17(5):359-371). Lack of proper adjuvant activation of APCs has also hampered other non-viral vector cancer vaccines, such as DNA vaccines. Oncolytic viruses preferentially replicate in dividing tumor cells over healthy tissue, whereupon 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 demonstrating clinical benefit in some melanoma patients, and with fewer immune toxicities than with other immunotherapies, its efficacy has been limited; there is a lack of distal tumor efficacy and broader application to other tumor types. Other oncolytic virus (OV)-based vaccines, such as those utilizing

[0434] paramyxovirus, reovirus and picornavirus, among others, have met with 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. Upon IV administration, the virus is rapidly diluted, thus requiring high titers that can lead to hepatotoxicity. If pre-existing immunity exists, the virus is rapidly neutralized in the blood, and acquired immunity then restricts repeat dosing (Maroun et al. (2017) Future Virol. 12(4): 193—213).

[0435] Of the limitations of virally-based vaccine vectors and oncolytic viruses, the greatest limitations can be the virus itself. Viral antigens have strikingly higher affinities to human T cell receptors (TCR) compared to tumor antigens (Aleksic et al. (2012) Eur J Immunol. 42(12):3174-3179). Tumor antigens, presented alongside of viral vector antigens by MHC-1 on the surface of even highly activated APCs, will be outcompeted for binding to TCRs, resulting in very poor antigen-specific anti-tumor immunity. A tumor-targeting immunostimulatory vector, as provided herein, that does not itself provide high affinity T cell epitopes can circumvent these limitations.

[0436] D. BACTERIAL CANCER IMMUNOTHERAPY

[0437] Provided herein are immunostimulatory bacteria that are modified so that they accumulate in tumor-resident immune cells, and do not infect epithelial or other cells. The immunostimulatory bacteria contain plasmids that encode and express, under control of a host-recognized promoter, and secrete, therapeutic products, such as immunostimulatory proteins that are part of a cytosolic DNA / RNA sensor pathway, leading to the expression of type I IFN. Thus, the immunostimulatory bacteria are cancer therapeutics that, by virtue of modification of the bacterial genome, and the encoded therapeutic product(s), deliver an immunotherapy directly to the tumor microenvironment. The bacteria and methods and uses provided herein solve prior problems encountered with other cancer immunotherapeutics. The

[0438] immunostimulatory proteins that are part of a cytosolic DNA / RNA sensor pathway leading to the expression of type I IFN, in addition to expression in the

[0439] immunostimulatory bacteria provided herein, can be encoded or provided in other delivey vehicles, such as exosomes, liposomes, oncolytic viruses, and gene therapy vectors.

[0440] 1. Bacterial Therapies

[0441] Acute inflammation associated with microbial infection has been

[0442] observationally linked with the spontaneous elimination of tumors for centuries. The recognition that bacteria have anticancer activity goes back to the 1800s, when several physicians observed regression of tumors in patients infected with Streptococcus pyogenes. William Coley began the first study using bacteria for the treatment of end stage cancers, and developed a vaccine composed of S. pyogenes and Serratia marcescens. This vaccine successfully was used to treat a variety of cancers, including sarcomas, carcinomas, lymphomas and melanomas. Since then, a number of bacteria, including species of Clostridium , Mycobacterium , Bifidobacterium , Listeria , such as, L. monocytogenes , and Escherichia species, have been studied as sources of anti-cancer vaccines (see, e.g. , Published International PCT Application Nos. WO 1999 / 013053 and 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; and Pawelek et al. (2003) Lancet Oncol.4.548-556).

[0443] Bacteria can infect animal and human cells, and some possess the innate ability to deliver DNA into the cytosol of cells. Bacteria also are suitable for therapy because they can be administered orally, they propagate readily in vitro and in vivo , and they can be stored and transported in a lyophilized state. Bacterial genetics readily are manipulated, and the complete genomes for many strains have been fully characterized (Feigner et al. (2016) mbio 7(5):e01220-16). As a result, bacteria have been used to deliver and express a variety of genes, including those that encode cytokines, angiogenesis inhibitors, toxins and prodrug-converting enzymes.

[0444] Salmonella , for example, has been used to express immune-stimulating molecules, such as 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), for treating tumors. Bacterial vectors also are cheaper and easier to produce than viral vectors, and bacterial delivery is favorable over viral delivery because it can be quickly eliminated by antibiotics if necessary, rendering it a safer alternative.

[0445] To be used, however, the strains must not be pathogenic, or not pathogenic after modification, for use as a therapeutic. For example, in the treatment of cancer, the therapeutic bacterial strains must be attenuated or rendered sufficiently non-toxic so as to not cause systemic disease and / or septic shock, but still maintain some level of infectivity to effectively colonize tumors. Genetically modified bacteria have been described that are to be used as antitumor agents to elicit direct tumoricidal effects and / or to deliver tumoricidal molecules (Clairmont, et al. (2000) J. Infect. Dis.

[0446] 181 : 1996-2002; Bermudes, 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). Among these are bioengineered strains of Salmonella enterica serovar Typhimurium (S. typhimurium). These bacteria accumulate preferentially >1, 000-fold greater in tumors than in normal tissues and disperse homogeneously in tumor tissues (Pawelek, J. et al. (1997) Cancer Res.

[0447] 57:4537-4544; Low, K. B. et al. (1999) Nat. Biotechnol. 17:37-41). Preferential replication allows the bacteria to produce and deliver a variety of anticancer therapeutic agents at high concentrations directly within the tumor, while minimizing toxicity to normal 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, S. N. et al.

[0448] (1992) Biotechnology 10:888-892; DiPetrillo, M. D. et al. (1999) Vaccine 18:449- 459; Hohmann, E. L. et al. (1996 ) J. Infect. Dis. 173:1408-1414; Sirard, J. C. et al.

[0449] (1999) Immunol. Rev. 171 :5-26). The S. typhimurium phoP / phoQ operon is a typical bacterial two-component regulatory system composed of a membrane-associated sensor kinase (PhoQ) and a cytoplasmic transcriptional regulator (PhoP: 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). PhoP / phoQ is required for virulence, and its deletion results in poor survival of this bacterium in macrophages and a 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 deletion strains have been employed as effective vaccine delivery vehicles (Galan, J. E. and Curtiss, R. III.

[0450] (1989) Microb Pathog 6:433-443; Fields, P. I. et al. ( 1986) I1roc Natl Acad Sci USA 83: 189-193; Angelakopoulos, H. and Hohmann, E. L. (2000) Infect Immun 68:213- 241). Attenuated Salmonellae have been used for targeted delivery of tumoricidal proteins (Bermudes, D. et al. (2002) Curr Opin Drug Discov Devel 5: 194-199;

[0451] Tjuvajev J. et al. (2001) J Control Release 74:313-315).

[0452] Bacterially-based cancer therapies have demonstrated limited clinical benefit. A variety of bacterial species, including Clostridium novyi (Dang et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98(26): 15155-15160; U.S. Patent Publications Nos.

[0453] 2017 / 0020931 and 2015 / 0147315; and U.S. Patent Nos. 7,344,710 and 3,936,354),

[0454] Mycobacterium bovis (U.S. Patent Publications Nos. 2015 / 0224151 and

[0455] 2015 / 0071873), Bifidobacterium bifidum (Kimura et al. (1980) Cancer Res. 40:2061- 2068), Lactobacillus casei (Yasutake et al. (1984) Med Microbiol Immunol.

[0456] 173(3): 113-125), Listeria monocytogenes (Le et al. (2012) Clin. Cancer Res.

[0457] 18(3):858-868; Starks et al. (2004) J. Immunol. 173:420-427; U.S. Patent Publication No. 2006 / 0051380) and Escherichia coli (U.S. Patent No. 9,320,787), have been studied as possible agents for anticancer therapy.

[0458] The Bacillus Calmette-Guerin (BCG) strain, for example, is approved for the treatment of bladder cancer in humans, and is more effective than intravesical chemotherapy, often being used as a first-line treatment (Gardlik et al. (2011) Gene therapy 18:425-431). Another approach utilizes Listeria monocytogenes , a live attenuated intracellular bacterium capable of inducing potent CD8+T cell priming to expressed tumor antigens in mice (Le et al. (2012) Clin. Cancer Res. 18(3):858-868). In a clinical trial of the Listeria- based vaccine incorporating the tumor antigen mesothelin, together with an allogeneic pancreatic cancer-based GVAX vaccine in a prime-boost approach, a median survival of 6.1 months was noted in patients with advanced pancreatic cancer, versus 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 larger phase 2b study, possibly pointing to the difficulties in attempting to induce immunity to a low affinity self-antigen such as mesothelin.

[0459] Bacterial strains can be modified as described herein. The strains can be attenuated or their cellular targets modified by standard methods and / or by deletion or modification of genes, and by alteration or introduction of genes that render the bacteria able to grow in vivo primarily in immunoprivileged environments, such as the TME, in tumor cells, in tumor-resident immune cells, and solid tumors. Starting strains for modification as described herein can be selected from among, for example, Shigella , Listeria , E. coli , Bifidobacteriae and Salmonella. For example, Shigella sonnei , Shigella flexneri , Shigella disenteriae , Listeria monocytogenes , Salmonella typhi , Salmonella typhimurium , Salmonella gallinarum , and Salmonella enteritidis. Other suitable bacterial species include Rickettsia , Klebsiella , Bordetella , Neisseria , Aeromonas, Franciesella , Corynebacterium , Citrobacter , Chlamydia , Haemophilus ,

[0460] Brucella , Mycobacterium , Mycoplasma , Legionella , Rhodococcus, Pseudomonas , Helicobacter , Vibrio, Bacillus, and Erysipelothrix. For example, Rickettsia Rikettsiae, Rickettsia prowazecki , Rickettsia tsutsugamuchi , Rickettsia mooseri, Rickettsia sibirica , Bordetella bronchiseptica , Neisseria meningitidis , Neisseria gonorrhoeae , Aeromonas eucrenophila , Aeromonas salmonicida , Francie sella tularensis ,

[0461] Corynebacterium pseudotuberculosis , Citrobacter freundii , Chlamydia pneumoniae , Haemophilus sornnus , Brucella abortus , Mycobacterium intracellulare , Legionella pneumophila , Rhodococcus equi , Pseudomonas aeruginosa , Helicobacter mustelae , Vibrio cholerae , Bacillus subtilis , Erysipelothrix rhusiopathiae , Yersinia

[0462] enterocolitica , Rochalimaea quintana , and Agrobacterium tumerfacium. Any known therapeutic, including immunostimulatory, bacteria can be modified as described herein.

[0463] 2. Comparison of the Immune Responses to Bacteria and Viruses

[0464] Bacteria, like viruses, have the advantage of being naturally

[0465] immunostimulatory. Bacteria and viruses contain conserved structures known as Pathogen- Associated Molecular Patterns (PAMPs), which 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 lead to pathogen clearance (Iwasaki and Medzhitov (2010) Science 327(5963):291-295). The manner in which the innate immune system is engaged by PAMPs, and from what type of infectious agent, determines the appropriate adaptive immune response to combat the invading pathogen.

[0466] A class of PRRs known as Toll Like Receptors (TLRs) recognize PAMPs derived from bacterial and viral origins, and are located in various compartments within the cell. TLRs bind a range 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.

[0467] Immunol. 17(4):338-344). Host surveillance of S. typhimurium for example, is largely mediated through TLR2, TLR4 and TLR5 (Arpaia et al. (2011) Cell 144(5):675-688). These TLRs signal through MyD88 and TRIF adaptor molecules to mediate induction of NF-KB dependent pro-inflammatory cytokines such as TNF-a, IL-6 and IFN-g (Pandey et. al. (2015) Cold Spring Harb Perspect Biol 7(l):a016246). Another category of PRRs are 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, resulting in the cleavage of caspase-1 and induction of the pro-inflammatory cytokines IL-Ib and IL-18, leading to pyroptotic cell death of infected macrophages (Fink et al. (2007) Cell Microbiol. 9(11):2562-2570).

[0468] While engagement of TLR2, TLR4, TLR5 and the inflammasome induces pro- inflammatory cytokines that mediate bacterial clearance, they activate a

[0469] predominantly NF-KB-driven signaling cascade that leads to recruitment and activation of neutrophils, macrophages and CD4+T cells, but not the DCs and CD8+T cells that are required for anti -tumor immunity (Lui et al. (2017) Signal Transduct Target Ther. 2: 17023). In order to activate CD8+T cell-mediated anti-tumor immunity, IRF3 / IRF7-dependent type I interferon signaling is critical for DC activation and cross-presentation of tumor antigens to promote CD8+T cell priming (Diamond et al. (2011) . / . Exp. Med. 208(10): 1989-2003; Fuertes et al. (2011) . / . Exp. Med. 208(10):2005-2016). Type I interferons (IFN-a, IFN-b) are the signature cytokines induced by two distinct TLR-dependent and TLR-independent signaling pathways. The TLR-dependent pathway for inducing IFN-b occurs following endocytosis of pathogens, whereby TLR3, 7, 8 and 9 detect pathogen-derived DNA and RNA elements within the endosomes. TLRs 7 and 8 recognize viral nucleosides and nucleotides, and synthetic agonists of these, such as resiquimod and imiquimod have been clinically validated (Chi et al. (2017) Frontiers in Pharmacology 8:304). Synthetic dsRNA, such as polyinosinic:polycytidylic acid (poly (I:C)) and poly ICLC, an analog that is formulated with poly L lysine to resist RNase digestion, is an agonist for TLR3 and MDA5 pathways and a powerful inducer of IFN-b (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-b via IRF3. Additionally, TLR4 has been shown to induce IFN-b via MyD88-independent TRIF activation of IRF3 (Owen et al. (2016) mBio.l. X e02051-15). It subsequently was shown that TLR4 activation of DCs was independent of type I IFN, so the ability of TLR4 to activate DCs via type I IFN is not likely biologically relevant (Hu et al. (2015) Proc. Natl. Acad. Sci. U.S.A. 112:45). Further, TLR4 signaling has not been shown to directly recruit or activate CD8+T cells.

[0470] Of the TLR-independent type I IFN pathways, one 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- 1), melanoma differentiation-associated gene 5 (MDA-5), and through the IFN-b promoter stimulator 1 (IPS-1; also known as mitochondrial antiviral-signaling protein or MAVS) adaptor protein-mediated phosphorylation of the IRF-3 transcription factor, leading to induction of IFN-b (Ireton and Gale (2011) Viruses 3 (6): 906-919). Synthetic RIG-I-binding elements have also been discovered unintentionally in common lentiviral shRNA vectors, in the form of an AA dinucleotide sequence at the U6 promoter transcription start site. Its subsequent deletion in the plasmid prevented confounding off-target type I IFN activation (Pebemard et al. (2004) Differentiation. 72: 103-111).

[0471] 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 that is now recognized as the 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, resulting in the induction of IFN-b and other pro-inflammatory cytokines and chemokines that strongly activate innate and adaptive immunity (Burdette et al. (2011) Nature 478(7370): 515-518). Sensing of cytosolic dsDNA through STING requires cyclic GMP-AMP synthase (cGAS), a host cell nucleotidyl transferase that directly binds dsDNA, and in response, synthesizes a cyclic dinucleotide (CDN) second messenger, cyclic GMP-AMP (cGAMP), which binds and activates STING (Sun et al. (2013) Science 339(6121):786-791; Wu et al. (2013) Science 339(6121):826-830). CDNs derived from bacteria such as c-di-AMP produced from intracellular Listeria monocytogenes can also directly bind murine STING, but only 3 of the 5 human STING alleles. Unlike the CDNs produced by bacteria, in which the two purine nucleosides are joined by a phosphate bridge with 3’ -3’ linkages, the internucleotide phosphate bridge in the cGAMP synthesized by mammalian cGAS is joined by a non-canonical 2’-3’ linkage. These 2’-3’ molecules bind to STING with 300-fold better affinity than bacterial 3’-3’ CDNs, and thus, are more potent physiological ligands of human STING (see, e.g ., Civril 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):pll; Ablasser et al. (2013) Nature 503(7477):530-534).

[0472] The cGAS / STING signaling pathway in humans has evolved to preferentially respond to viral pathogens over bacterial pathogens, and this can explain why previous bacterial vaccines harboring host tumor antigens have made for poor 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, with TLR-dependent type I IFN production by plasmacytoid DCs operating only when the STING pathway has been virally- inactivated (Hervas-Stubbs et al. (2014) . / . Immunol. 193: 1151-1161). Further, for bacteria such as S. typhimurium , while capable of inducing IFN-b via TLR4, CD8+T cells are neither induced nor required for clearance or protective immunity (Lee et al. (2012) Immunol Lett. 148(2): 138-143). The lack of physiologically relevant CD8+T epitopes for many strains of bacteria, including S. typhimurium , has impeded bacterial vaccine development and protective immunity to subsequent infections, even from the same genetic strains (Lo et al. (1999) J. Immunol. 162:5398-5406). Bacterially-based cancer immunotherapies are biologically limited in their ability to induce type I IFN to recruit and activate CD8+T cells, which is necessary to promote tumor antigen cross-presentation and durable anti-tumor immunity. The immunostimulatory bacteria provided herein, however, are engineered to solve this problem. The

[0473] immunostimulatory bacteria provided herein induce viral-like TLR-independent type I IFN signaling, rather than TLR-dependent bacterial immune signaling, which preferentially induces CD8+T cell mediated anti-tumor immunity.

[0474] STING activates innate immunity in response to sensing nucleic acids in the cytosol. Downstream signaling is activated through binding of CDNs, which are synthesized by bacteria or by the host enzyme cGAS in response to binding to cytosolic dsDNA. Bacterial and host-produced CDNs have distinct phosphate bridge structures, which differentiates their capacity to activate STING. IFN-b is the signature cytokine of activated STING, and virally-induce type I IFN, rather than bacterially-induced IFN, is required for effective CD8+T cell mediated anti-tumor immunity. Immunostimulatory bacteria provided herein include those that are STING agonists and those that express STING.

[0475] 3. Salmonella Therapy

[0476] Salmonella is exemplary of a bacterial genus that can be used as a cancer therapeutic. The Salmonella exemplified herein is an attenuated species or is one that, by virtue of the modifications described herein, for use as a cancer therapeutic, has reduced toxicity.

[0477] a. Tumor-tropic Bacteria

[0478] A number of bacterial species have demonstrated preferential replication within solid tumors when injected from a distal site. These include, but are not limited to, species of Salmonella , Bifodobacterium , Clostridium , and Escherichia. The natural tumor-homing properties of the bacteria combined with the host’s innate immune response to the bacterial infection is thought to mediate the anti-tumor response. This tumor tissue tropism has been shown to reduce the size of tumors to varying degrees. One contributing factor to the tumor tropism of these bacterial species is the ability to replicate in anoxic or hypoxic environments. A number of these naturally tumor- tropic bacteria have been further engineered to increase the potency of the antitumor response (reviewed in Zu et al. (2014) Crit Rev Microbiol. 40(3):225-235; and Feigner et al. (2017) Microbial Biotechnology 10(5): 1074-1078).

[0479] b. Salmonella enterica serovar Typhimurium

[0480] Salmonella enterica serovar Typhimurium (S. typhimurium ) is exemplary of a bacterial species for use as an anti-cancer therapeutic. One approach to using bacteria to stimulate host immunity to cancer has been through the Gram-negative facultative anaerobe S. typhimurium , which preferentially accumulates in hypoxic and necrotic areas in the body, including tumor microenvironments. S. typhimurium accumulates in these environments due to the availability of nutrients from tissue necrosis, the leaky tumor vasculature, and their increased likelihood to survive in the immune system- evading tumor microenvironment (Baban et al. (2010) Bioengineered Bugs 1(6):385- 294). S. typhimurium is able to grow under both aerobic and anaerobic conditions; therefore, it is able to colonize small tumors that are less hypoxic, and large tumors that are more hypoxic. S. typhimurium is a Gram-negative, facultative pathogen that is transmitted via the fecal-oral route. It causes localized gastrointestinal infections, but also enters the bloodstream and lymphatic system after oral ingestion, infecting systemic tissues such as the liver, spleen and lungs. Systemic administration of wild-type S. typhimurium overstimulates TNF-a induction, leading to a cytokine cascade and septic shock, which, if left untreated, can be fatal. As a result, pathogenic bacterial strains, such as S. typhimurium , must be attenuated to prevent systemic infection, without completely suppressing their ability to effectively colonize tumor tissues. Attenuation is often achieved by mutating a cellular structure that can elicit an immune response, such as the bacterial outer membrane, or limiting its ability to replicate in the absence of supplemental nutrients.

[0481] S. typhimurium is an intracellular pathogen that is rapidly taken up by myeloid cells, such as macrophages, or it can induce its own uptake in non-phagocytic cells, such as epithelial cells. Once inside cells, it can replicate within a Salmonella containing vacuole (SCV) and can also escape into the cytosol of some epithelial cells. Many of the molecular determinants of S. typhimurium pathogenicity have been identified and the genes are clustered in Salmonella pathogenicity islands (SPIs). The two best characterized pathogenicity islands are SPI-1, which is responsible for mediating bacterial invasion of non-phagocytic cells, and SPI-2 which is required for replication within the SCV (Agbor and McCormick (2011) Cell Microbiol.

[0482] 13(12): 1858-1869). Both of these pathogenicity islands encode macromolecular structures called type three secretion systems (T3SS) that can translocate effector proteins across the host membrane (Galan and Wolf-Watz (2006) Nature 444:567- 573).

[0483] c. Bacterial Attenuation

[0484] Therapeutic bacteria for administration as a cancer treatment should be modified so that they do not cause diseases. Various methods to achieve this are known in the art. Auxotrophic mutations, for example, render bacteria incapable of synthesizing an essential nutrient, and deletions / mutations in genes such as aro , pur, gua, thy , nad and asd (U.S. Patent Publication No. 2012 / 0009153) are widely used. Nutrients produced by the biosynthesis pathways involving these genes are often unavailable in host cells, and as such, bacterial survival is challenging. For example, attenuation of Salmonella and other species can be achieved by deletion of the aroA gene, which is part of the shikimate pathway, connecting glycolysis to aromatic amino acid biosynthesis (Feigner et al. (2016 )MBio 7(5):e01220-16). Deletion of aroA therefore results in bacterial auxotrophy for aromatic amino acids and subsequent attenuation (U.S. Patent Publication Nos. 2003 / 0170276, 2003 / 0175297,

[0485] 2012 / 0009153 and 2016 / 0369282; International Application Publication Nos. WO 2015 / 032165 and WO 2016 / 025582). Similarly, other enzymes involved in the biosynthesis pathway for aromatic amino acids, including aroC and aroD have been deleted to achieve attenuation (U.S. Patent Publication No. 2016 / 0369282;

[0486] International Application Publication No. WO 2016 / 025582). For example, S.

[0487] typhimurium strain SL7207 is an aromatic amino acid auxotroph (aroA mutant); strains A1 and Al-R are leucine-arginine auxotrophs. VNP20009 is a purine auxotroph purl mutant). As shown herein, it is also auxotrophic for the

[0488] immunosuppressive nucleoside adenosine.

[0489] Mutations that attenuate bacteria also include, but are not limited to, mutations in genes that alter the biosynthesis of lipopolysaccharide, such as rfaL, rfaG , rfaH , rfaD , rfaP , rFb , rfa, msbB , htrB,firA,pagL,pagP , IpxR, arnT , ep / A , and IpxT;

[0490] mutations that introduce a suicide gene, such as sacB , nuk, hok , gef kil or phi A ;

[0491] mutations that introduce a bacterial lysis gene, such as hly and cly mutations in virulence factors, such as isyA, pag , prg, iscA , virG, pic and act mutations that modify the stress response, 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 Publication Nos. 2012 / 0009153, 2003 / 0170276, 2007 / 0298012; U.S. Patent No. 6,190,657;

[0492] International Application Publication No. WO 2015 / 032165; Feigner 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

[0493] 79(12): 5027-5038; Kong et al. (2012) Proc. Natl. Acad. Sci. USA 109(47): 19414- 19419). Ideally, the genetic attenuations comprise gene deletions rather than point mutations to prevent spontaneous compensatory mutations that might result in reversion to a virulent phenotype. i. msbBr Mutants

[0494] The enzyme lipid A biosynthesis myristoyl transferase, encoded by the msbB gene in S. typhimurium , catalyzes the addition of a terminal myristyl group to the lipid A domain of lipopoly saccharide (LPS) (Low e / al. (1999) Nat. Biotechnol. 17(1):37- 41). Deletion of msbB thus alters the acyl composition of the lipid A domain of LPS, the major component of the outer membranes of Gram-negative bacteria. This modification significantly reduces the ability of the LPS to induce septic shock, attenuating the bacterial strain and reducing the potentially harmful production of TNFa, thus, lowering systemic toxicity. S. typhimurium msbB mutants maintain their ability to preferentially colonize tumors over other tissues in mice and retain anti tumor activity, thus, increasing the therapeutic index of Salmonella- based

[0495] immunotherapeutics (see, e.g ., U.S. Patent Publication Nos. 2003 / 0170276,

[0496] 2003 / 0109026, 2004 / 0229338, 2005 / 0225088 and 2007 / 0298012).

[0497] For example, deletion of msbB in the S. typhimurium strain VNP20009 results in production of a predominantly penta-acylated LPS, which is less toxic than native hexa-acylated LPS, and allows for systemic delivery without the induction of 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 the Salmonella strains, that dramatically reduce virulence, and thereby provide for lower toxicity, and permit administration of higher doses.

[0498] ii. purl Mutants

[0499] Immunostimulatory bacteria that can be attenuated by rendering them auxotrophic for one or more essential nutrients, such as purines (for example, adenine), nucleosides (for example, adenosine) or amino acids (for example, arginine and leucine), are employed. 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 tumor

[0500] microenvironments. Hence, strains of immunostimulatory bacteria described herein are attenuated because they require adenosine for growth, and they preferentially colonize TMEs, which, as discussed below, have an abundance of adenosine.

[0501] Phosphoribosylaminoimidazole synthetase, an enzyme encoded by the purl gene (synonymous with the purM gene), is involved in the biosynthesis pathway of purines. Disruption of the purl gene thus renders the bacteria auxotrophic for purines. In addition to being attenuated, purl mutants are enriched in the tumor environment and have significant anti -tumor activity (Pawelek et al. (1997) Cancer Research 57:4537-4544). It was previously described that this colonization results from the high concentration of purines present in the interstitial fluid of tumors as a result of their rapid cellular turnover. Since the purl bacteria are unable to synthesize purines, they require an external source of adenine, and it was thought that this would lead to their restricted growth in the purine-enriched tumor microenvironment (Rosenberg et al. (2002) J Immunotherapy 25(3):218-225). While the VNP20009 strain was initially reported to contain a deletion of the purl gene (Low et al. (2003) Methods in

[0502] Molecular Medicine Vol. 90, Suicide Gene Therapy Al -59), subsequent analysis of the entire genome of VNP20009 demonstrated that the purl gene is not deleted, but is disrupted by a chromosomal inversion (Broadway et al. (2014) Journal of

[0503] Biotechnology 192: 177-178). The entire gene is contained within two parts of the VNP20009 chromosome that is flanked by insertion sequences (one of which has an active transposase).

[0504] It is shown herein, that, purl mutant S. typhimurium strains are auxotrophic for the nucleoside adenosine, which is highly enriched in tumor microenvironments. Hence, when using VNP20009, it is not necessary to introduce any further modification to achieve adenosine auxotrophy. For other strains and bacteria, the purl gene can be disrupted as it has been in VNP20009, or it can contain a deletion of all or a portion of the purl gene to prevent reversion to a wild-type gene.

[0505] iii. Combinations of Attenuating Mutations

[0506] A bacterium with multiple genetic attenuations by means of gene deletions on disparate regions of the chromosome is desirable for bacterial immunotherapies because the attenuation can be increased, while decreasing the possibility of reversion to a virulent phenotype by acquisition of genes by homologous recombination with a wild-type genetic material. Restoration of virulence by homologous recombination would require two separate recombination events to occur within the same organism. Ideally, the combination of attenuating mutations selected for use in an

[0507] immunotherapeutic agent increases the tolerability without decreasing the potency, thereby increasing the therapeutic index. For example, as discussed below, disruption of the msbB and purl genes in S. typhimurium strain VNP20009, has been used for tumor-targeting and growth suppression, and elicits low toxicity in animal models (Clairmont et al. (2000) . / .

[0508] Infect. Dis. 181 : 1996-2002; Bermudes et al. (2000) Cancer Gene Therapy: Past Achievements and Future Challenges , edited by Habib Kluwer Academic / Plenum

[0509] 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. U.S.A. 104(31): 12879-12883; Luo et al. (2002) Oncology Research 12:501-508). VNP20009, however, does not show the same tumor accumulation and anti-tumor activity in human trials. Higher doses, which are required to manifest any anti-tumor activity, thus, are not possible due to toxicity. The immunostimulatory bacteria provided herein, which contain combinations of genetic modifications that, for example, reduce virulence, increase tolerability, decrease or eliminate bacterial infection of epithelial (and other non-immune) cells, increase accumulation in tumor- resident immune cells, and reduce cell death of tumor-resident immune cells, among other desirable properties that improve the therapeutic index, address this problem.

[0510] iv. VNP20009 and Other Attenuated S. typhimurium Strains

[0511] Exemplary of a therapeutic bacterium that can be modified as described herein is the strain designated as VNP20009 (ATCC # 202165, YS1646). The clinical candidate, VNP20009 (ATCC # 202165, YS1646), was at least 50,000-fold attenuated for safety by deletion of both the msbB and purl genes (Clairmont et al. (2000) J. Infect. Dis. 181 : 1996-2002; Low et al. (2003) Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy Al -59; Lee et al. (2000) International Journal of

[0512] Toxicology 19: 19-25). Similar strains of Salmonella that are attenuated also are contemplated. As described above, deletion of msbB alters the composition of the lipid A domain of lipopolysaccharide, the major component of Gram-negative bacterial outer membranes (Low et al. (1999) Nat. Biotechnol. 17(1):37-41). This prevents lipopolysaccharide-induced septic shock, attenuating the bacterial strain and lowering systemic toxicity, while reducing the potentially harmful production of TNFa (Dinarello, C. A. (1997) Chest 112(6 Suppl):321 S-329S; Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Deletion of the purl gene renders the bacteria auxotrophic for purines, which further attenuates the bacteria and enriches it in the tumor microenvironment (Pawelek et al. (1997) Cancer Res. 57:4537-4544; Broadway et al. (2014) J. Biotechnology 192: 177-178).

[0513] The accumulation of VNP20009 in tumors results from a combination of factors including: the inherent invasiveness of the parental strain, ATCC #14028, its ability to replicate in hypoxic environments, and its requirement for high

[0514] concentrations of purines that are present in the interstitial fluid of tumors. It also is shown herein that VNP20009 also is auxotrophic for the nucleoside adenosine, which can accumulate to pathologically high levels in the tumor microenvironment and contribute to an immunosuppressive tumor microenvironment (Peter Vaupel and Amulf Mayer Oxygen Transport to Tissue XXXVII, Advances in Experimental Medicine and Biology 876 chapter 22, pp. 177-183). When VNP20009 was administered into mice bearing syngeneic or human xenograft tumors, the bacteria accumulated preferentially within the extracellular components of tumors at ratios exceeding 300-1000 to 1, reduced TNFa induction, and demonstrated tumor growth inhibition as well as prolonged survival compared to control mice (Clairmont et al. (2000) J. Infect. Dis. 181 : 1996-2002). Results from the Phase 1 clinical trial in humans, however, revealed that while VNP20009 was relatively safe and well tolerated, poor accumulation was observed in human melanoma tumors, and very little anti-tumor activity was demonstrated (Toso et al. (2002) J. Clin. Oncol.

[0515] 20(1): 142-152). Higher doses, which would be required to affect any anti-tumor activity, were not possible due to toxicity that correlated with high levels of pro- inflammatory cytokines.

[0516] Other strains of S. typhimurium can be used for tumor-targeted delivery and therapy, such as, for example, leucine-arginine auxotroph A-l (Zhao et al. (2005) Proc. Natl. Acad. Sci. USA 102(3):755-760; Yu et al. (2012) Scientific Reports 2:436; U.S. Patent No. 8,822,194; U.S. Patent 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 Publication Nos. 2012 / 0009153, 2016 / 0369282 and

[0517] 2016 / 0184456) and its obligate anaerobe derivative YB1 (International Application

[0518] Publication No. WO 2015 / 032165; Yu et al. (2012) Scientific Reports 2:436;

[0519] Leschner et al. (2009) PLoS ONE 4(8):e6692; Yu et al. (2012) Scientific Reports 2:436); aroA / aroD mutant A typhimurium strain BRD509, a derivative of the SL1344 (wild-type) strain (Yoon et al. (2017) European . / . of Cancer 70:48-61); asct / cya / crp mutant S. typhimurium strain c4550 (Sorenson et al. (2010) Biology:

[0520] Targets & Therapy 4:61-73) and phoP / phoQ S. typhimurium strain LH430

[0521] (International Application Publication No. WO 2008 / 091375).

[0522] The strain VNP20009 failed to show a clinical benefit in a study involving patients with advanced melanoma, but the treatment was safely administered to advanced cancer patients. A maximum tolerated dose (MTD) was established. Hence, this strain, as well as other similarly engineered bacterial strains, can be used as a starting material for tumor-targeting, therapeutic delivery vehicles. Modifications provided herein provide a strategy to increase efficacy, by increasing the anti-tumor efficiency and / or the safety and tolerability of the therapeutic agent.

[0523] v. S. typhimurium Engineered To Deliver

[0524] Macromolecules

[0525] S. typhimurium also has been modified to deliver the tumor-associated antigen (TAA) survivin (SVN) to APCs to prime adaptive immunity (U.S. Patent Publication No. 2014 / 0186401; Xu et al. (2014) Cancer Res. 74(21):6260-6270). SVN is an inhibitor of apoptosis protein (LAP) which prolongs cell survival and provides cell cycle control, and is overexpressed in all solid tumors and poorly expressed in normal tissues. This technology employs the Salmonella Pathogenicity Island 2 (SPI-2) and its type III secretion system (T3SS) to deliver the TAAs into the cytosol of APCs, which then are activated to induce TAA-specific CD8+T cells and anti-tumor immunity (Xu et al. (2014) Cancer Res. 74(21):6260-6270). Similar to the Listeria- based TAA vaccines, this approach has shown promise in mouse models, but has yet to demonstrate effective tumor antigen-specific T cell priming in humans. In addition to gene delivery, S. typhimurium also has been used for the delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) for cancer therapy. For example, attenuated S. typhimurium have been modified to express certain shRNAs, such as those that target STAT3 and IDOl (International Application Publication No. WO 2008 / 091375; and U.S. Patent No. 9,453,227).

[0526] VNP20009 transformed with an shRNA plasmid against the immunosuppressive gene indolamine deoxygenase (IDO), successfully silenced 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). Combining this vector with the co-administration of PEGPH20 (an enzyme that depletes extracellular hyaluronan), showed positive results in the treatment of pancreatic ductal

[0527] adenocarcinoma tumors (Manuel et al. (2015) Cancer Immunol. Res. 3(9): 1096-1107; U.S. Patent Publication No. 2016 / 0184456). In another study, an S. typhimurium strain attenuated by a phoP / phoQ deletion and expressing a signal transducer and activator of transcription 3 (STAT3)-specific shRNA, was found to inhibit tumor growth and reduce the number of metastatic organs, extending the life of C57BL6 mice (Zhang et al. (2007) Cancer Res. 67(12):5859-5864). In another example, S. typhimurium strain SL7207 has been used for the delivery of shRNA targeting CTNNBl, the gene that encodes b-catenin (Guo et al. (2011) Gene therapy 18:95- 105; U.S. Patent Publication Nos. 2009 / 0123426, 2016 / 0369282), while S.

[0528] 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 Publication Nos. 2009 / 0208534, 2014 / 0186401 and 2016 / 0184456; International Application Publication Nos. WO 2008 / 091375 and WO 2012 / 149364). siRNAs targeting the autophagy genes Atg5 and Beclinl have been delivered to tumor cells using S.

[0529] typhimurium strains Al-R and VNP20009 (Liu et al. (2016) Oncotarget 7(16):22873- 22882). Improvement of such strains is needed so that they more effectively stimulate the immune response, and have other advantageous properties, such as the

[0530] immunostimulatory bacteria provided herein. Further and alternative modifications of various bacteria have been described in published International PCT Application Publication No. WO 2019 / 014398 and U.S. Publication No. 2019 / 0017050 Al. The bacteria described in each of these publications, also described herein, can be modified as described herein to further improve the immunostimulatory and tumor targeting properties.

[0531] The bacteria can be modified as described herein to have reduced

[0532] inflammatory effects, and thus, to be less toxic. As a result, for example, higher dosages can be administered. Any of these strains of Salmonella , as well as other species of bacteria, known to those of skill in the art and / or listed above and herein, can be modified as described herein, such as by introducing adenosine auxotrophy. Exemplary are the S. typhimurium species described herein.

[0533] The bacterial strains provided herein are engineered to deliver therapeutic molecules / products. The strains herein deliver immunostimulatory proteins, including modified gain-of-function variants of cytosolic DNA / RNA sensors that can constitutively evoke / induce type I IFN expression, and other immunostimulatory proteins, such as cytokines, that promote an anti-tumor immune response in the tumor microenvironment. The strains also can include genomic modifications that reduce pyroptosis of phagocytic cells, thereby providing for a more robust immune response, and / or reduce or eliminate the ability to infect / invade epithelial cells, but retain the ability to infect / invade phagocytic cells, so that they accumulate more effectively in tumors and in tumor-resident immune cells. The bacterial strains encode therapeutic products. Accumulation in tumor-resident immune cells allows the encoded therapeutic products to be expressed and secreted into the tumor microenvironment, increasing the therapeutic efficacy.

[0534] 4. Enhancements of Immunostimulatory Bacteria to Increase Therapeutic Index and Expression in Tumor-Resident Immune Cells

[0535] Provided herein are enhancements to immunostimulatory bacteria that reduce toxicity and improve the anti-tumor activity. Exemplary of such enhancements are the following. They are described with respect to Salmonella , particularly S.

[0536] typhimurium ; it is understood that the skilled person can effect similar enhancements in other bacterial species and other Salmonella strains

[0537] a. asd Gene Deletion

[0538] The asd gene in bacteria encodes an aspartate-semialdehyde dehydrogenase. asd mutants of S. typhimurium have an obligate requirement for diaminopimelic acid (DAP) which is required for cell wall synthesis and will undergo lysis in environments deprived of DAP. This DAP auxotrophy can be used for plasmid selection and maintenance of plasmid stability in vivo , without the use of antibiotics, when the asd gene is complemented in trans on a plasmid. Non-antibiotic-based plasmid selection systems are advantageous and allow for: 1) the use of administered antibiotics as a rapid clearance mechanism in the event of adverse symptoms, and 2) antibiotic-free scale up of production, where such use is commonly avoided. The asd gene complementation system provides for such selection (Galan et al. (1990) Gene 28:29-35). The use of the asd gene complementation system to maintain plasmids in the tumor microenvironment is expected to increase the potency of S. typhimurium engineered to deliver plasmids encoding genes, and therapeutic products / proteins, such as the STING proteins, and other immunostimulatory proteins, as described herein.

[0539] An alternative use for an asd mutant of S. typhimurium is to exploit the DAP auxotrophy to produce an autolytic (or suicidal) strain for delivery of macromolecules to infected cells without the ability to persistently colonize host tumors. Deletion of the asd gene makes the bacteria auxotrophic for DAP when grown in vitro or in vivo. An example described herein (see, e.g., Example 3), provides an asd deletion strain that is auxotrophic for DAP and contains a plasmid that encodes a therapeutic product, and that does not contain an asd complementing gene, resulting in a strain that is defective for replication in vivo. This strain is propagated in vitro in the presence of DAP and grows normally, and then is administered as an

[0540] immunotherapeutic agent to a mammalian host, where DAP is not present. The suicidal strain is able to invade host cells but is not be able to replicate due to the absence of DAP in mammalian tissues, lysing automatically and delivering its cytosolic contents (e.g, plasmids or proteins).

[0541] In examples provided herein, an asd gene deleted strain of VNP20009 was further modified to express an LLO protein lacking its endogenous periplasmic secretion signal sequence (cytoLLO), causing it to accumulate in the cytoplasm of the Salmonella. LLO is a cholesterol-dependent pore forming hemolysin from Listeria monocytogenes that mediates phagosomal escape of bacteria. When the autolytic strain is introduced into tumor bearing mice, the bacteria are taken up by phagocytic immune cells and enter the Salmonella containing vacuole (SCY). In this environment, the lack of DAP will prevent bacterial replication, and result in autolysis of the bacteria in the SCV. Lysis of the suicidal strain will then allow for release of the plasmid and the accumulated LLO that will form pores in the cholesterol- containing SVC membrane, and allow for delivery of the plasmid into the cytosol of the host cell. Here, gene products encoded on the plasmid, that are under control of a eukaryotic promoter, can be expressed by the host cell machinery

[0542] b. Adenosine Auxotrophy

[0543] Metabolites derived from the tryptophan and ATP / adenosine pathways are major drivers in forming an immunosuppressive environment within the tumor.

[0544] Adenosine, which exists in the free form inside and outside of cells, is an effector of immune function. Adenosine decreases T-cell receptor induced activation ofNF-kB, and inhibits IL-2, IL-4, and IFN-g. Adenosine decreases T-cell cytotoxicity, increases T-cell anergy, and increases T-cell differentiation to Foxp3+or Lag-3+regulatory T- cells (T-regs). In NK cells, adenosine decreases IFN-g production, and suppresses NK cell cytotoxicity. Adenosine blocks neutrophil adhesion and extravasation, decreases phagocytosis, and attenuates levels of superoxide and nitric oxide. Adenosine also decreases the expression of TNF-a, IL-12, and MIP-la (CCL3) on macrophages, attenuates MHC Class II expression, and increases levels of IL-10 and IL-6.

[0545] Adenosine immunomodulation activity occurs after its release into the extracellular space of the tumor and activation of adenosine receptors (ADRs) on the surfaces of target immune cells, cancer cells or endothelial cells. The high adenosine levels in the tumor microenvironment result in local immunosuppression, which limits the capacity of the immune system to eliminate cancer cells.

[0546] Extracellular adenosine is produced by the sequential activities of membrane associated ectoenzymes, CD39 and CD73, which are expressed on tumor stromal cells, together producing adenosine by phosphohydrolysis of ATP or ADP produced from dead or dying cells. CD39 converts extracellular ATP (or ADP) to 5'AMP, which is converted to adenosine by CD73. Expression of CD39 and CD73 on endothelial cells is increased under the hypoxic conditions of the tumor

[0547] microenvironment, thereby increasing levels of adenosine. Tumor hypoxia can result from inadequate blood supply and disorganized tumor vasculature, impairing delivery of oxygen (Carroll and Ashcroft (2005 ) Expert. Rev. Mol. Med. 7(6): 1-16). Hypoxia, which occurs in the tumor microenvironment, also inhibits adenylate kinase (AK), which converts adenosine to AMP, leading to very high extracellular adenosine concentrations. The extracellular concentration of adenosine in the hypoxic tumor microenvironment has been measured at 10-100 mM, which is up to about 100-1000 fold higher than the typical extracellular adenosine concentration of approximately 0.1 mM (Vaupel et al. (2016) Adv Exp Med Biol . 876: 177-183; Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Since hypoxic regions in tumors are distal from microvessels, the local concentration of adenosine in some regions of the tumor can be higher than others.

[0548] To direct effects to inhibit the immune system, adenosine also can control cancer cell growth and dissemination by effects on cancer cell proliferation, apoptosis and angiogenesis. For example, adenosine can promote angiogenesis, primarily through the stimulation of A2Aand A2Breceptors. Stimulation of the receptors on endothelial cells can regulate the expression of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on endothelial cells, maintain vascular integrity, and promote vessel growth (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857).

[0549] Activation of one or more of A2A, A2Bor A3on various cells by adenosine can stimulate the production of the pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), or angiopoietin 2 (Antonioli etal. (2013) Nat. Rev. Can. 13:842-857).

[0550] Adenosine also can directly regulate tumor cell proliferation, apoptosis and metastasis through interaction with receptors on cancer cells. For example, studies have shown that the activation of Ai and A2Areceptors promote tumor cell

[0551] proliferation in some breast cancer cell lines, and activation of A2Breceptors have cancer growth-promoting properties in colon carcinoma cells (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Adenosine also can trigger apoptosis of cancer cells, and various studies have correlated this activity to activation of the extrinsic apoptotic pathway through A3or the intrinsic apoptotic pathway through A2Aand A2B(Antonioli et al. (2013)). Adenosine can promote tumor cell migration and metastasis, by increasing cell motility, adhesion to the extracellular matrix, and expression of cell attachment proteins and receptors to promote cell movement and motility. The extracellular release of adenosine triphosphate (ATP) occurs from stimulated immune cells and damaged, dying or stressed cells. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome, when stimulated by this extracellular release of ATP, activates caspase-1 and results in the secretion of the cytokines IL-Ib and IL-18, which in turn activate innate and adaptive immune responses (Stagg and Smyth (2010) Oncogene 29:5346-5358). ATP is catabolized into adenosine by the enzymes CD39 and CD73. Activated adenosine acts as a highly immunosuppressive metabolite via a negative-feedback mechanism and has a pleiotropic effect against multiple immune cell types in the hypoxic tumor microenvironment (Stagg and Smyth (2010) Oncogene 29:5346-5358). Adenosine receptors A2Aand A2Bare expressed on a variety of immune cells and are stimulated by adenosine to promote cAMP- mediated signaling changes, resulting in immunosuppressive phenotypes of T-cells, B-cells, NK cells, dendritic cells, mast cells, macrophages, neutrophils, and NKT cells. As a result of this, adenosine levels can accumulate to over one hundred times their normal concentration in pathological tissues, such as solid tumors, which have been shown to overexpress ecto-nucleotidases, such as CD73. Adenosine has also been shown to promote tumor angiogenesis and development. An engineered bacterium that is auxotrophic for adenosine would thus exhibit enhanced tumor targeting and colonization.

[0552] Immunostimulatory bacteria, such as Salmonella typhi , can be made auxotrophic for adenosine by deletion of the tsx gene (Bucarey et al. (2005) Infection and Immunity 73(10):6210-6219) or by deletion of purD (Husseiny (2005) Infection and Immunity 73(3):1598-1605). In the Gram negative bacteria Xanthomonas oryzae, a purD gene knockout strain was shown to be auxotrophic for adenosine (Park et al. (2007) FEMS Microbiol Lett 276:55-59). As exemplified herein, S. typhimurium strain VNP20009, is auxotrophic for adenosine due to its purl deletion, hence, further modification to render it auxotrophic for adenosine is not required. Hence, embodiments of the immunostimulatory bacterial strains, as provided herein, are auxotrophic for adenosine. Such auxotrophic bacteria selectively replicate in the tumor microenvironment, further increasing accumulation and replication of the administered bacteria in tumors, and decreasing the levels of adenosine in and around tumors, thereby reducing or eliminating the immunosuppression caused by accumulation of adenosine. Exemplary of such bacteria, provided herein, is a modified strain of S. typhimurium containing purljmsbB mutations to provide adenosine auxotrophy. Other genomic mutations also can be included to impart other advantageous properties to the bacteria, as discussed herein.

[0553] c. Flagellin Deficient Strains

[0554] Flagella are organelles on the surface of bacteria that are composed of a long filament attached via a hook to a rotary motor that can rotate in a clockwise or counterclockwise manner to provide a means for locomotion. Flagella in S.

[0555] typhimurium are important for chemotaxis and for establishing an infection via the oral route, due to the ability to mediate motility across the mucous layer in the gastrointestinal tract. While flagella have been demonstrated to be required for chemotaxis to and colonization of tumor cylindroids 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), flagella are not required for tumor colonization in animals when the bacteria are administered intravenously (Stritzker el al. (2010) International Journal of Medical Microbiology 300:449-456). Each flagellar filament is composed of tens of thousands of flagellin subunits. The S. typhimurium chromosome contains two genes ,fliC and fljB, that encode antigenically distinct flagellin monomers. Mutants defective for both fliC and fljB are nonmotile and avirulent when administered via the oral route of infection, but maintain virulence when administered parenterally.

[0556] Flagellin is a major pro-inflammatory determinant of Salmonella (Zeng et al. (2003) J. Immunol. 171 :3668-3674), and is directly recognized by TLR5 on the surface of cells, and by NLRC4 in the cytosol (Lightfield et al. (2008) Nat Immunol. 9(10): 1171-1178). Both pathways lead to pro-inflammatory responses resulting in the secretion of cytokines, including IL-Ib, IL-18, TNF-a and IL-6. Attempts have been made to make Salmonella- based cancer immunotherapy more potent by increasing the pro-inflammatory response to flagellin by engineering the bacteria to secrete Vibrio vulnificus flagellin B, which induces greater inflammation than flagellin encoded by fliC and fljB (Zheng et al. (2017) Sci. Transl. Med. 9(376):eaak9537).

[0557] Provided are immunostimulatory bacteria, such as the Salmonella species S. typhimurium , engineered to lack both flagellin subunits fliC and fljB , to reduce pro- inflammatory signaling. For example, as shown herein, a Salmonella strain lacking msbB , which results in reduced TNF-alpha induction, is combined with fliC and fljB knockouts. The resulting Salmonella strain has a combined reduction in TNF-alpha induction and reduction in TLR5 recognition. These modifications, msbB , fliC and fljR, can be combined with a bacterial plasmid, optionally containing CpGs, and also a cDNA expression cassette to provide expression of a heterologous protein(s) under the control of a eukaryotic promoter, such as, for example, STING pathway gain-of- function protein variants, immunostimulatory cytokines, and / or also inhibitory RNAi molecule(s). The resulting bacteria have reduced proinflammatory signaling, and robust anti -turn or activity.

[0558] Elimination of the flagella imparts additional advantageous properties that increase the therapeutic index of the bacteria. For example, as shown herein (see, e.g ., Example 6), elimination of the flagella (i.e., in Salmonella, fliC / fljB j, decreases pyroptosis in murine macrophages and in human monocytes, results in an inability to infect epithelial cells, and restricts uptake of the bacteria to tumor-resident

[0559] immune / myeloid cells.

[0560] As described below and elsewhere herein, deletion of the flagella can be combined with one or more other genomic modifications that impart advantageous properties that improve the therapeutic index of the bacteria, including, for example, a set, msbB , purl, pagP , csgD , adrA , and / or other modifications as described herein.

[0561] Such modified bacteria can be transformed with a plasmid encoding therapeutic products that increase the anti -turn or immune response in the subject, including, for example, cytosolic DNA / RNA sensors and gain-of-function mutants thereof, as well as immunostimulatory proteins, such as cytokines.

[0562] For example, as provided herein, a fliC and fljB double mutant was constructed in the asd deleted strain of S. typhimurium strain VNP20009. VNP20009, which is attenuated for virulence by disruption of purl / purM , also was engineered to contain an msbB deletion, that results in production of a lipid A subunit of LPS that is less toxigenic than wild-type lipid A. This results in reduced TNF-a production in a mouse model after intravenous administration, compared to strains with wild-type lipid A. Also, a fliC and fljB double mutant was constructed on a wild-type strain of S. typhimurium containing the asd, purl / purM and msbB deletions. The resulting strains are exemplary of strains that are attenuated for bacterial inflammation by modification of lipid A to reduce TLR2 / 4 signaling, and deletion of the flagellin subunits to reduce TLR5 recognition and inflammasome induction. Deletion of the flagellin subunits combined with modification of the LPS allows for greater tolerability in the host, and directs the immunostimulatory response towards production of immunostimulatory proteins and / or delivery of RNA interference against desired targets in the TME, which elicits an anti-tumor response and promotes an adaptive immune response to the tumor.

[0563] d. Deletion of Genes in the LPS Biosynthetic Pathway

[0564] The lipopolysaccharide (LPS) of Gram-negative bacteria is the major component of the outer leaflet of the bacterial membrane. It is composed of three major parts, lipid A, a nonrepeating core oligosaccharide, and the O antigen (or O polysaccharide). O antigen is the outermost portion on LPS and serves as a protective layer against bacterial permeability, however, the sugar composition of O antigen varies widely between strains. The lipid A and core oligosaccharide vary less, and are more typically conserved within strains of the same species. Lipid A is the portion of LPS that contains endotoxin activity. It is typically a disaccharide decorated with multiple fatty acids. These hydrophobic fatty acid chains anchor 5 the LPS into the bacterial membrane, and the rest of the LPS projects from the cell surface.

[0565] The lipid A domain is responsible for much of the toxicity of Gram-negative bacteria. Typically, LPS in the blood is recognized as a significant pathogen associated molecular pattern (PAMP), and induces a profound pro-inflammatory response. LPS is the ligand for a membrane-bound receptor complex comprising CD14, MD2 and TLR4. TLR4 is a transmembrane protein that can signal through the MyD88 and TRIF pathways to stimulate the NFKB pathway and result in the production of pro-inflammatory cytokines, such as TNF-a and IL-Ib, the result of which can be endotoxic shock, which can be fatal. LPS in the cytosol of mammalian cells can bind directly to the CARD domains of caspases 4, 5, and 11, leading to autoactivation and pyroptotic cell death (Hagar et al. (2015) Cell Research 25: 149- 150).

[0566] The composition of lipid A and the toxigenicity of lipid A variants is well documented. For example, a monophosphorylated lipid A is much less inflammatory than lipid A with multiple phosphate groups. The number and length of the acyl chains on lipid A also can have a profound impact on the degree of toxicity.

[0567] Canonical lipid A from E. coli has six acyl chains, and this hexa-acylation is potently toxic. S. typhimurium lipid A is similar to that of E. coli ; it is a glucosamine disaccharide that carries four primary and two secondary hydroxyacyl chains (Raetz and Whitfield (2002 ) Annu. Rev. Biochem. 71 :635-700). As described above, msbR mutants of S. typhimurium cannot undergo the terminal myristoylation of LPS, and produce predominantly penta-acylated lipid A that is significantly less toxic than hexa-acylated lipid A. The modification of lipid A with palmitate is catalyzed by palmitoyl transferase (PagP). Transcription of the pagP gene is under control of the phoP / phoQ system, which is activated by low concentrations of magnesium, e.g., inside the SCV. Thus, the acyl content of S. typhimurium is variable, and with wild- type bacteria, it can be hexa- or penta-acylated. The ability of S. typhimurium to palmitate its lipid A increases resistance to antimicrobial peptides that are secreted into phagolysozomes.

[0568] In wild-type S. typhimurium , expression of pagP results in a lipid A that is hepta-acylated. In an msbB mutant (in which the terminal acyl chain of the lipid A cannot be added), the induction of pagP results in a hexa-acylated LPS (Kong et al. (2011) Infection and Immunity 79(12):5027-5038). Hexa-acylated LPS has been shown to be the most pro-inflammatory. While other groups have sought to exploit this pro-inflammatory signal, for example, by deletion of pagP to allow only hexa- acylated LPS to be produced (Feigner et al. (2016) Gut Microbes 7(2): 171-177; Feigner et al. (2018) Oncoimmunology 7(2): el382791), this can lead to poor tolerability, due to the TNF-a-mediated pro-inflammatory nature of the LPS and paradoxically less adaptive immunity (Kocijancic et al. (2017) Oncotarget

[0569] 8(30):49988-50001).

[0570] LPS is a potent TLR4 agonist that induces TNF-a and IL-6. The dose-limiting toxicities in the I.V. VNP20009 clinical trial (Toso et al. (2002) J. Clin. Oncol.

[0571] 20(1): 142-152) at 1E9 CFU / m2were cytokine mediated (fever hypotension), with TNF-a levels > 100,000 pg / ml and IL-6 levels > 10,000 pg / ml in serum at 2 hours. Despite the msbB deletion in VNP20009 and its reduced pyrogenicity, the LPS still can be toxic at high doses, possibly due to the presence of hexa-acylated LPS. Thus, a pagP / msbB strain is better tolerated at higher doses, as it cannot produce hexa- acylated LPS, and will allow for dosing in humans at or above 1E9 CFU / m2. Higher dosing can lead to increased tumor colonization, enhancing the therapeutic efficacy of the immunostimulatory bacteria.

[0572] Herein, Salmonella bacteria, such as S. typhimurium , are engineered to lack both flagellin subunits fliC and fljB , to reduce pro-inflammatory signaling. For example, as shown herein, a Salmonella strain lacking msbB , which results in reduced TNF-alpha induction, is combined with fliC and fljB knockouts. This results in a Salmonella strain that has a combined reduction in TNF-alpha induction and reduction in TLR5 recognition. These modifications can be combined with pagP and other genomic modifications discussed herein, and the resulting bacterial strain can be transformed with an immunostimulatory plasmid (encoding immunostimulatory protein(s)), optionally containing CpGs. The resulting bacteria have reduced pro- inflammatory signaling, but robust anti-tumor activity.

[0573] Exemplified herein, are live attenuated Salmonella strains, such as the exemplary strain of S. typhimurium , that only can produce penta-acylated LPS, that contain a deletion of the msbB gene (that prevents the terminal myristoylation of lipid A, as described above), and that further are modified by deletion of pagP (preventing palmitoylation). A strain modified to produce penta-acylated LPS will allow for lower levels of pro-inflammatory cytokines, improved stability in the blood and resistance to complement fixation, increased sensitivity to antimicrobial peptides, enhanced tolerability, and increased anti-tumor immunity when further modified to express a therapeutic product(s), such as heterologous immune-stimulatory proteins and / or interfering RNAs against, for example, immune checkpoints.

[0574] As provided herein, for example, a pagP mutant also can be constructed on an asd, msbB , purl / purM and fliC fljB deleted strain of S. typhimurium VNP20009, or other strains as described herein, or wild-type S. typhimurium. The resulting strains are exemplary of strains that are attenuated for bacterial inflammation by modification of lipid A to reduce TLR2 / 4 signaling, and deletion of the flagellin subunits to reduce TLR5 recognition and inflammasome induction, and deletion of pagP to produce penta-acylated LPS. Deletion of the flagellin subunits combined with modification of the LPS allows for greater tolerability in the host, and greater stability in the blood and resistance to complement fixation, providing for improved trafficking to the tumor site, in order to direct the immunostimulatory response towards production of any gene product, such as immunostimulatory proteins, and / or delivery of RNA interference against desired targets in the TME to elicit an anti-tumor response and promote an adaptive immune response to the tumor.

[0575] e. Deletions in Genes Required for Biofilm Formation

[0576] Bacteria and fungi are capable of forming multicellular structures called biofilms. Bacterial biofilms are encased within a mixture of secreted and cell wall- associated polysaccharides, glycoproteins, and glycolipids, as well as extracellular DNA, known collectively as extracellular polymeric substances. These extracellular polymeric substances protect the bacteria from multiple insults, such as cleaning agents, antibiotics, and antimicrobial peptides. Bacterial biofilms allow for colonization of surfaces, and are a cause of significant infection of prosthetics, such as injection ports and catheters. Biofilms also can form in tissues during the course of an infection, which leads to increases in the duration of bacterial persistence and shedding, and limits the effectiveness of antibiotic therapies. Chronic persistence of bacteria in biofilms is associated with increased turn ori genesis, for example in S. typhi infection of the gall bladder (Di Domenico et al. (2017) Int. J Mol. Sci. 18: 1887).

[0577] S. typhimurium biofilm formation is regulated by CsgD. CsgD activates the csgBAC operon, which results in increased production of the curb fimbrial subunits CsgA and CsgB (Zakikhani et al. (2010) Molecular Microbiology 77(3):771-786). CsgA is recognized as a PAMP by TLR2 and induces production of IL-8 from human macrophages (Tukel et al. (2005) Molecular Microbiology 58(l):289-304). Further, CsgD indirectly increases cellulose production by activating the adrA gene that encodes for di-guanylate cyclase. The small molecule cyclic di-guanosine

[0578] monophosphate (c-di-GMP) generated by AdrA is a ubiquitous secondary messenger found in almost all bacterial species. The AdrA-mediated increase in c-di-GMP enhances expression of the cellulose synthetase gene bcsA , which in turn increases cellulose production via stimulation of the bcsABZC and bcsEFG operons. Reduction in the capability of immunostimulatory bacteria, such as S. typhimurium , to form biofilms can be achieved through deletion of genes involved in biofilm formation, such as, for example, csgD , csgA, csgB, adrA, bcsA, bcsB , bcsZ , bcsE, bcsF , bcsG, dsbA or r / s / rii (Anwar et al. (2014) PLoS One 9(8):el06095).

[0579] S. typhimurium can form biofilms in solid tumors as protection against phagocytosis by host immune cells. Salmonella mutants that cannot form biofilms are taken up more rapidly by host phagocytic cells and are cleared from infected tumors (Crull et al. (2011) Cellular Microbiology 13(8): 1223-1233). This increase in intracellular localization within phagocytic cells can reduce the persistence of extracellular bacteria, and enhance the effectiveness of plasmid delivery, expression and release of encoded therapeutic products into the TME, as well as gene knockdown by RNA interference, as described herein. Immunostimulatory bacteria engineered to reduce biofilm formation, will increase clearance rate from tumors / tissues and therefore increase the tolerability of the therapy, and will prevent colonization of prosthetics in patients, thereby increasing the therapeutic benefit of these strains. Adenosine mimetics can inhibit S. typhimurium biofilm formation, indicating that the high adenosine concentration in the tumor microenvironment can contribute to tumor- associated biofilm formation (Koopman et al. (2015) Antimicrob Agents Chemother 59:76 -84). As provided herein, live attenuated strains of bacteria, such as S.

[0580] typhimurium , that contain a purl disruption (and therefore, colonize adenosine-rich tumors), and are also prevented from forming biofilms by deletion of one or more genes required for biofilm formation, are engineered to deliver plasmids encoding therapeutic products, such as cytosolic DNA / RNA sensors and gain-of-function variants thereof, and other immunostimulatory proteins, such as cytokines, and interfering RNA, to stimulate a robust anti-tumor immune response.

[0581] The adrA gene encodes a di-guanylate cyclase that produces c-di-GMP, which is required for S. typhimurium biofilm formation. c-di-GMP binds to and is an agonist for the host cytosolic protein STING. Immunostimulatory bacteria that are reduced in c-di-GMP production via the deletion of adrA is counterintuitive, but bacterial mutants, such as S. typhimurium mutants, that are unable to form biofilms (including adrA mutants), have demonstrated reduced therapeutic potential in mouse tumor models (Crull et al. (2011) Cellular Microbiology 13(8): 1223-1233). Several human alleles of STING are refractory to binding bacterially-produced 3’ 3’ CDNs (Corrales et al. (2015) Cell Reports 11 : 1022-1023). As described herein, bacterial strains, such as S. typhimurium strains, that are engineered to be adenosine auxotrophic, and are reduced in their ability to induce pro- inflammatory cytokines by modification of the LPS and / or deletion of flagellin, and / or deletion of genes required for biofilm formation, are further modified to deliver interfering RNAs, and other therapeutic, anti-cancer products, such as immunostimulatory proteins, including cytosolic DNA / RNA sensors and gain-of- function variants thereof ( e.g ., STING and others) and cytokines, to promote robust anti-tumor immune responses.

[0582] f. Salmonella Engineered to Escape the Salmonella Containing Vacuole (SCV)

[0583] Salmonella , such as S. typhimurium , are intracellular pathogens that replicate primarily in a membrane bound compartment called a Salmonella containing vacuole (SCV). In some epithelial cell lines, and at a low frequency, S. typhimurium have been shown to escape into the cytosol where they can replicate. Salmonella engineered to escape the SCV with higher efficiency will be more efficient at delivering

[0584] macromolecules, such as plasmids, to the host cell cytosol, as the lipid bilayer of the SCV is a potential barrier. Plasmid release into the host cytosol allows for the expression of therapeutic products encoded on the plasmid, that are under the control of host-recognized regulatory signals, such as eukaryotic promoters, increasing the efficiency of production and delivery of the therapeutic products to the TME, particularly when the bacteria are phagocytosed by tumor-resident immune cells, and improving the therapeutic index of the bacteria.

[0585] Provided herein are Salmonella strains and methods that have enhanced frequency of SCV escape. As discussed below and elsewhere herein, this is achieved by deletion of genes required for Salmonella induced filament (SIF) formation. These mutants have an increased frequency of SCV escape and can replicate in the cytosol of the host cell. For example, enhanced plasmid delivery using a sifA mutant of S. typhimurium has been demonstrated. The sifA gene encodes an SPI-2 T3SS-2 secreted effector protein that mimics or activates a 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 the escape of S. typhimurium by prevention of SIF formation releases live bacteria into the cytosol, where they can replicate.

[0586] Another method to enhance S. typhimurium escape from the SCV and increase the delivery of macromolecules such as plasmids to the cytosol, is the expression of a heterologous hemolysin that results in pore formation in, or rupture of, the SCV membrane. One such hemolysin is the Listeriolysin O protein (LLO) from Listeria monocytogenes , which is encoded by the hlyA gene. LLO is a cholesterol- dependent pore-forming cytolysin that is secreted from L. monocytogenes and is primarily responsible for phagosomal escape and entry into the cytosol of host cells. Secretion of LLO from S. typhimurium can result in bacterial escape and lead to replication in the cytosol. To prevent intact S. typhimurium from escaping the SCV and replicating in the cytosol, the nucleotides encoding the secretion signal sequence can be removed from the gene, producing cytoLLO. In this manner, the active LLO is contained within the cytoplasm of the S. typhimurium and LLO is only released when the bacteria undergo lysis (for example, due to the lack of intracellular DAP in an asct strain). Bacterial lysis in the SCV allows for the release of the plasmid and

[0587] accumulated cytoLLO, which will form pores in the SCV, allowing for the delivery of the plasmid into the host cell cytosol, where the encoded therapeutic product(s) can be expressed.

[0588] As provided herein, Salmonella strains, such as the S. typhimurium strain

[0589] VNP20009, engineered to express cytoLLO to enhance delivery of plasmids for expression of therapeutic products, such as STING proteins and variants thereof, and other immunostimulatory proteins, can increase the therapeutic potency of the immunostimulatory bacteria. This is advantageous, where the bacteria are engineered to accumulate in tumor-resident immune cells, as herein, whereby the expressed therapeutic products are released directly into the tumor microenvironment.

[0590] g. Deletions of SPI-1 and SPI-2 Genes and / or Other Genes to Eliminate the Ability of the Bacteria to Infect Epithelial Cells, Including Deletion of Flagella

[0591] As described above, pathogenesis, in certain bacterial species, including

[0592] Salmonella species, such as S. typhimurium , involves a cluster of genes referred to as Salmonella pathogenicity islands (SPIs). S. typhimurium is an intracellular pathogen that is rapidly taken up by myeloid cells, such as macrophages, or it can induce its own uptake in non-phagocytic cells, such as epithelial cells. Once inside cells, it can replicate within a Salmonella containing vacuole (SCV) and can also escape into the cytosol of some epithelial cells. The two best characterized pathogenicity islands are SPI-1, which is responsible for mediating bacterial invasion of non-phagocytic cells, such as epithelial cells, and SPI-2, which is required for replication within the SCV (Agbor and McCormick (2011) Cell Microbiol. 13(12): 1858—1869). SPI-1 and SPI-2 encode macromolecular structures called type three secretion systems (T3SS) that can translocate effector proteins across the host membrane (Galan and Wolf-Watz (2006) Nature 444:567-573).

[0593] i. Salmonella Pathogenicity Island 1 (SPI-1)

[0594] SPI-l-Dependent Host Cell Invasion

[0595] The invasion-associated Salmonella pathogenicity island 1 (SPI-1), including the type 3 secretion system (T3SS), is responsible for the translocation of effector proteins into the cytosol of host cells, causing actin rearrangements that lead to the uptake of Salmonella. Salmonella invades non-phagocytic intestinal epithelial cells using a type 3 secretion system (T3SS) encoded by SPI-1, which forms a needle-like structure that injects effector proteins directly into the cytosol of host cells. These effector proteins lead to rearrangement of the eukaryotic cell cytoskeleton to facilitate invasion of the intestinal epithelium, and also induce proinflammatory cytokines. The SPI-1 locus includes 39 genes that encode components of this invasion system (see, e.g., Kimbrough et al. (2002) Microbes Infect. 4(l):75-82). SPI-1 genes comprise a number of operons, including: sitABCD , sprB , avrA, hilC , orgABC, prgKJIH, hill), hi l A , iagB, sptP , sicC, iacP, sipADCB , sic A , spaOPQRS, invFGEABCIJ, and invH. The operons and genes and their functions are described and depicted, for example, in Kimbrough et al. ((2002) Microbes Infect. 4(l):75-82). SPI-1 genes include, but are not limited to: avrA , hilA , hill), invA, invB, invC, invE , invF, invG, invH , invl , inv.J, iacP, iagB, spaO, spaP, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgl, prgj, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and spill T3SSs are complexes that play a large role in the infectivity of Gram-negative bacteria, by injecting bacterial protein effectors directly into host cells in an ATP- dependent manner. T3SS complexes cross the inner and outer bacterial membranes and create a pore in eukaryotic cell membranes upon contact with a host cell. They consist of an exportation apparatus, a needle complex and a translocon at the tip of the needle (see, e.g., Kimbrough et al. (2002) Microbes Infect. 4(l):75-82). The needle complex includes the needle protein Prgl, a basal body, which anchors the complex in the bacterial membranes and consists of the proteins PrgH, PrgK and InvG, and other proteins, including InvH, PrgJ (rod protein) and InvJ. The translocon, which forms the pore in the host cell, is a complex of the proteins SipB, SipC and SipD. The exportation apparatus, which allows for the translocation of the effector proteins, is comprised of the proteins SpaP, SpaQ, SpaR, SpaS, InvA, InvC and OrgB. A cytoplasmic sorting platform, which establishes the specific order of protein secretion, is composed of the proteins SpaO, OrgA and OrgB (see, e.g. , Manon et al. (2012), Salmonella , Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0596] The effectors translocated into the host cell by T3SS-1 (T3SS of SPI-1) include SipA, SipC, SopB, SopD, SopE, SopE2 and SptP, which are essential for cell invasion. For example, S. typhimurium sip A mutants exhibit 60-80% decreased invasion, sipC deletion results in a 95% decrease in invasion, and sopB deletion results in a 50% decrease in invasion (see, e.g., Manon et al. (2012), Salmonella , Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364). Other effectors include AvrA, which controls Salmonella- induced inflammation. Chaperones, which bind secreted proteins and maintain them in a conformation that is competent for secretion, include SicA, InvB and SicP. Transcriptional regulators include HilA, HilD, InvF, SirC and SprB. Unclassified T3SS SPI-1 proteins, which have various functions in type III secretion, include OrgC, InvE, Invl, IacP and IagB (see, e.g. , Kimbrough et al. (2002) Microbes Infect. 4(l):75-82).

[0597] The SPI-1 T3SS is essential for crossing the gut epithelial layer, but is dispensable for infection when bacteria are injected parenterally. The injection of some proteins (e.g, Prgl and PrgJ) and the needle complex itself also can induce inflammasome activation and pyroptosis of phagocytic cells. This pro-inflammatory cell death can limit the initiation of a robust adaptive immune response by directly inducing the death of antigen-presenting cells (APCs), as well as modifying the cytokine milieu to prevent the generation of memory T-cells. Thus, the inactivation of SPI-1 -dependent invasion, through the inactivation or knockout of one or more genes involved in SPI-1, eliminates the ability of the bacteria to infect epithelial cells, but does not affect their ability to infect or invade phagocytic cells, including phagocytic immune cells, such as tumor-associated myeloid cells. These SPI-1 genes include, but are not limited to, one more of: avrA, hi I A , hill), invA, invB, invC, invE , invF, invG, invH , invl , invJ , iacP, iagB, spaO, spaP , spaQ, spaR, spaS, orgA, orgB , orgC, prgPI , prgl,prgj , prgK , .v / cri , s / c , sip A, sipB, sipC , szpZ), sz>C, sqp5, sop / ), sopE , sopE2, sprB, and .s / ; / / J.

[0598] SPI-l-Independent Host Cell Invasion

[0599] Salmonella mutants lacking the T3SS-1 have been shown to invade numerous cell lines / types, by a T3SS-1 independent invasion mechanism, involving several proteins, including the invasins Rck, PagN and HlyE. The rck operon contains 6 open reading frames: pefl, srgl), srgA , srgB, rck and srgC. pefl encodes a transcriptional regulator of the pef operon, which is involved in the biosynthesis of the Pef fimbriae. These fimbriae are involved in biofilm formation, adhesion to murine small intestine and fluid accumulation in the infant mouse. SrgA oxidizes the disulfide bond of PefA, the major structural subunit of the Pef fimbriae. srgD encodes a putative

[0600] transcriptional regulator; SrgD together with Pefl work to induce a synergistic negative regulation of flagellar gene expression. srgB encodes a putative outer membrane protein, and srgC encodes a putative transcriptional regulator (see, e.g. , Manon et al. (2012), Salmonella , Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0601] Rck is a 17 kDa outer membrane protein encoded by the large virulence plasmid of S. Enteritidis and S. Typhimurium, that induces adhesion to and invasion of epithelial cells, and confers a high level of resistance to neutralization by complement, by preventing the formation of the membrane attack complex. An rck mutant exhibited a 2-3 fold decrease in epithelial cell invasion compared to the wild- type strain, while Rck overexpression leads to increased invasion. Rck induces cell entry by a receptor-mediated process, promoting local actin remodeling and weak and closely adherent membrane extensions. Thus, Salmonella can enter cells by two distinct mechanisms: the Trigger mechanism mediated by the T3SS-1 complex, and a Zipper mechanism induced by Rck (see, e.g., Manon et al. (2012), Salmonella , Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364). The invasin PagN is an outer membrane protein that has also been shown to play a role in Salmonella invasion. pagN expression is regulated by phoP. Specific stimuli, for example, acidified macrophage phagosome environments or low Mg2+concentrations, are sensed by PhoQ, which then activates PhoP to regulate specific genes. It has been shown that the deletion of pagN in S. typhimurium results in a 3- fold decrease in the invasion of enterocytes, without altering cell adhesion. Although the PagN-mediated entry mechanism is not fully understood, it has been shown that actin polymerization is required for invasion. Studies have shown that PagN is required for Salmonella survival in BALB / c mice, and that a pagN mutant is less competitive for colonizing the spleen of mice than the parent strain. Because pagN is activated by PhoP, it is mostly expressed intracellularly, where the SPI-1 island encoding T3SS-1 is downregulated. It is thus possible that bacteria exiting epithelial cells or macrophages have an optimal level of PagN expression, but have low T3SS-1 expression, which can mediate subsequent interactions with other cells encountered following host cell destruction, indicating a role for PagN in Salmonella pathogenesis (see, e.g. , Manon et al. (2012), Salmonella , Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0602] hlyE shares more than 90% sequence identity with the E. coli HlyE (ClyA) hemolysin. The HlyE protein lyses epithelial cells when exported from bacterial cells via outer membrane vesicle release, and is involved in epithelial cell invasion. HlyE also is involved in the establishment of systemic Salmonella infection (see, e.g. , Manon et al. (2012), Salmonella , Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0603] As a result, elimination of the bacterium’s ability to infect epithelial cells also can be achieved by engineering the immunostimulatory bacteria herein to contain knockouts or deletions / disruptions of genes encoding proteins involved in SPI-1- independent invasion, such as one or more of the genes rck , pagN , hlyE , pefh srgD , srgA , srgB , and srgC.

[0604] The immunostimulatory bacteria provided herein include those with deletion or disruption of the MIA gene and / or other genes in the T3SS pathway. When these bacteria are administered, such as intravenously or intratumorally, infection is focused towards phagocytic cells, such as macrophages and dendritic cells, that do not require the SPI-1 T3SS for uptake. This enhances the safety profile of the immunostimulatory bacteria provided herein. It prevents off-target cell invasion and prevents fecal-oral transmission. In addition to reducing the uptake of Salmonella by non-phagocytic cells, such as epithelial cells, deletion or disruption of genes in this pathway also prolongs the longevity of the phagocytic cells, by preventing inflammasome activation and pyroptosis in macrophages, thus, inducing less cell death in human macrophages, compared to bacteria that do not contain a deletion in this pathway. For example, deletion of genes in the SPI-1 pathway (such as, for example, the needle and rod proteins) can prevent pyroptosis by preventing inflammasome activation, but maintains TLR5 signaling. This, in turn, permits prolonged secretion of encoded proteins, such as the STING proteins or other therapeutic / anti-cancer products encoded by the immunostimulatory bacteria provided herein, and permits macrophage trafficking to tumors, thus improving the efficacy of the immunostimulatory bacteria.

[0605] As described herein, provided are immunostimulatory bacteria that are modified so that they do not infect epithelial cells, but retain the ability to infect phagocytic cells, including tumor-resident immune cells, thereby effectively targeting the immunostimulatory bacteria, and the encoded therapeutic products, to the tumor microenvironment. This is achieved by deleting or knocking out any of the proteins in SPI-1, including, but not limited to, deletions of one more of: avrA, hi l A , hill), invA, invB, invC, invE , invF, invG, invH , invl , invJ , iacP, iagB, spaO, spaP , spaQ, spaR, spaS, orgA, orgB , orgC, prgH , prgl , prgj , prgK , sic A, sicP, sip A, sipB, sipC , sipD, sirC, sopB, sopD , sopE, sopE2, sprB , and sptP, as well as one or more of rck , pagN , hlyE , pefl, srgD, srgA, srgB, rck and srgC.

[0606] The immunostimulatory bacteria that do not infect epithelial cells can be further modified as described herein, to encode therapeutic products that stimulate the immune system, including, for example, products that induce type I interferon (e.g, cytosolic DNA / RNA sensors and GOF variants thereof), and also to encode immunostimulatory proteins, such as cytokines. The bacteria generally have an asd deletion to render them unable to replicate in a mammalian host. For example, provided are strains of S. typhimurium modified by deletion of one or more SPI-1 genes, and also modified by one or more of a purl deletion, an msbB deletion, and an asd deletion, and further modified by delivering plasmids encoding therapeutic products, such as proteins that stimulate the immune system, such as cytosolic DNA / RNA sensors and gain-of-function mutants thereof, that induce type I interferon, and / or immunostimulatory cytokines.

[0607] For example, bacteria with deletions of a regulatory gene ( e.g ., hilA or invF) required for expression of the SPI-1 -associated type 3 secretion system (T3SS-1), a T3SS-1 structural gene (e.g., invG or prgH ), and / or a T3SS-1 effector gene (e.g., sipA or avrA) are provided. As discussed above, this secretion system is responsible for injecting effector proteins into the cytosol of non-phagocytic host cells, such as epithelial cells, that cause the uptake of the bacteria; deletion of one or more of these genes eliminates infection / invasion of epithelial cells. Deletion of one or more of the genes, such as hilA, provides immunostimulatory bacteria that can be administered intravenously or intratumorally, resulting in infection of phagocytic cells, which do not require the SPI-1 T3SS for uptake, and also prolongs the longevity of these phagocytic cells. The hilA mutation also reduces the quantity of pro-inflammatory cytokines, increasing the tolerability of the therapy, as well as the quality of the adaptive immune response.

[0608] Additionally or alternatively, the immunostimulatory bacteria can contain knockouts or deletions in genes to inactivate products involved in SPI-1 -independent infection / invasion, such as one or more of the genes pagN , hlyE , pefl, srgD, srgA, srgB , and srgC, reducing or eliminating the bacterium’s ability to infect epithelial cells.

[0609] As described herein, genes involved in the SPI-1 pathway, and bacterial flagella, activate the inflammasome in phagocytic cells (immune cells), triggering pyroptosis. Knocking out or disrupting SPI-1 genes and genes that encode flagella, decreases or eliminates pyroptosis, and also, eliminates infection of epithelial cells, resulting in increased infection of phagocytic cells. Thus, the immunostimulatory bacteria can contain knockouts or deletions to inactivate products of genes that induce cell death of tumor-resident immune cells, such as genes that encode proteins that are directly recognized by the inflammasome; these include fljB,fliC,prgI and prgj. As shown herein (see, e.g., Example 6), elimination of the flagella (i.e., in Salmonella, fliC / fljBr), decreases pyroptosis in murine macrophages and in human monocytes, results in an inability to infect epithelial cells, and restricts uptake of the bacteria to tumor-resident immune / myeloid cells.

[0610] Hence, provided are immunostimulatory bacteria that accumulate in phagocytic cells, particularly tumor-resident immune cells, in which they express products encoded on plasmids that are controlled by eukaryotic regulatory signals, such as RNA polymerase II. Products include those that evoke immune responses, such as through pathways that increase expression of type I interferons, which increase the host immune response in the tumor microenvironment. The

[0611] immunostimulatory bacteria also can encode other products, including

[0612] immunostimulatory proteins, such as IL-2, further enhancing the immune response in the tumor microenvironment.

[0613] ii. Salmonella Pathogenicity Island 2 (SPI-2)

[0614] Salmonella also have a Salmonella pathogenicity island 2 (SPI-2), encoding another T3SS that is activated following entry of the bacterium into the host cell, and interferes with phagosome maturation, resulting in the formation of a specialized Salmonella-containing vacuole (SCV), where the Salmonella resides during intracellular survival and replication. SPI-2 T3SS effectors include SseB, SseC, SseD and SpiC, which are responsible for assembly of the F-actin coat around intracellular bacteria; this actin coat promotes fusion of the SCV with actin-containing or actin- propelled vesicles, and prevents it from fusing with unfavorable compartments. SifA is responsible for the formation of Salmonella- induced filaments (SIFs), which are tubules that connect the individual SCVs in the infected cell. SifA is essential to maintaining the integrity of the SCV, and sifA mutants are released into the cytosol of host cells. SseF and SseG are components of the SPI-2 T3SS that are involved in SCV positioning and cellular trafficking processes that direct materials required for the bacterium’s survival and replication, to the SCV. SseF and SseG also are involved in SIF formation. Other SPI-2 T3SS effectors include PipB2, SopD2, and SseJ, which are involved in SIF and SCV formation, and maintenance of vacuole integrity; SpvC, SseL, and SspHl, which are involved in host immune signaling; and SteC, SspH2, SrfH / Ssel and SpvB, which are involved in the formation of the SCV F-actin meshwork, in the migration of infected phagocytes, in the inhibition of actin polymerization, and in P-body disassembly in infected cells (Cobum et al. (2007) Clinical Microbiology Reviews 20(4):535-549; Figueira and Holden (2012)

[0615] Microbiology 158: 1147-1161).

[0616] The immunostimulatory bacteria herein can include deletions or

[0617] modifications in any of the SPI-2 T3SS genes that affect the formation or integrity of the SCV and associated structures, such as SIFs. These mutants have an increased frequency of SCV escape and can replicate in the cytosol. For example,

[0618] immunostimulatory bacteria, such as Salmonella species, engineered to escape the SCV are more efficient at delivering macromolecules, such as plasmids, to the host cell cytosol, as the lipid bilayer of the SCV is a potential barrier. Enhancing the escape of the bacteria from the SCV by prevention of SIF formation releases live bacteria into the cytosol, where they can replicate and express the encoded therapeutic products or proteins under control of the host cell machinery (i.e., under the control of eukaryotic regulatory elements, such as eukaryotic promoters). This enhances the therapeutic efficacy of the bacteria, and is achieved by deletion or mutation of genes required for Salmonella induced filament (SIF) formation, including, for example, sifA , sseJ, sseL , sopD2,pipB2 , sseF, sseG, spvB, and s / e A .

[0619] The immunostimulatory bacteria that can escape the SCV can be further modified as described herein to encode products that stimulate the immune system, including, for example, products that induce type I interferon, and also to encode cytokines. The bacteria generally have an asd deletion to render them unable to replicate in a mammalian host.

[0620] h. Endonuclease (endA) Mutations to Increase Plasmid

[0621] Delivery

[0622] The endA gene (for example, SEQ ID NO:250) encodes an endonuclease (for example, SEQ ID NO:251) that mediates degradation of double stranded DNA (dsDNA) in the periplasm of Gram negative bacteria. Most common strains of laboratory E. coli are endA , as a mutation in the endA gene allows for higher yields of plasmid DNA. This gene is conserved among species. To facilitate intact plasmid DNA delivery, the endA gene of the engineered immunostimulatory bacteria is deleted or mutated to prevent its endonuclease activity. Exemplary of such mutations is an E208K amino acid substitution (Durfee, et al. (2008) J Bacteriol. 190(7):2597- 2606) or a corresponding mutation in the species of interest. endA , including E208, is conserved among bacterial species, including Salmonella (see, e.g, SEQ ID NO:251). Thus, the E208K mutation can be used to eliminate endonuclease activity in other species, including Salmonella species. Those of skill in the art can introduce other mutations or deletions to eliminate endA activity. Effecting this mutation, or deleting or disrupting the gene to eliminate activity of the endA in the immunostimulatory bacteria herein, such as in Salmonella , increases the efficiency of intact plasmid DNA delivery, thereby increasing expression of the encoded therapeutic product(s) and enhancing anti-tumor efficacy.

[0623] i. RIG-I Binding Sequences

[0624] As discussed above, type I interferons (IFN-a, IFN-b) are the signature cytokines induced by distinct TLR-dependent and TLR-independent signaling pathways. Of the TLR-independent type I IFN pathways, one is mediated by host recognition of single- stranded (ss) and double-stranded (ds) RNA in the cytosol.

[0625] These are sensed by RNA helicases, including retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and through the IFN-b promoter stimulator 1 (IPS-1) adaptor protein-mediated phosphorylation of the IRF-3 transcription factor, leading to induction of type I IFN (Ireton and Gale (2011) Viruses 3(6):906-919). RIG-I recognizes dsRNA and ssRNA bearing 5’ -triphosphates. This moiety can directly bind RIG-I, or be synthesized from a poly(dA-dT) template by the poly DNA-dependent RNA polymerase III (Pol III) (Chiu, Y. H. 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 prevents type I IFN activation (Pebernard et al. (2004) Differentiation. 72:103-111). A RIG-I binding sequence can be included in the plasmids provided herein; this inclusion can increase

[0626] immunostimulation, by inducing type I IFN production, that increases anti-tumoral activity of the immunostimulatory bacteria herein.

[0627] j. DNase II Inhibition

[0628] Another nuclease responsible for degrading foreign and self DNA is DNase II, an endonuclease, which resides in the endosomal compartment and degrades DNA following apoptosis. Lack of DNase II ( Dnase2a in mice) results in the accumulation of endosomal DNA that escapes to the cytosol and activates cGAS / STING signaling (Lan Y. Y. et al. (2014) Cell Rep. 9(1): 180-192). DNase II-deficiency in humans presents with autoimmune type I interferonopathies. In cancer, dying tumor cells that are engulfed by tumor-resident macrophages prevent cGAS / STING activation, and potential autoimmunity, through DNase II digestion of DNA within the endosomal compartment (Ahn et al. (2018) Cancer Cell 33:862-873). Hence, embodiments of the immunostimulatory bacterial strains, as provided herein, which encode products that can inhibit DNase II in the tumor microenvironment, can provoke accumulation of endocytosed apoptotic tumor DNA in the cytosol, where it can act as a potent cGAS / STING agonist.

[0629] k. RNase H2 Inhibition

[0630] While TREX1 (three-prime repair exonuclease 1) and DNase II function to clear aberrant DNA accumulation, RNase H2 functions similarly to eliminate pathogenic accumulation of RNA:DNA hybrids in the cytosol . Deficiencies in RNase H2 also contribute to the autoimmune phenotype of Aicardi-Goutieres syndrome (Rabe, B. (2013) J Mol. Med. 91 : 1235-1240). Loss of RNase H2 and subsequent accumulation of RNA:DNA hybrids or genome-embedded ribonucleotide substrates has been shown to activate cGAS / STING signaling. (MacKenzie et al. (2016) EMBO J. Aprl5;35(8):831-44). Hence, embodiments of the immunostimulatory bacterial strains that encode products that inhibit or reduce expression of RNase H2, thereby inhibiting RNase H2, result in tumor-derived RNA:DNA hybrids and derivatives thereof, which activate cGAS / STING signaling and enhance anti-tumor immunity.

[0631] 1. Stabilin-l / CLEVER-1 Inhibition

[0632] Another molecule expressed primarily on monocytes, and involved in regulating immunity, is stabilin-1 (gene name STAB1 , also known as CLEVER- 1 , FEEL-1 ). Stabilin-1 is a type I transmembrane protein that is upregulated on endothelial cells and macrophages following inflammation, and in particular, on tumor-associated macrophages (Kzhyshkowska et al. (2006) J. Cell. Mol. Med.

[0633] 10(3):635-649). Upon inflammatory activation, stabilin-1 acts as a scavenger and aids in wound healing and apoptotic body clearance, and can prevent tissue injury, such as liver fibrosis (Rantakari et al. (2016 ) Proc. Natl. Acad. Sci. USA 113(33):9298-9303). Upregulation of stabilin-1 directly inhibits antigen-specific T-cell responses, and knockdown by siRNA in monocytes was shown to enhance their pro-inflammatory function (Palani, S. et al. (2016 ) J. Immunol. 196: 115-123). Hence, embodiments of the immunostimulatory bacterial strains that encode products that inhibit or reduce expression of Stabilin-l / CLEVER-1 in the tumor microenvironment, enhance the pro- inflammatory functions of tumor-resident macrophages.

[0634] m. CpG Motifs and CpG Islands

[0635] Unmethylated cytidine-phosphate-guanosine (CpG) motifs are prevalent in bacterial, but not vertebrate, genomic DNA. Pathogenic DNA and synthetic oligodeoxynucleotides (ODNs) containing CpG motifs activate host defense mechanisms, leading to innate and acquired immune responses. The unmethylated CpG motifs contain a central unmethylated CG dinucleotide plus flanking regions. In humans, four distinct classes of CpG ODNs have been identified, based on differences in structure and the nature of the immune response they induce. K-type ODNs (also referred to as B-type) contain from 1 to 5 CpG motifs, typically on a phosphorothioate backbone. D-type ODNs (also referred to as A-type) have a mixed

[0636] phosphodiester / phosphorothioate backbone and have a single CpG motif, flanked by palindromic sequences that permits the formation of a stem-loop structure, as well as poly G motifs at the 3' and 5' ends. C-type ODNs have a phosphorothioate backbone and contain multiple palindromic CpG motifs that can form stem loop structures or dimers. P-Class CpG ODNs have a phosphorothioate backbone and contain multiple CpG motifs with double palindromes that can form hairpins at their GC-rich 3 ' ends (Scheiermann and Klinman (2014) Vaccine 32(48):6377-6389). For purposes herein, the CpGs are encoded in the plasmid DNA; they can be introduced as a motif, or in a gene.

[0637] 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 recognizes hypomethylated CpG motifs in the DNA of prokaryotes that do not occur naturally in mammalian DNA (McKelvey el al. (2011) J. Autoimmunity 36:76-86). Recognition of CpG motifs upon phagocytosis of pathogens into endosomes in immune cell subsets induces IRF7- dependent type I interferon signaling and activates innate and adaptive immunity.

[0638] Immunostimulatory bacteria, such as Salmonella species, such as S.

[0639] typhimurium strains, carrying plasmids containing CpG islands / motifs, are provided herein. These bacteria can activate TLR9 and induce type I IFN-mediated innate and adaptive immunity. As exemplified herein, bacterial plasmids that contain

[0640] hypomethylated CpG islands can elicit innate and adaptive anti-tumor immune responses that, in combination with the encoded products, such as the gain-of- function variant STING proteins, can have synergistic or enhanced anti-tumor activity. For example, the asd gene (see, e.g ., SEQ ID NO:48) encodes a high frequency of hypomethylated CpG islands. CpG motifs can be included in

[0641] combination with any of the therapeutic products, such as STING proteins and mutants thereof, described in or apparent from the description herein, in the immunostimulatory bacteria, and thereby enhance or improve the anti -turn or immune response, by modulating TLRs, such as TLR9.

[0642] Immunostimulatory CpGs can be included in the plasmids, by including a nucleic acid, typically from a bacterial gene (e.g, asd), that encodes a gene product, and also by adding a nucleic acid that includes CpG motifs. The plasmids herein can include CpG motifs. Exemplary CpG motifs are known (see, e.g, U.S. Patent Nos. 8,232,259, 8,426,375 and 8,241,844). These include, for example, synthetic immunostimulatory oligonucleotides, between 10 and 100, 10 and 20, 10 and 30, 10 and 40, 10 and 50, or 10 and 75 base pairs long, with the general formula:

[0643] (CpG)n, where n is the number of repeats.

[0644] Generally, at least one or two repeats are used; non-CG bases can be interspersed. Those of skill in the art are very familiar with the general use of CpG motifs for inducing an immune response by modulating TLRs, particularly TLR9.

[0645] 5. Modifications That Increase Uptake of Gram-Negative Bacteria, such as Salmonella , by Immune Cells, and Reduce Immune Cell Death

[0646] The immunostimulatory bacteria provided herein, such as the exemplary strains of S. typhimurium, can be modified to increase uptake by immune cells, such as tumor-resident immune cells, and to decrease uptake by non-immune cells, such as epithelial cells. The bacteria also can be modified to decrease immune cell death, such as by decreasing macrophage pyroptosis. Numerous modifications of the bacterial genome can do one or both of increasing infection of immune cells and decreasing pyroptosis. The immunostimulatory bacteria provided herein include such

[0647] modifications, for example, deletions and / or disruptions of genes involved in the SPI- 1 T3SS pathway, such as disruption or deletion of hilA, rod protein and / or needle protein, and / or disruption / deletion of other bacterial genes, encoding flagellin. These modifications allow the bacteria to accumulate in tumor-resident immune cells, where they can express the encoded therapeutic product(s) and release them directly into the tumor microenvironment, enhancing the therapeutic efficacy. Additionally, prolonging the life of tumor-resident macrophages, e.g ., by decreasing pyroptosis, allows for the efficient production of the encoded therapeutic products, and activation of an immune response in the tumor microenvironment, further enhancing the anti tumor therapeutic efficacy of the bacteria.

[0648] a. Bacterial Uptake by Immune cells

[0649] The genome of the immunostimulatory bacteria provided herein can be modified to increase or promote infection of immune cells, particularly immune cells in the tumor microenvironment, such as phagocytic cells. This includes reducing infection of non-immune cells, such as epithelial cells, or increasing infection of immune cells. The invasive phenotype of Gram-negative bacteria, such as Salmonella , can result from the activity of genes encoded in pathways that promote the invasion of host cells. The invasion-associated Salmonella pathogenicity island 1 (SPI-1) of Salmonella is exemplary. SPI-1 includes the type 3 secretion system (T3SS), that is responsible for translocation of effector proteins into the cytosol of host cells. These proteins can cause actin rearrangements that lead to the uptake of Salmonella. T3SS effectors mediate the uptake of S. typhimurium into non-phagocytic host cells, such as epithelial cells. The SPI-1 T3SS has been shown to be essential for crossing the gut epithelial layer, but is dispensable for infection when bacteria are injected

[0650] parenterally, for example. SPI-1 mutants have defects in epithelial cell invasion, dramatically reducing oral virulence, but are taken up normally by phagocytic cells, such as macrophages (Kong el al. (2012) Proc. Natl. Acad. Sci. U.S.A.

[0651] 109(47): 19414-19419).

[0652] The immunostimulatory bacteria, such as S. typhimurium strains, provided herein, can be engineered with mutations in SPI-1 T3SS genes, preventing their uptake by epithelial cells, and focusing them to immune cells, such as macrophages, such as tumor-associated macrophages, enhancing the anti-tumor immune response. Additionally, as shown herein (see, e.g. , Example 6), elimination of the flagella, results in an inability to infect epithelial cells, and restricts uptake of the bacteria to tumor-resident immune / myeloid cells. Thus, in embodiments herein, the

[0653] immunostimulatory bacteria can be modified by deletion or disruption of genes in the SPI-1 T3SS and / or by deletion or disruption of genes encoding the flagella, to prevent or reduce infection of non-phagocytic cells ( e.g ., epithelial cells) and increase or restrict infection to tumor-resident myeloid cells. Such bacteria also can be modified with plasmids encoding therapeutic product(s), such as those that induce type I IFN, and immunostimulatory cytokines, further enhancing the anti-tumor immune response and the therapeutic efficacy by expressing the therapeutic products in tumor-resident immune cells.

[0654] b. Macrophage Pyroptosis

[0655] The macrophage NLRC4 inflammasome, which plays a role in the innate immune and antimicrobial responses, is a large multi-protein complex that recognizes cytosolic pathogens and provides for the autocatalytic activation of caspase-1.

[0656] Activation of caspase-1 induces maturation and release of the pro-inflammatory cytokines IL-Ib and IL-18, and triggers pyroptosis, a rapid inflammatory form of macrophage cell death. This pro-inflammatory cell death can limit the initiation of a robust adaptive immune response by directly inducing the death of antigen-presenting cells (APCs), as well as modifying the cytokine milieu to prevent the generation of memory T-cells.

[0657] Infection by certain Gram-negative bacteria encoding type 3 or 4 secretion systems, such as Salmonella typhimurium and Pseudomonas aeruginosa , triggers the activation of the NLRC4 inflammasome upon recognition of bacterial ligands, such as needle protein, rod protein and flagellin, following translocation into the host cell cytosol by the Salmonella pathogenicity island-1 type III secretion system (SPI-1

[0658] T3SS). Pyroptosis is not limited to macrophages; caspase-1 -dependent death has been observed in dendritic cells following infection with Salmonella (Li et al. (2016) Scientific Reports 6:37447; Chen et al. (2014) Cell Reports 8:570-582; (Fink and Cookson (2007) Cellular Microbiology 9(11):2562-2570)). As shown herein, the knock-out of genes in the Salmonella genome that are involved in the induction of pyroptosis enhances the anti-tumor immune response. This prevents the loss of immune cells, including macrophages, following bacterial infection. For example, genes encoding HilA, rod protein (PrgJ), needle protein (Prgl), flagellin and / or QseC can be knocked out / disrupted in the immunostimulatory bacteria provided herein.

[0659] i. Flagellin

[0660] As discussed above, for some bacteria species, such as Salmonella , flagellin, in addition to SPI-1 T3SS, is necessary for triggering pyroptosis in macrophages, and can be detected by, and activate, the macrophage NLRC4 inflammasome. Flagellin, which is the major component of flagellum, is recognized by TLR5. Salmonella encodes two flagellin genes, fliC and fljB,· elimination of flagellin subunits decreases pyroptosis in macrophages. For example, S. typhimurium with deletions in fliC and fljB resulted in significantly reduced IL-Ib secretion compared to the wild-type strain, whereas cellular uptake and intracellular replication of the bacterium remained unaffected. This demonstrates that flagellin plays a significant role in inflammasome activation. Additionally, S. typhimurium strains engineered to constitutively express FliC were found to induce macrophage pyroptosis (see, e.g ., Li et al. (2016) Scientific Reports 6:37447; Fink and Cookson (2007) Cellular Microbiology 9(11):2562-2570; and Winter et al. (2015) Infect. Immun. 83(4): 1546- 1555).

[0661] The genome of the immunostimulatory bacteria herein can be modified to delete, disrupt or mutate the flagellin genes fliC and fljB in S. typhimurium , leading to decreased cell death of tumor-resident immune cells, such as macrophages, and enhancing the anti-tumor immune response of the immunostimulatory bacteria.

[0662] ii. SPI-1 Proteins

[0663] SPI-1 proteins also activate the NLRC4 inflammasome in macrophages, activating caspase-1 and leading to cell death via pyroptosis. These effectors include, but are not limited to, rod protein (PrgJ) and needle protein (Prgl), for example.

[0664] Rod protein (PrgJ)

[0665] The NLRC4 inflammasome also detects aflagellated S. typhimurium. The flagellin-independent response is due to the detection of PrgJ, which is the SPI-1 T3SS rod protein in S. typhimurium. Delivery of purified PrgJ protein to the macrophage cytosol results in rapid NLRC4-dependent caspase-1 activation, as well as secretion of IL-Ib, similar to the effects induced by flagellin (Miao et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107(7):3076-3080). Thus, the mutation or knockout of the gene encoding PrgJ in S. typhimurium can reduce macrophage pyroptosis, which enhances the anti-tumor immune effect of the immunostimulatory bacteria, by preserving immune cells that are susceptible to being killed by the bacteria.

[0666] Needle protein (Prgl)

[0667] Prgl, which is the SPI-1 T3SS needle protein in S. typhimurium , also is recognized by, and activates, the NLRC4 inflammasome. The delivery of S.

[0668] typhimurium Prgl to the cytosol of human primary monocyte-derived macrophages results in IL-Ib secretion and subsequent cell death. A Salmonella mutant that expresses Prgl, but not flagellin, was shown to activate the inflammasome in primary monocyte-derived macrophages at later time points than strains expressing flagellin (Kortmann el al. (2015) J. Immunol. 195:815-819). The immunostimulatory bacteria provided herein, thus, can be modified to mutate or delete the gene encoding the needle protein in S. typhimurium , preventing immune cell pyroptosis, and enhancing the anti-tumor immune effect.

[0669] iii. QseC

[0670] The sensor protein QseC is a highly conserved membrane histidine sensor kinase that is found in many Gram-negative bacteria, and that responds to the environment and regulates the expression of several virulence factors. These virulence factors include, for example, the flhDC gene that encodes the master regulator of flagellum biosynthesis in S. typhimurium ; the sopB gene, which encodes a protein that plays a role in the invasion of non-phagocytic cells, the early maturation and regulation of trafficking of the Salmonella- containing vacuole (SCV), and the inhibition of SCV-lysosome fusion; and the sifA gene, which is required for SCV maintenance and membrane integrity.

[0671] It has been shown that selective inhibition of QseC by LED209 inhibits bacterial virulence without suppressing S. typhimurium growth, by inhibiting the

[0672] QseC-mediated activation of virulence-related gene expression (e.g, flhDC, sifA and sopB ), and partially protects mice from death following infection with S. typhimurium or Francisella tularensis. QseC blockade was found to inhibit caspase-1 activation, IL-Ib release, and S. typhimurium- induced pyroptosis of macrophages, by inhibiting excess inflammasome activation in the infected macrophages. Inhibition of QseC also suppresses flagellar gene expression and motility, and suppresses the invasion and replication capacities of S. typhimurium in epithelial cells (Li et al. (2016) Scientific Reports 6:37447). Thus, modification of the immunostimulatory bacteria herein, to mutate or knockout the gene encoding QseC, can enhance the anti-tumor immune response by focusing S. typhimurium infection to non-epithelial cells, and by reducing cell death in immune cells, such as by preventing pyroptosis in macrophages.

[0673] 6. Bacterial Culture Conditions

[0674] Culture conditions for bacteria can influence their gene expression. It has been documented that S. typhimurium can induce rapid pro-inflammatory caspase- dependent cell death of macrophages, but not epithelial cells, within 30 to 60 min of infection, by a mechanism involving the SPI-1 and its associated T3SS-1 (Lundberg et. al (1999) Journal of Bacteriology 181(11):3433-3437). It is now known that this cell death is mediated by activation of the inflammasome that subsequently activates caspase-1, which promotes the maturation and release of IL-Ib and IL-18 and initiates a novel form of cell death called pyroptosis (Broz and Monack (2011) Immunol. Rev. 243(1): 174-190). This pyroptotic activity can be induced by using log phase bacteria, whereas stationary phase bacteria do not induce this rapid cell death in macrophages. The SPI-1 genes are induced during the log phase of bacterial growth. Thus, by harvesting S. typhimurium , to be used therapeutically, at the 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 critical for establishing appropriate anti-tumor responses. In addition, limiting the secretion of pro-inflammatory cytokines, such as IL-Ib and IL-18, will improve the tolerability of administered S. typhimurium therapy. As provided herein, immunostimulatory S. typhimurium , harvested at stationary phase, will be used to induce anti-tumor responses.

[0675] 7. Increased Tumor Colonization

[0676] VNP20009 is an attenuated S. typhimurium-based microbial cancer therapy that was developed for the treatment of cancer. VNP20009 is attenuated through deletion of the genes msbB and purl (purM). The purl deletion renders the microbe auxotrophic for purines or adenosine. Deletion of the msbB gene reduces the toxicity associated with bacterial lipopolysaccharide (LPS), by preventing the addition of a terminal myristyl group to the lipid A domain, and producing a less toxic form of lipid A (Kahn et al. (1998) Mol. Microbiol. 29:571-579). There is a difference between mouse and humans in the ability of VNP20009 to colonize tumors. Systemic administration of VNP20009 resulted in colonization of mouse tumors; whereas systemic administration of VNP20009 in human patients resulted in very little tumor colonization. It was shown that in mice, VNP20009 exhibited a high degree of tumor colonization after systemic administration (see, e.g ., Clairmont et al. (2000) J Infect. Dis. 181 : 1996-2002; and Bermudes et al. (2001) Biotechnol Genet Eng Rev. 75:219-33). In a Phase 1 Study in advanced melanoma patients, however, very little VNP20009 was detected in human tumors after a 30- minute intravenous infusion (see, Toso et al. (2002) . Clin. Oncol. 20: 142-52). Patients that entered into a follow-up study evaluating a longer, four-hour infusion of VNP20009, also demonstrated a lack of detectable VNP20009 after tumor biopsy (Heimann et al. (2003 ) J. Immunother. 26: 179-180). Following intratumoral administration, colonization of a derivative of VNP20009 was detected (Nemunaitis et al. (2003) Cancer Gene Ther. 10:737 -44). Direct intratumoral administration of VNP20009 to human tumors resulted in tumor colonization, indicating that human tumors can be colonized at a high level, and that the difference in tumor colonization between mice and humans occurs only after systemic administration.

[0677] Strains, such as VNP20009, are inactivated by human complement, which leads to low tumor colonization. Strains that provide improved resistance to complement are provided herein. These strains contain modifications in the bacterial genome, and also can carry a plasmid, typically in low or medium copy number, to optionally encode genes to provide for replication ( asd under the control of a eukaryotic promoter), and nucleic acid(s) encoding a therapeutic product(s), such as, but not limited to, cytokines, gain-of-function mutants of proteins that stimulate production of type I interferon, and other such therapeutic genes / products, as described elsewhere herein.

[0678] The table below summarizes the bacterial genotypes / modifications, their functional effects, and the effects / benefits.

[0679]

[0680] Strains provided herein are AFLG so that they have no flagella, and / or ApagP. Additionally, the strains are one or more of Apurl ( ApurM ), AmsbB, and Aasd (in the bacterial genome). The plasmid is modified to encode products under control of host- recognized promoters ( e.g. , eukaryotic promoters, such as RNA polymerase II promoters, including those from eukaryotes, and animal viruses). The plasmids optionally can encode asd to permit replication in vivo , as well as nucleic acids with other beneficial functions (e.g, CpGs) and gene products as described elsewhere herein.

[0681] The immunostimulatory bacteria are derived from suitable bacterial strains, including attenuated and wild-type or other non-attenuated strains. Bacterial strains can be attenuated strains, or strains that are attenuated by standard methods, or that, by virtue of the modifications provided herein, are attenuated in that their ability to colonize is limited primarily to immunoprivileged tissues and organs, particularly immune and tumor cells, including solid tumors. Bacteria include, but are not limited to, for example, strains of Salmonella, Shigella, Listeria, E. coli, and Bifidobacteriae . For example, species include Shigella sonnei, Shigella flexneri, Shigella disenteriae, Listeria monocytogenes , Salmonella typhi , Salmonella typhimurium , Salmonella gallinarum , and Salmonella enteritidis. Other suitable bacterial species include Rickettsia , Klebsiella , Bordetella , Neisseria , Aeromonas, Francisella ,

[0682] Coryne bacterium, Citrobacter , Chlamydia , Haemophilus , Brucella , Mycobacterium , Mycoplasma , Legionella , Rhodococcus, Pseudomonas , Helicobacter , Vibrio , Bacillus , and Erysipelothrix. For example, Rickettsia Rikettsiae , Rickettsia prowazekii ,

[0683] Rickettsia tsutsugamuchi , Rickettsia mooseri, Rickettsia sibirica , Bordetella bronchiseptica , Neisseria meningitidis , Neisseria gonorrhoeae , Aeromonas eucrenophila , Aeromonas salmonicida , Francisella tularensis , Corynebacterium pseudotuberculosis , Citrobacter freundii , Chlamydia pneumoniae , Haemophilus sornnus , Brucella abortus , Mycobacterium intracellulare , Legionella pneumophila , Rhodococcus equi , Pseudomonas aeruginosa , Helicobacter mustelae , Vibrio cholerae , Bacillus subtilis , Erysipelothrix rhusiopathiae , Yersinia enterocolitica , Rochalimaea quintana , and Agrobacterium tumerfacium.

[0684] Exemplary of the immunostimulatory bacteria provided herein are species of

[0685] Salmonella. Exemplary of bacteria for modification as described herein are wild-type strains of Salmonella , such as the strain that has all of the identifying characteristics of the strain deposited in the ATCC as accession #14028. Engineered strains of Salmonella typhimurium , such as strain YS1646 (ATCC Catalog # 202165; also referred to as VNP20009, see, also International PCT Application Publication No.

[0686] WO 99 / 13053) that is engineered with plasmids to complement an asr / gene knockout and antibiotic-free plasmid maintenance, are provided. The strains then are modified to delete the flagellin genes and / or to delete pagP. The strains also are rendered auxotrophic for purines, particularly adenosine, and are asr / and msbB . The <m / gene can be provided on a plasmid for replication in the eukaryotic host. These deletions and plasmids are described elsewhere herein. Any of the nucleic acids encoding therapeutic products and immunostimulatory proteins and other products, described elsewhere herein and / or known to those of skill in the art, can be included on the plasmid. The plasmid generally is present in low to medium copy number as described elsewhere herein. Therapeutic products include gain-of-function mutants of cytosolic DNA / RNA sensors, that can constitutively evoke / induce type I IFN expression, and other immunostimulatory proteins, such as cytokines, that promote an anti-tumor immune response in the tumor microenvironment, and other such products described herein.

[0687] E. NON-HUMAN STING PROTEINS AND GAIN-OF-FUNCTION MUTATIONS IN PROTEINS THAT STIMUUATE THE IMMUNE RESPONSE IN THE TUMOR MICROENVIRONMENT

[0688] Provided are immunostimulatory bacteria that contain sequences of nucleotides that encode gene products that are therapeutic, particularly anti-cancer products, including products that promote or stimulate an anti-tumor or anti-viral immune response. Included among the therapeutic products are products, referred to as cytosolic DNA / RNA sensors, that evoke immune responses when exposed to nucleic acids, such as RNA, DNA, nucleotides, dinucleotides, cyclic nucleotides, cyclic dinucleotides, and other such molecules, in the cytosol of cells. The

[0689] immunostimulatory bacteria herein encode modified products that have increased activity or that constitutively evoke immune responses, and do not require the presence of the DNA / RNA products in the cytosol. Exemplary are encoded proteins that include gain-of-function mutations that increase immune responses in the tumor microenvironment. Not only are immunostimulatory bacteria provided, but also, other delivery vehicles can be used to deliver nucleic acids encoding such

[0690] immunostimulatory proteins, or to deliver the encoded proteins. These delivery vehicles include exosomes, vectors, and viruses. For example, oncolytic viruses also can be modified to express the gain-of-function products, particularly oncolytic viruses, such as vaccinia virus, that are cytoplasmic viruses. The encoded gain-of- function products can be delivered in exosomes, liposomes, and other suitable vehicles, generally targeted to tumors.

[0691] The immunostimulatory bacteria that encode the gain-of-function products

[0692] (and / or other therapeutic products) include immunostimulatory bacteria that preferentially infect tumors, including tumor-resident immune cells, and / or immunostimulatory bacteria in which the genome is modified so that the bacteria induces less cell death in tumor-resident immune cells, whereby the

[0693] immunostimulatory bacteria accumulate in tumor cells and tumor-resident immune cells, to thereby deliver the constitutively active proteins and / or other therapeutic products to the cells and the tumor microenvironment, to stimulate the immune response against the tumor. The immunostimulatory bacteria further can encode a tumor antigen in the subject to enhance the response against the particular tumor. Any of the immunostimulatory bacteria provided herein and described above and below can be modified to encode such a gain-of-function product. The product is encoded on a plasmid under control of a promoter, and any other desired regulatory sequences recognized in a eukaryotic, such as a human, or other animal, or mammalian, subject. Generally, the nucleic acid encoding the gain-of-function product is under the control of an RNA polymerase II promoter.

[0694] The therapeutic products, including the gain-of-function variants that include STING proteins and other proteins in the type I interferon signaling pathway as described herein, and other anti-cancer products, are expressed under control of a eukaryotic promoter. Promoters include, for example, the EF-1 alpha promoter, CMV, SV40, PGK, EIF4A1, CAG, CD68 and synthetic MND promoters; viral promoters, such as O, MSCV and TLR promoters, and a respiratory syncytial virus (RSV) promoter; cellular promoters, such as EIF-la; inducible chimeric promoters, such as tet-CMV; and tissue-specific promoters (Chang el al. (2013) Cold Spring Harb Protoc; dok lO. l 101 / pdb.prot075853).

[0695] Additionally, any of the bacteria described herein for modification, such as any of the strains of Salmonella , Shigella , E. coli , Bifidobacteriae , Rickettsia , Vibrio , Listeria , Klebsiella , Bordetella , Neisseria , Aeromonas, Francisella , Cholera ,

[0696] Coryne bacterium, Citrobacter , Chlamydia , Haemophilus , Brucella , Mycobacterium ,

[0697] Mycoplasma , Legionella , Rhodococcus, Pseudomonas , Helicobacter , Bacillus , and Erysipelothrix , or attenuated strains thereof or modified strains thereof, exosomes, liposomes and oncolytic viruses, can be modified by introducing a plasmid

[0698] containing, or encoding on a plasmid in the bacteria, nucleic acids encoding the gain- of-function product(s) under control of an RNA polymerase promoter recognized by the host. The gain-of-function products are expressed in the infected subject’s cells. The immunostimulatory bacteria include those that are modified, as described herein, to accumulate in, or to preferentially infect, tumors and tumor-resident immune cells. For example, immunostimulatory bacteria that encode gain-of-function products leading to the expression of, or the constitutive expression of, type I interferon (IFN), such as IFN-beta, further are modified to have reduced ability or no ability to infect epithelial cells, but are able to infect phagocytic cells, including tumor-resident immune cells, and / or the bacteria are modified so that they do not kill the infected phagocytic cells.

[0699] The immunostimulatory bacteria herein can encode products, referred to as cytosolic DNA / RNA sensors, that evoke immune responses when exposed to nucleic acids, such as RNA, DNA, nucleotides, dinucleotides, cyclic nucleotides, cyclic dinucleotides, and other such molecules, in the cytosol of cells. The

[0700] immunostimulatory bacteria herein, encode modified products that constitutively evoke immune responses, and do not require the presence of the DNA / RNA and other nucleotides in the cytosol. Exemplary of such are components of pathways that induce type I interferon expression. The products contemplated herein include modified forms of these DNA / RNA sensors, that have constitutive activity or increased activity (gain-of-function products), such that type I interferon(s) is / are expressed or produced in the absence of nucleotides, dinucleotides, cyclic nucleotides, cyclic dinucleotides, and other such ligands, in the cytosol of cells. Expression of these modified products in cells, particularly in tumor cells and tumor-resident immune cells, leads to constitutive expression of type I interferons, including interferon-b, in the tumor microenvironment. Because the immunostimulatory bacteria, and also oncolytic viruses (and other delivery vehicles as described herein), that express these gain-of- function products accumulate in or preferentially infect tumor cells and tumor- resident immune cells, the products are expressed in the tumor microenvironment, resulting in increased immune responses in the tumor microenvironment, and enhanced therapeutic efficacy.

[0701] Exemplary gene products that can be encoded in the immunostimulatory bacteria and other vehicles, include, but are not limited to, proteins that sense or are involved in innate pathways that recognize cytosolic DNA / RNA and activate type I interferon production. Proteins involved in innate DNA / RNA recognition that activate type I interferon include, but are not limited to: STING, RIG-I, MDA-5, IRF-3, IRF- 7, TRIM56, RIP1 / RIPK1, Sec5 / EXOC2, TRAF2, TRAF3, TRAF6, STAT1,

[0702] LGP2 / DHX58, DDX3 / DDX3X, DHX9 / DDX9, DDX1, DDX21, DHX15 / DDX15, DHX33 / DDX33, DHX36 / DDX36, DDX60, and SNRNP200. Gain-of-function mutations in any of these proteins that result in constitutive type I interferon expression are known, or can be identified, and can be delivered by the immunostimulatory bacteria, or other vectors, and delivery vehicles, such as exosomes or liposomes, to the tumor microenvironment, such as by infection of cells or targeting and binding to tumor cells. The gain-of-function mutations include those identified from individuals with disorders resulting from constitutive type I interferon expression. Exemplary of gain-of-function products are those that occur in subjects with interferonopathies. As noted above, mutations can be identified by screening to generate gain-of-function products as well.

[0703] The immunostimulatory bacteria herein encode such proteins, such as STING, including non-human STING proteins that have lower NF-kB signaling activity than the NF-KB signaling activity of human STING, and variants of the STING proteins and other DNA / RNA sensors that constitutively evoke immune responses, and do not require the presence of the DNA / RNA or other nucleotide ligands in the cytosol. Exemplary of such are components of pathways that induce type I interferon expression.

[0704] The nucleic acids encoding the identified gain-of-function mutant products can be further modified to improve properties for expression. Modifications include, for example, codon optimization to increase transcriptional efficiency in a

[0705] mammalian, particularly human, subject, such as reduction of GC content or CpG dinucleotide content, removal of cryptic splicing sites, negative CpG islands, replacement of the Shine-Dalgamo (SD) sequence, and replacement of TATA box and / or terminal signals to increase transcriptional efficiency. Also, codons can be optimized for increasing translation efficiency by altering codon usage bias, decreasing GC content, decreasing mRNA secondary structure, removing premature PolyA sites, removing RNA instability motifs (ARE), reducing stable free energy of mRNA, modifying internal chi sites and ribosomal binding sites, and reducing RNA secondary structures.

[0706] 1. Type I Interferons and Pathways

[0707] Type I interferon induction pathways, mediated by host recognition of nucleic acids, such as single-stranded and double-stranded RNA, and of cyclic di-nucleotides and other such forms of nucleic acids, are known to induce type I UN. There also are Toll-Like Receptor (TLR)-independent type I UN pathways, mediated by host recognition of single- stranded (ss) and double-stranded (ds) RNA in the cytosol. These nucleic acids are sensed by RNA helicases, including retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and through promoter stimulator 1 (IPS-1) adaptor protein-mediated phosphorylation of the IRF-3 transcription factor, leading to induction of IFN-beta (Ireton and Gale (2011) Viruses 3(6):906-919). As discussed herein, proteins in these pathways can be modified, or can exist as variants, that result in constitutive expression of type I interferons (also referred to as interferon type 1), which include IFN-a and IFN-b. Provided herein are immunostimulatory bacteria and other delivery vehicles, including exosomes, liposomes and oncolytic viruses, that encode the variant proteins. These delivery vehicles can be used to treat cancers by directly administering to subjects and / or by administering them to cells, allogeneic or autologous, for use in cell therapy protocols.

[0708] Type I interferons (IFNs; also referred to as interferon type 1), include IFN-a and IFN-b, and are pleiotropic cytokines with antiviral, antitumor and

[0709] immunoregulatory activities. IFN-b is produced by most cell types; IFN-a primarily is produced by hematopoietic cells, particularly plasmacytoid dendritic cells. Type I IFNs are produced following the sensing of pathogen-associated molecular patterns (PAMPs), including microbial and virus nucleic acids and LPS (lipopolysaccharides), by pattern recognition receptors (PRRs) and by cytokines. They are involved in the innate immune response against pathogen, including viral, infection, and are potent immunomodulators that promote antigen presentation, mediate DC maturation, activate cytotoxic T lymphocytes (CTLs), natural killer (NK) cells and macrophages, and activate the adaptive immune system by promoting the development of high- affinity antigen-specific T and B cell responses and immunological memory.

[0710] Type I IFNs exhibit anti -proliferative and pro-apoptotic effects on tumors and have anti-angiogenic effects on tumor neovasculature. They induce the expression of MHC class I molecules on tumor cell surfaces, increase the immunogenicity of tumor cells, and activate cytotoxicity against them. Type I IFN has been used as a therapeutic for treatment of cancers and viral infections. For example, IFN-a (sold under the trademark Intron® / Roferon®-A) is approved for the treatment of hairy cell leukemia, malignant melanoma, AIDS-related Kaposi’s sarcoma, and follicular non- Hodgkin’s lymphoma; it also is used in the treatment of chronic myelogenous leukemia (CML), renal cell carcinoma, neuroendocrine tumors, multiple myeloma, non-follicular non-Hodgkin’s lymphoma, desmoid tumors and cutaneous T-cell lymphoma (Ivashkiv and Donlin (2014) Nat. Rev. Immunol. 14(l):36-49; Kalliolias and Ivashkiv (2010) Arthritis Research & Therapy 12(Suppl 1): S 1 ; Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893).

[0711] Expression of type I interferons in tumors and the tumor microenvironment is among the immune responses that the immunostimulatory bacteria and other delivery vehicles herein are designed to evoke. Inducing or evoking type I interferon provides anti-tumor immunity for the treatment of cancer.

[0712] 2. Type I Interferonopathies and Gain-of-Function Mutants

[0713] The induction of type I interferons (IFNs), proinflammatory cytokines and chemokines is necessary for mounting an immune response that prevents or inhibits infection by pathogens. This response also can be effective as an anti-tumor agent.

[0714] The immunostimulatory bacteria and other delivery vehicles provided herein encode proteins that constitutively induce type I IFNs. Among these proteins are those that occur in individuals with various diseases or disorders that involve the over production of immune response modulators. For example, over-production or excessive production, or defective negative regulation of type I IFNs and pro- inflammatory cytokines, can lead to undesirable effects, such as inflammatory and autoimmune diseases. Disorders involving the overproduction, generally chronic, of type I IFNs and pro-inflammatory cytokines, are referred to as interferonopathies (see, e.g ., Lu and MacDougall (2017) Front. Genet. 8: 118; and Konno et al. (2018) Cell Reports 23 : 1112-1123). Disorders and clinical phenotypes associated with type I interferonopathies include Aicardi-Goutieres syndrome (AGS), STING-associated vasculopathy with onset in infancy (SAVI), Singleton-Merten syndrome (SMS), atypical SMS, familial chilblain lupus (FCL), systemic lupus erythematosus (SLE), bilaterial striatal necrosis (BSN), cerebrovascular disease (CVD), dyschromatosis symmetrica hereditaria (DSH), spastic paraparesis (SP), X-linked reticulate pigmentary disorder (XLPDR), proteasome-associated auto-inflammatory syndrome (PRAAS), intracranial calcification (ICC), Mendelian susceptibility to mycobacterial disease (MSMD), and spondyloenchondrodysplasia (SPENCD) (see, e.g. , Rodero et al. (2016) J. Exp. Med. 213(12):2527-2538). These phenotypes are associated with particular genotypes, involving mutations in genes that lead to constitutive activities of products involved in the induction of type I IFNs.

[0715] The sustained activation of interferon signaling can be due to: 1) loss-of- function mutations leading to increased cytosolic DNA ( e.g ., mutations in TREX1 and SAMHD1 ) or increased cytosolic RNA / DNA hybrids (e.g., mutations in RNASEH2A,

[0716] RNASEH2B, RNASEH2C and POLA1); 2) loss-of-function mutations resulting in a defect in RNA editing and abnormal sensing of self-nucleic acid RNA species in the cytosol (e.g, mutations mADARl); 3) gain-of-function mutations leading to constitutive activation of cytosolic IFN signaling pathways / increased sensitivity to cytosolic nucleic acid ligands (e.g, mutations in RIG-I, MDA5 and STING); 4) loss- of-function mutations leading to aberrant RNA signaling via MAVS caused by a disturbance of the unfolded protein response (e.g, mutations in SKIV2L); 5) loss-of- function mutations in molecules responsible for limiting IFN receptor (IFNARl / 2) signaling, leading to uncontrolled IFN-stimulated gene (ISG) production (e.g, mutations in USP18 and ISG 15); 6) proteasomal dysfunction, leading to increased IFN signaling through an unknown mechanism (e.g, mutations in PSMA3, PSMB4 and PSMB8); and 7) loss-of-function mutations in TRAP / A CP 5 and Clq, where the mechanisms leading to type I IFN signaling remain unclear (Rodero et al. (2016) . / . Exp. Med. 213(12):2527-2538).

[0717] Of interest herein are mutations that lead to gain-of-function. There are known mutations in STING, MDA5 and RIG-I, associated with gain-of-function (GOF), resulting in the constitutive activation of the encoded proteins and / or enhanced sensitivity or increased affinity or binding to endogenous ligands. GOF mutations in STING, for example, are linked to SAVI and FCL; GOF mutations in MDA5 are linked to AGS and SMS; and GOF mutations in RIG-I are linked to atypical SMS.

[0718] The immunostimulatory bacteria, and oncolytic viruses, provided herein that encode these proteins with gain-of-function mutations, exploit the constitutive activation of these proteins to increase production of type I IFNs and pro- inflammatory cytokines. Tumor-targeting immunostimulatory bacteria, as well as oncolytic viruses and other delivery vehicles, are provided herein that encode STING, MDA5 and / or RIG-I with gain-of-function mutations. Such immunostimulatory bacteria and other delivery vehicles, increase the production of type I IFNs and pro- inflammatory cytokines in the tumor microenvironment, potentiating the anti-tumor immune response and improving the therapeutic efficacy of the immunostimulatory bacteria. The gene encoding STING is referred to as IMEM173 , the gene encoding MDA5 is IFIH1 , and the gene encoding RIG-I is DDX58. There are numerous alleles for each gene, and known mutations that can occur in genes with any of the alleles, resulting in gain-of-function. The mutations listed below can occur singly or can be used in any combination. Other mutations that result in gain-of-function can be identified by routine screening / mutation protocols. The table below lists exemplary g...

Claims

WHAT IS CLAIMED:

1. A modified Stimulator of Interferon Genes (STING) protein from a non-human species, where the non-human STING is one that has lower NF-KB signaling activity compared to the NF-KB signaling activity of human STING, and, optionally, higher type I interferon (IFN) pathway signaling activity compared to human STING, wherein:the non-human STING protein is modified to include a mutation or mutations so that it has increased activity or acts constitutively in the absence of cytosolic nucleic acids;the mutations are insertions, deletions, and / or replacements of amino acids; andthe STING protein optionally has a deletion of the TRAF6 binding site.

2. A modified Stimulator of Interferon Genes (STING) protein from a non-human species, or a chimeric human STING protein, or variants of the chimeric STING protein, wherein the modified STING protein and variant of the chimeric STING protein comprise one or more mutation(s) associated with gain-of-function (GOF) that result(s) in the constitutive activation of the encoded STING protein and / or enhanced sensitivity, or increased affinity or binding to endogenous ligands, wherein:the STING protein is modified by one or more of an insertion, deletion, and replacement of an amino acid or amino acids;the chimeric human STING protein comprises a portion of a human STING protein and a portion of the non-human STING protein;the non-human or chimeric STING protein has IFN-beta signaling activity, and attenuated nuclear factor kappa-light-chain-enhancer of activated B cell (NF-KB) signaling activity, compared to the NF-KB signaling activity of human STING; and the mutation or mutations resulting in a variant or modified protein result in increased STING activity or constitutive activity in inducing IFN-beta production.

3. The modified STING protein of claim 1 or claim 2, wherein the unmodified human STING protein comprises the sequence of amino acids set forth in any of SEQ ID N0s:305-309, or is a human allelic variant thereof with at least 98%sequence identity to the sequence of amino acids set forth in any of SEQ ID NOs:305- 309.

4. The modified STING protein of any of claims 1-3, wherein:the STING protein is a chimera comprising replacement of a C-terminal tail (CTT) region in a STING protein from a first species with the CTT of a STING protein from a second species;the STING protein of the second species has lower NF-KB signaling activity than the NF-KB signaling activity of human STING; andthe TRAF6 binding site in the CTT optionally is deleted.

5. The modified STING protein of any of claims 1-4, wherein the mutation or mutations is / are any that correspond to those associated with the human auto-inflammatory disease STING-associated vasculopathy with onset in infancy (SAVI).

6. A modified Stimulator of Interferon Genes (STING) protein that is a chimera, comprising replacement of the CTT (C-terminal tail) region in a STING from a first species with the CTT of STING from a second species, wherein:the STING protein of the second species has lower NF-KB signaling activity than the NF-KB signaling activity of human STING; andthe TRAF6 binding site in the CTT is deleted.

7. The modified STING protein of any of claims 2-6, wherein:the chimera comprises a portion of a human STING protein and a portion of a non-human STING protein; andthe human STING protein comprises the sequence of amino acids set forth in any of SEQ ID N0s:305-309, or is a human allelic variant thereof with at least 98% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOs:305- 309.

8. The modified STING protein of any of claims 2 and 4-7 that is a chimera comprising portions of STING proteins from two species, whereby the resulting STING protein has IFN-beta signaling activity, wherein the first species is human, and the second species is selected from among Tasmanian devil, marmoset, cattle, cat, ostrich, boar, bat, manatee, crested ibis, coelacanth, mouse and ghost shark.

9. The modified STING protein of any of claims 1-8, wherein the type I IFN signaling activity of non-human STING is at least or at least about 30% that of a wild type human STING protein.

10. The modified STING protein of any of claims 1-9, wherein the NF-KB signaling activity of the non-human STING is less than 30%, less than 20%, less than15%, less than 10%, or less than 5% that of wild type human STING NF-KB signaling activity.

11. The modified STING protein of any of claims 1-10, wherein the non human species or second species is selected from among Tasmanian devil, marmoset, cattle, cat, ostrich, boar, bat, manatee, crested ibis, coelacanth, mouse and ghost shark.

12. The modified STING protein of any of claims 1-11, wherein the modification of STING is a mutation or mutations that correspond, by reference to and alignment with human STING, to a mutation that occurs in an interferonopathy, wherein the sequence of human STING with which alignment is effected is set forth in any of SEQ ID N0s:305-309.

13. The modified STING protein of any of claims 1-12 that comprises replacement of the C-terminal tail (CTT) with the CTT from a second STING protein that has reduced NF-KB signaling activity compared to the NF-KB signaling activity of human STING.

14. The modified STING protein of any of claims 1-13, wherein theTRAF6 binding site in the CTT is deleted.

15. The modified STING protein of claim 13 or claim 14, wherein the second CTT is from a Tasmanian devil, marmoset, cattle, cat, ostrich, boar, bat, manatee, crested ibis, coelacanth, mouse or ghost shark STING protein.

16. The modified STING protein of claim 13 or claim 14, wherein the second CTT is from a Tasmanian devil, marmoset, cattle, cat, ostrich, boar, bat, manatee, crested ibis, coelacanth, mouse or ghost shark STING protein, and it replaces the human STING CTT.

17. The modified STING protein of any of claims 13-16, wherein the replacing CTT is selected from among the following species and has a sequence:Tasmanian RQEEFAIGPKRAMTVTTSSTLSQEPQLLISGMEQPLSLRTDGF SEQ ID NO:353, devilMarmoset EEEEVTVGSLKTSEVPSTSTMSQEPELLISGMEKPLPLRSDLF SEQ ID NO:354,Cow EREVTMGSTETSVMPGSSVLSQEPELLISGLEKPLPLRSDVF SEQ ID NO:355,Cat EREVTVGSVGTSMVRNPSVLSQEPNLLISGMEQPLPLRTDVF SEQ ID NO:356,Ostrich RQEEYTVCDGTLCSTDLSLQISESDLPQPLRSDCL SEQ ID NO:357,Boar EREVTMGSAETSVVPTSSTLSQEPELLISGMEQPLPLRSDIF SEQ ID NO:358,Bat EKEEVTVGTVGTYEAPGSSTT .HOF.PF.I J JSGMDQPT .PI .RTDIF SEQ ID NO:359,Manatee EREEVTVGSVGTSVVPSPSSPSTSSLSQEPKLLISGMEQPLPLR SEQ ID NO:360,TDVFCrested ibis CHEEYT VYEGNQPHNP STTT .HSTF.I NI .OISF.SDI POPI .RSDCF SEQ ID NO:361,Coelacanth QKEEYFMSEQTQPN S S STSCL STEPQLMISDTD APHTLKRQ V C SEQ ID NO:362,(variant 1)Coelacanth QKEEYFMSEQTQPNSSSTSCLSTEPQLMISDTDAPHTLKSGF SEQ ID NO:363,(variant 2)Ghost LTEYPVAEPSNANETDCMSSEPHLMISDDPKPLRSYCP SEQ ID NO:365, and sharkMouse F.KF.F.VTMNAPMTSVAPPPSVI .SOF.PRI J JSGMDQPT .PI .RTDI J SEQ ID NO:366, or allelic variants of each of these sequences having at least 98% sequence identity thereto.

18. The modified STING protein of any of claims 4-17, wherein the human STING CTT comprises the sequenceEKEE VT V GSLKT S A VP STS TM S QEPELLIS GMEKPLPLRTDF S (SEQ IDNO:352), or is an allelic variant having at least 98% sequence identity thereto.

19. The modified STING protein of any of claims 4-18, wherein the modified STING protein is a chimera in which the human STING CTT is replaced with a CTT from the Tasmanian devil STING.

20. The modified STING protein of claim 19, wherein the C-terminal tail(CTT) from the Tasmanian devil STING comprises the sequence:RQEEFAIGPKRAMTVTTSSTLSQEPQLLISGMEQPLSLRTDGF (SEQ IDNO:353), or is an allelic variant having at least 98% sequence identity thereto.

21. The modified STING protein of any of claims 1-20, comprising deletion of the TRAF6 binding site.

22. The modified STING protein of claim 21, wherein the STING is a human STING, and the TRAF6 binding site comprises the amino acid residues DFS at the C-terminus.

23. The modified STING protein of any of claims 1-22, comprising a modification that increases type I interferon signaling activity or renders the activity constitutive in the absence of cytosolic nucleic acids.

24. The modified STING protein of claim 23, wherein the modification corresponds, by reference to and alignment with human STING, to a mutation thatoccurs in an interferonopathy, wherein the human STING has the sequence set forth in any of SEQ ID N0s:305-309.

25. The modified STING protein of any of claims 1-24, wherein the modification that confers increased activity or constitutive activity is one or more amino acid replacements that correspond(s) to one or more of S102P, V147L,V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A,S358A / E360A / S366A, D231 A / R232A / K236A / R238A, S358A, E360A, S366A,R238A, R375A, and S324A / S326A, with reference to the sequence of human STING, as set forth in any one of SEQ ID N0s:305-309.

26. The modified STING protein of any of claims 1-25, wherein the non human STING protein is a Tasmanian Devil STING protein of SEQ ID NO:331, or an allelic variant thereof having at least 98% sequence identity to the STING protein ofSEQ ID NO:331.

27. The modified STING protein of any of claims 1-26, comprising a replacement corresponding to C206Y or R284G, with reference to the sequence of human STING as set forth in any of SEQ ID N0s:305-309.

28. The modified STING protein of claim 27, comprising the sequence of amino acids set forth in SEQ ID NO: 332 or 333.

29. The modified STING protein of any of claims 1-25, wherein the sequences of the non-human species STING proteins are those set forth in SEQ ID NOs: 331, 338 and 341-350, or the non-human STING proteins are allelic variants of any of the STING proteins set forth in SEQ ID NOs: 331, 338 and 341-350 having at least 98% sequence identity therewith.

30. An immunostimulatory bacterium, comprising a plasmid encoding the modified STING protein of any of claims 1-29, or encoding a STING protein from a non-human species, wherein the STING protein has type I IFN signaling activity, and attenuated NF-KB signaling activity compared to the NF-KB signaling activity of human STING.

31. The immunostimulatory bacterium of claim 30, wherein the non human species is selected from among Tasmanian devil, marmoset, cattle, cat, ostrich, boar, bat, manatee, crested ibis, coelacanth, mouse and ghost shark.

32. An immunostimulatory bacterium, comprising a plasmid encoding a gain-of-function variant of an immunostimulatory protein that, in unmodified form, is part of a cytosolic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN).

33. The immunostimulatory bacterium of claim 32, wherein:the gain-of function (GOF) variant is a constitutively active variant of an immunostimulatory protein that, in humans, promotes or causes interferonopathies; andthe genome of the immunostimulatory bacterium is modified so that the bacterium preferentially infects tumor-resident immune cells, and / or the genome of the immunostimulatory bacterium is modified so that it induces less cell death in tumor-resident immune cells (decreases pyroptosis), whereby the immunostimulatory bacterium accumulates in tumors or in the tumor microenvironment or in tumor- resident immune cells to thereby deliver the constitutively active immunostimulatory protein to the host cell to stimulate or induce expression of type I IFN.

34. The immunostimulatory bacterium of claim 32 or claim 33, wherein the GOF variant protein comprises a mutation that eliminates a phosphorylation site in the immunostimulatory protein, to thereby reduce nuclear factor kappa-light-chain- enhancer of activated B cell (NF-KB) signaling.

35. The immunostimulatory bacterium of any of claims 32-34, wherein the immunostimulatory protein that induces type I IFN is STING, RIG-I, IRF-3, IRF-7 or MDA5.

36. An immunostimulatory bacterium, comprising a plasmid encoding an immunostimulatory protein that induces expression of type I IFN, or a variant thereof that has increased activity or constitutive activity, wherein the immunostimulatory protein is STING, RIG-I, IRF-3, IRF-7 or MDA5.

37. The immunostimulatory bacterium of any of claims 32-36, wherein the immunostimulatory protein is a variant of STING, RIG-I, IRF-3, IRF-7 or MDA5 that comprises a gain-of-function mutation resulting in increased expression of type I IFN.

38. The immunostimulatory bacterium of any of claims 32-37, wherein the immunostimulatory protein is a variant of STING, RIG-I, IRF-3, IRF-7 or MDA5, in which one or more serine (S) or threonine (T) residue(s) that is / are phosphorylated as a consequence of viral infection, is / are replaced with an aspartic acid (D) residue, whereby the resulting variant protein is a phosphomimetic that constitutively induces type I IFN.

39. The immunostimulatory bacterium of claim 38, wherein:the variant protein is IRF-3 and comprises one or more replacement(s) at residues at positions corresponding to positions 385, 386, 396, 398, 402, 404 and 405, with reference to SEQ ID NO: 312; andthe residues are replaced with aspartic acid residues.

40. The immunostimulatory bacterium of claim 39, wherein the variant IRF-3 protein has the replacement S396D, with reference to SEQ ID NO:312.

41. The immunostimulatory bacterium of claim 39, wherein the variant IRF-3 protein comprises the replacements S396D / S398D / S402D / T404D / S405D, with reference to SEQ ID NO:312, or conservative replacements thereof.

42. The immunostimulatory bacterium of claim 39, wherein the the variant IRF-3 protein comprises the replacements S396D / S398D / S402D / T404D / S405D, with reference to SEQ ID NO: 312.

43. The immunostimulatory bacterium of any of claims 32-42, wherein the immunostimulatory protein is a variant that, when expressed in a subject, leads to constitutive expression of type I IFN.

44. The immunostimulatory bacterium of any of claims 30-43, wherein the modified immunostimulatory protein or modified STING protein has at least 95% sequence identity with the corresponding unmodified protein, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications selected from among insertions, deletions, and replacements.

45. The immunostimulatory bacterium of any of claims 32-44, wherein the immunostimulatory protein is a variant that, when expressed in a subject, leads to constitutive expression of type I IFN.

46. The immunostimulatory bacterium of any of claims 32-44, wherein the immunostimulatory protein, in its unmodified form, senses or interacts directly orindirectly with cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides to induce expression of type I IFN, and the variant protein induces expression of type I IFN in the absence of the sensing or interacting with the cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides.

47. The immunostimulatory bacterium of claim 46, wherein theimmunostimulatory protein is a gain-of-function (GOF) variant that does not require cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides to result in expression of type I IFN.

48. The immunostimulatory bacterium of any of claims 32-47, wherein the immunostimulatory protein, in unmodified form, activates or induces expression of type I IFN.

49. The immunostimulatory bacterium of any of claims 32-48, wherein the unmodified form of the immunostimulatory protein is involved in innate cytosolic DNA / RNA recognition that activates type I IFN.

50. The immunostimulatory bacterium of claim 49, wherein the type I IFN is an interferon-a or interferon-b.

51. The immunostimulatory bacterium of any of claims 32-50, wherein the immunostimulatory protein is selected from among STING, RIG-I, MDA-5, IRF-3, IRF-7, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, and SNRNP200.

52. The immunostimulatory bacterium of any of claims 32-50, wherein the immunostimulatory protein is selected from among TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, and SNRNP200.

53. The immunostimulatory bacterium of any of claims 32-52, wherein the immunostimulatory protein that is part of a cytosolic DNA / RNA sensor pathway is a variant that comprises a gain-of-function mutation, or a mutation whereby expression of type I interferon, or other immune mediator / modulator, is constitutive.

54. The immunostimulatory bacterium of claim 53, wherein:the immunostimulatory protein that senses cytosolic DNA / RNA is a variantSTING, MDA5, RIG-I, IRF-7 or IRF-3 protein;unmodified STING has the sequence set forth in any of SEQ ID N0s:305-309, unmodified MDA5 has the sequence set forth in SEQ ID NO: 310, unmodified RIG-I has the sequence set forth in SEQ ID NO:311, unmodified IRF-7 has the sequence set forth in SEQ ID NO:313, and unmodified IRF-3 has the sequence set forth in SEQ ID NO:312; andthe modified immunostimulatory protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications selected from among replacements, deletions, and insertions.

55. The immunostimulatory bacterium of any of claims 32-54, wherein the immunostimulatory protein is selected from among STING, MDA5, IRF-3, IRF-7, and RIG-I, and comprises a gain-of-function mutation(s) that renders the STING, MDA5, IRF-3, IRF-7, or RIG-I constitutively active, whereby expression of type I IFN is constitutive.

56. The immunostimulatory bacterium of claim 54 or claim 55, wherein the modifications, which are amino acid replacements, are selected as follows:a) in STING, with reference to SEQ ID N0s:305-309, one or more selected from among: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A,R310A / E316A, E316A, E316N, E316Q, S272A, R375A, R293A / T294A / E296A,D231A, R232A, K236A, Q273A, S358A / E360A / S366A,D231 A / R232 A / K236 A / R238 A, S358A, E360A, S366A, R238A, and S324A / S326A; b) in MDA5, with reference to SEQ ID NO:310, one or more of: T33 II, T331R, A489T, R822Q, G821S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F, and A452T;c) in RIG-I, with reference to SEQ ID NO:311, one or both of E373 A andC268F;d) in IRF-3, with reference to SEQ ID NO: 312, S396D;e) in IRF-7, with reference to SEQ ID NO:313, one or more of:S477D / S479D, S475D / S477D / S479D, andS475D / S476D / S477D / S479D / S483D / S487D; andf) conservative amino acids replacements that increase activity or render type I interferon expression constitutive.

57. The immunostimulatory bacterium of any of claims 54-56, wherein the immunostimulatory protein is a variant STING that contains one or more amino acid replacement(s) selected, with reference to SEQ ID N0s:305-309, from among:S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231 A / R232A / K236A / R238A, S358A, E360A, S366A,R238A, R375A, and S324A / S326A.

58. An immunostimulatory bacterium, comprising nucleic acids encoding a modified STING protein, which is an immunostimulatory protein that, inunmodified form, senses cytosolic RNA / DNA, wherein the STING protein is encoded on a plasmid and the encoding nucleic acid is operatively linked to regulatory sequences recognized by a eukaryotic host.

59. The immunostimulatory bacterium of claim 58, wherein the modified STING proteindoes not require cyclic dinucleotides (CDNs) for activity.

60. The immunostimulatory bacterium of claim 58 or claim 59, comprising a sequence of nucleotides encoding a modified STING protein, wherein:the modification(s) in STING render its activity constitutive so that it does not require cGAMP for activity;the modified STING protein is encoded by a modified TMEM173 gene;the modification(s) comprise insertions, deletions or replacements of amino acid(s); andthe modified STING protein has enhanced immunostimulatory activity compared to the unmodified STING protein.

61. The immunostimulatory bacterium of claim 59 or claim 60, wherein the amino replacement(s) in STING, with reference to SEQ ID N0s:305-309, is one or more selected from among: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A,S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A,R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A,D231 A / R232 A / K236 A / R238 A, S358A, E360A, S366A, R238A, R375A, andS324A / S326A, and conservative amino acid replacements thereof.

62. The immunostimulatory bacterium of any of claims 30-61, comprising a mutation in the genome of the bacterium that reduces toxicity or infectivity of non- immune cells in a host.

63. The immunostimulatory bacterium of any of claims 30-62, wherein the genome of the immunostimulatory bacterium is modified whereby the bacterium is flagellin (fliC / fljBt ) and / or pagF .

64. The immunostimulatory bacterium of any of claims 30-63 wherein the genome of the immunostimulatory bacterium is modified whereby the bacterium is flagellin- (fliC / fljBr).

65. The immunostimulatory bacterium of any of claims 30-64, wherein the genome of the immunostimulatory bacterium is modified whereby the bacterium is pagP / msbB .

66. The immunostimulatory bacterium of any of claims 30-63, wherein the bacterium is flagellin (fliC / fljB ) and pagP~.

67. The immunostimulatory bacterium of any of claims 30-66, wherein the immunostimulatory bacterium is aspartate-semialdehyde dehydrogenase ( asc ).

68. The immunostimulatory bacterium of any of claims 30-66 that is aspartate-semialdehyde dehydrogenase (asct), wherein the bacterium is asct by virtue of disruption or deletion of all or a portion of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (asct), whereby endogenous asd is not expressed.

69. The immunostimulatory bacterium of any of claims 30-68 that encodes aspartate-semialdehyde dehydrogenase (asct) on the plasmid under control of a bacterial promoter.

70. The immunostimulatory bacterium of any of claims 30-69 that is msbB .

71. The immunostimulatory bacterium of any of claims 30-70 that is purl (purM).

72. The immunostimulatory bacterium of any of claims 30-71 that is asct, purl , msbB , flagellin (fliC / fljR), and pagP~.

73. The immunostimulatory bacterium of any of claims 30-72, wherein one or more genes or operons involved in SPI-1 -dependent invasion are deleted or inactivated, whereby the immunostimulatory bacterium does not invade or infect epithelial cells.

74. The immunostimulatory bacterium of claim 73, wherein one or more of aw A, hi I A , hill), invA, invB, invC, invE , invF, invG, invH , invl , invJ , iacP, iagB, spaO, spaQ, spaR, spaS, orgA, orgB , orgC, prgH, prgl, prgj, prgK , .v / cri , sicP, sip A, sipB, sipC , .SY / Y / J, SZVC, .so / ; / / , sop / ), sopE , sopE2, sprB, and .s / ; / / Jis deleted or inactivated.

75. The immunostimulatory bacterium of any of claims 30-72, wherein one or more genes involved in SPI-1 -independent invasion are deleted or inactivated,, wherein:the immunostimulatory bacterium does not invade or infect epithelial cells; andthe one or more genes is / are selected from among pagN, hlyE, pefl, srgl), srgA , srgB , and srgC.

76. The immunostimulatory bacterium of any of claims 30-75, comprising: a modification of the bacterial genome, whereby the bacterium induces less cell death in tumor-resident immune cells; and / ora modification of the bacterial genome, whereby the bacterium preferentially infects tumor-resident immune cells compared to other cell types.

77. The immunostimulatory bacterium of any of claims 30-76, wherein the bacterium encodes asd on a plasmid, and its expression is under the control of a promoter expressed in a tumor microenvironment (TME) of a eukaryotic subject.

78. The immunostimulatory bacterium of any of claims 30-77 that is auxotrophic for adenosine and adenine.

79. The immunostimulatory bacterium of any of claims 30-77 that is auxotrophic for adenosine.

80. The immunostimulatory bacterium of any of claims 30-79 that is auxotrophic for adenosine and lacks flagella, wherein the wild-type bacterium has flagella.

81. The immunostimulatory bacterium of any of claims 30-80, comprising a mutation in the genome of the bacterium that reduces toxicity or infectivity of non- immune cells in a host.

82. The immunostimulatory bacterium of any of claims 30-81, wherein the bacterium is pagP~.

83. The immunostimulatory bacterium of any of claims 30-82, wherein the immunostimulatory bacterium is flagellin fliC / fljB ) and / or pagP , and one or more of purl (purM ), msbB purl) , adrA , csgD , qseC , and hi l A , IppA , or IrrB .

84. The immunostimulatory bacterium of any of claims 30-83, wherein the immunostimulatory bacterium is hilA pagP~, and flagellin (fliC / fljB ).

85. The immunostimulatory bacterium of any of claims 30-84 that comprises a nucleic acid encoding a CpG motif, wherein the nucleic acid encoding the CpG motif is included in or is part of a bacterial gene that is encoded on the plasmid.

86. The immunostimulatory bacterium of claim 85, wherein the gene that comprises CpGs is asd , wherein the asd is encoded on the plasmid.

87. The immunostimulatory bacterium of any of claims 30-86 that is flagellin deficient, wherein the wild-type bacterium comprises flagella.

88. The immunostimulatory bacterium of any of claims 30-87, wherein the plasmid comprises a nucleic acid encoding cytoLLO, which is listeriolysin O (LLO) protein lacking the signal sequence.

89. The immunostimulatory bacterium of any of claims 30-88 that comprises a DNA nuclear targeting sequence (DTS) encoded on the plasmid.

90. The immunostimulatory bacterium of claim 89, wherein the DTS is an SV40 DTS.

91. The immunostimulatory bacterium of any of claims 30-90 that has a deletion or modification in the gene encoding endonuclease I ( endA ), whereby end A activity is inhibited or eliminated.

92. The immunostimulatory bacterium of any of claims 30-91, wherein the plasmid comprises one or more of a CpG motif, an asd gene selectable marker for plasmid maintenance and a DNA nuclear targeting sequence.

93. The immunostimulatory bacterium of any of claims 30-92, wherein the immunostimulatory bacterium comprises a purl deletion, an msbB deletion, an asd deletion, and an adrA deletion.

94. The immunostimulatory bacterium of any of claims 30-93, wherein the immunostimulatory bacterium comprises:one or more of a mutation in a gene that alters the biosynthesis oflipopolysaccharide, selected from among one or more of rfaL, rfaG , rfaH rfaD , rfaP , rFb , rfa, msbB , htrB,firA,pagL,pagP , IpxR, arnd , eplA , and IpxT ; and / orone or more of a mutation that introduces a suicide gene and is selected from among one or more of sacB , nuk, hok , gef kil and phi A ; and / orone or more of a mutation that introduces a bacterial lysis gene and is selected from among one or both of hly and cly and / ora mutation in one or more virulence factor(s) selected from among isyA,pag , prg, iscA, virG,plc and acl and / orone or more of a mutation in a gene that modifies the stress response, selected from among recA , htrA , htpR , hsp and groEL; and / ora mutation in min that disrupts the cell cycle; and / orone or more of a mutation in a gene that disrupts or inactivates regulatory functions, selected from among cya, crp , phoP / phoQ and ompR.

95. The immunostimulatory bacterium of any of claims 30-94 that is msbB / asd IpurP.

96. The immunostimulatory bacterium of any of claims 30-95 in which prgH and / or prgK are inactivated or deleted.

97. The immunostimulatory bacterium of any of claims 30-96, wherein the nucleic acid encoding the immunostimulatory protein or STING protein on the plasmid is operatively linked to nucleic acid encoding a secretory signal, whereby, upon expression in a host, the product is secreted.

98. The immunostimulatory bacterium of any of claims 30-97, wherein the plasmid encoding the immunostimulatory protein is present in low copy number or medium copy number.

99. The immunostimulatory bacterium of any of claims 30-98, wherein the plasmid comprises a medium-to-low copy number origin of replication.

100. The immunostimulatory bacterium of any of claims 30-98, wherein the plasmid comprises a low copy number origin of replication.

101. The immunostimulatory bacterium of any of claims 30-100, wherein the plasmid is present in low copy number.

102. The immunostimulatory bacterium of any of claims 98-101, wherein medium copy number is less than 150 or less than about 150 and more than 20 or about 20 or is between 20 or 25 and 150.

103. The immunostimulatory bacterium of claim 101, wherein low copy number is less than 25 or less than 20 or less than about 25 or less than about 20 copies.

104. The immunostimulatory bacterium of any of claims 30-103, wherein the origin of replication in the plasmid is selected from among the origins derived from pBR322, pl5A, pSClOl, pMBl, colEl, colE2, pPSlO, R6K, Rl, RK2, and pUC.

105. The immunostimulatory bacterium of any of claims 30-104, wherein the nucleic acid encoding the immunostimulatory protein or STING protein, whose unmodified form senses cytosolic RNA / DNA, is on a plasmid and is operatively linked to regulatory sequences recognized by a eukaryotic host.

106. The immunostimulatory bacterium of any of claims 30-105, wherein the plasmid encodes the immunostimulatory protein or STING under control of a eukaryotic promoter.

107. The immunostimulatory bacterium of claim 105 or claim 106, wherein the regulatory sequences comprise an RNA polymerase II promoter or an RNA polymerase III promoter.

108. The immunostimulatory bacterium of claim 105 or claim 106, wherein the regulatory sequences comprise a terminator and / or promoters selected from among SV40, hGH, BGH, chicken beta-globulin, and rbGlob (rabbit globulin) genes.

109. The immunostimulatory bacterium of claim 107, wherein the promoter is an RNA polymerase II promoter.

110. The immunostimulatory bacterium of claim 107, wherein the promoter is an RNA polymerase II promoter that is a viral promoter or a mammalian RNA polymerase II promoter.

111. The immunostimulatory bacterium of claim 110, wherein the promoter is a viral promoter selected from among a cytomegalovirus (CMV) promoter, an SV40 promoter, an Epstein Barr virus (EBV) promoter, a herpes virus promoter, a respiratory syncytial virus (RSV) promoter, and an adenovirus promoter.

112. The immunostimulatory bacterium of any of claims 105-110, wherein the promoter that controls expression of one or more of the encoded heterologous proteins on the plasmid is an elongation factor-1 (EF-1) alpha promoter, or a UBC promoter, or a PGK promoter, an MND promoter, or a C AGG promoter.

113. The immunostimulatory bacterium of any of claims 105-110, wherein the promoter is an EF-1 alpha promoter, a CMV promoter, an SV40 promoter, a PGK promoter, an MND promoter, an EIF4A1 promoter, a CAG promoter, or a CD68 promoter.

114. The immunostimulatory bacterium of claim 113, wherein the promoter is an EF-1 alpha promoter.

115. The immunostimulatory bacterium of any of claims 105-113, wherein the promoter is a viral promoter that is a late promoter.

116. The immunostimulatory bacterium of any of claims 30-115, wherein the bacterium is a Gram-negative bacterium.

117. The immunostimulatory bacterium of any of claims 30-116, wherein the bacterium is Rickettsia rikettsiae , Rickettsia prowazekii , Rickettsia tsutsugamuchi ,Rickettsia mooseri, Rickettsia sibirica , Bordetella bronchiseptica , Neisseria meningitidis , Neisseria gonorrhoeae , Aeromonas eucrenophila , Aeromonas salmonicida , Franciesella tularensis , Corynebacterium pseudotuberculosis ,Citrobacter freundii , Chlamydia pneumoniae , Haemophilus sornnus , Brucella abortus , Mycobacterium intr acellular e, Legionella pneumophila , Rhodococcus equi ,Pseudomonas aeruginosa , Helicobacter mustelae , Vibrio cholerae , Bacillus subtilis , Erysipelothrix rhusiopathiae , Yersinia enterocolitica , Rochalimaea quintana , orAgrobacterium tumerfacium , or an attenuated strain thereof or a modified strain thereof of any of the preceding list of bacterial strains.

118. The immunostimulatory bacterium of any of claims 30-116, wherein the bacterium is a strain of Salmonella , Shigella , E. coli , Bifidobacteriae , Rickettsia , Vibrio , Listeria , Klebsiella , Bordetella , Neisseria , Aeromonas, Francisella , Cholera ,Coryne bacterium, Citrobacter , Chlamydia , Haemophilus , Brucella , Mycobacterium , Mycoplasma , Legionella , Rhodococcus, Pseudomonas , Helicobacter , Bacillus , or Erysipelothrix , or an attenuated strain thereof or a modified strain thereof of any of the preceding list of bacterial strains.

119. The immunostimulatory bacterium of any of claims 30-119 that is an attenuated bacterium.

120. The immunostimulatory bacterium of any of claims 30-120 that is a strain of Salmonella.

121. The immunostimulatory bacterium of claim 120 that is a Salmonella typhimurium strain.

122. The immunostimulatory bacterium of claim 120 or claim 121, wherein the unmodified Salmonella is a wild-type strain.

123. The immunostimulatory bacterium of claim 120 or claim 121, wherein the unmodified Salmonella strain is attenuated.

124. The immunostimulatory bacterium of any of claims 30-123, wherein the immunostimulatory bacterium is derived from an attenuated Salmonella typhimurium strain selected from among strains designated as AST- 100, VNP20009, YS1646 (ATCC #202165), RE88, SL7207, c 8429, c 8431, and c 8468, or is derived from a wild-type bacterium that has all of the identifying characteristics of the strain deposited as ATCC Accession No. 14028, or is strain ATCC 14028.

125. The immunostimulatory bacterium of any of claims 30-124 that comprises, encoded on the plasmid, a combination of:a) nucleic acid encoding one or more of a gain-of-function mutant of STING, RIG-I, MDA5, IRF3, and IRF7; andb) nucleic acid encoding one or more of IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-15, IL-15 / IL-15R alpha chain complex, IL-2 that has attenuated binding to IL-2Ra, IL-2 modified so that it does not bind to IL-2Ra, IL-18, IL-36 gamma,CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, proteins that are involved in or that effect or potentiate recruitment / persistence of T cells, CD40, CD40 Ligand (CD40L), 0X40, 0X40 Ligand (OX40L), 4-1BB, 4-1BB Ligand (4-1BBL), members of the B7-CD28 family, a TGF-beta polypeptide antagonist, and members of the tumor necrosis factor receptor (TNFR) superfamily.

126. The immunostimulatory bacterium of claim 125 that comprises nucleic acid encoding two of the proteins in a) and two of the proteins in b).

127. The immunostimulatory bacterium of claim 125 or claim 126, wherein different promoters and terminators control expression of each protein.

128. The immunostimulatory bacterium of any of claims 125-127, wherein the genes are expressed from a single promoter and comprise a single terminator, and the sequence encoding each protein comprises an Internal Ribosome Entry Site (IRES), whereby the encoded nucleic acid is polycistronic.

129. The immunostimulatory bacterium of any of claims 125-128, wherein the promoters are selected from among EF-1 alpha, CMV, or GAPDH promoters, and the terminators are selected from among SV40, hGH, BGH, and rbGlob.

130. The immunostimulatory bacterium of any of claims 30-129, wherein the plasmid that encodes the protein comprises a construct that includes an enhancer, a promoter, the open reading frame encoding the protein, and a poly A tail.

131. The immunostimulatory bacterium of any of claims 30-130, wherein the plasmid encoding the protein comprises a construct that includes an enhancer, a promoter, an IRES, the open reading frame encoding the protein, and a polyA tail.

132. The immunostimulatory bacterium of any of claims 30-131, wherein the plasmid encoding the protein comprises a construct that includes an enhancer, a promoter, an IRES, a localization sequence, the open reading frame encoding the protein, and a polyA tail.

133. The immunostimulatory bacterium of any of claims 30-132, wherein the construct on the plasmid encoding the protein comprises a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) or a Hepatitis B virus Posttranscriptional Regulatory Element (HPRE).

134. The immunostimulatory bacterium of any of claims 30-133 that has a deletion in the SPI-2 complex.

135. The immunostimulatory bacterium of any of claims 30-134, wherein the bacterium is treated with polymoxin to reduce pyrogenicity.

136. A delivery vehicle, comprising nucleic acids encoding the modified STING protein of any of claims 1-29.

137. A delivery vehicle, comprising nucleic acids encoding a STING protein from a non-human species, wherein the STING protein has type I IFN signaling activity, and attenuated NF-KB signaling activity compared to the NF-KB signaling activity of human STING.

138. The delivery vehicle of claim 136 or claim 137, wherein the human STING protein comprises the sequence set forth in any of SEQ ID N0s:305-309, or is a human allelic variant thereof with at least 98% sequence identity to the sequence of amino acids set forth in any of SEQ ID N0s:305-309.

139. The delivery vehicle of any of claims 136-138, wherein the type I IFN signaling activity is at least or at least about 30% of the type I IFN signaling activity of wild type human STING.

140. The delivery vehicle of any of claims 136-139, wherein the NF-KB signaling activity is less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of the NF-KB signaling activity of human STING.

141. The delivery vehicle of any of claims 136-140, wherein the non-human species is Tasmanian devil, marmoset, cattle, cat, ostrich, boar, bat, manatee, crested ibis, coelacanth, mouse, zebrafish, or ghost shark.

142. A delivery vehicle, comprising nucleic acids encoding animmunostimulatory protein that, when expressed in a subject, leads to constitutive expression of type I IFN, wherein the delivery vehicle is selected from among a nanoparticle, a liposome, an exosome, a bacterium, a virus, a cell, and a microvesicle.

143. The delivery vehicle of claim 142, wherein the immunostimulatory protein is in a signaling pathway that senses or interacts directly or indirectly with cytosolic nucleic acids, nucleotides, dinucleotides, cyclic nucleotides, or cyclic dinucleotides.

144. The delivery vehicle of claim 143, wherein the immunostimulatory protein that is in a pathway that senses or interacts directly or indirectly with cytosolicnucleic acids, nucleotides, dinucleotides, cyclic nucleotides, or cyclic dinucleotides, activates type I IFN.

145. The delivery vehicle of any of claims 142-144, wherein theimmunostimulatory protein is involved in innate DNA / RNA recognition that activates type I interferon, and is selected from among STING, RIG-I, MDA-5, IRF-3, IRF-7, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, and SNRNP200.

146. The delivery vehicle of any of claims 142-145, wherein theimmunostimulatory protein is selected from among STING, MDA5, IRF-3, IRF-7 and RIG-1.

147. The delivery vehicle of claim 146, wherein:The unmodified STING protein has the sequence set forth in any of SEQ ID N0s:305-309;the unmodified MDA5 protein has the sequence set forth in SEQ ID NO: 310; the unmodified RIG-I protein has the sequence set forth in SEQ ID NO: 311 ; the unmodified IRF-3 protein has the sequence set forth in SEQ ID NO:312; andthe unmodified IRF-7 protein has the sequence set forth in SEQ ID NO:313.

148. The delivery vehicle of any of claims 142-147, wherein theimmunostimulatory protein contains an amino acid replacement s) that render(s) its activity constitutive.

149. The delivery vehicle of any of claims 142-148, wherein theimmunostimulatory protein, when it occurs in a subject, results in an interferonopathy.

150. The delivery vehicle of any of claims 142-149, wherein theimmunostimulatory protein is selected from among a variant STING, variant MDA5, variant IRF-3, variant IRF-7, and variant RIG-I that comprises a gain-of-function mutation or mutations.

151. The delivery vehicle of claim 150, wherein the gain-of-function mutation(s) results in constitutive expression of type I interferon.

152. The delivery vehicle of claim 150 or claim 151, wherein the mutations are as follows:a) in STING, with reference to SEQ ID N0s:305-309, one or more selected from among: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A,R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231 A,R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A, and S324A / S326A;b) in MDA5, with reference to SEQ ID NO:310, one or more of: T33 II, T331R, A489T, R822Q, G821S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F, and A452T;c) in RIG-I, with reference to SEQ ID NO:311, one or both of E373 A andC268F;d) in IRF-3, with reference to SEQ ID NO:312, S396D; ande) in IRF-7, with reference to SEQ ID NO:313, one or more of:S477D / S479D, S475D / S477D / S479D, andS475D / S476D / S477D / S479D / S483D / S487D.

153. The delivery vehicle of claim 152, wherein unmodified STING has the sequence set forth in any of SEQ ID N0s:305-309, unmodified MDA5 has the sequence set forth in SEQ ID NO: 310, unmodified RIG-I has the sequence set forth in SEQ ID NO:311, unmodified IRF-3 has the sequence set forth in SEQ ID NO:312, and unmodified IRF-7 has the sequence set forth in SEQ ID NO: 313, and each modified protein has at least 95% sequence identity to the unmodified protein.

154. The delivery vehicle of any of claims 136-153 that is a cell, an exosome, an oncolytic virus or viral vector, a liposome or other lipid-based vehicle, or an immunostimulatory bacterium.

155. The delivery vehicle of claim 154, wherein the delivery vehicle is a cell that is a stem cell or an immune cell.

156. The delivery vehicle of any of claims 136-154 that is an exosome.

157. The delivery vehicle of any of claims 136-154 that is an oncolytic virus.

158. The delivery vehicle of any of claims 136-154 that is an oncolytic virus selected from among an oncolytic adenovirus, adeno-associated virus, retrovirus,herpes simplex virus, rhabdovirus, papillomavirus, vesicular stomatitis virus, measles virus, Newcastle disease virus, picomavirus, Sindbis virus, poxvirus, parvovirus, reovirus, coxsackievirus, influenza virus, mumps virus, poliovirus, Seneca Valley Virus, Sendai Virus, Dengue Virus, poliovirus, semliki forest virus, alphavirus, and flavivirus.

159. The delivery vehicle of claim 158, wherein the oncolytic virus is a vaccinia virus, a herpes virus, a measles virus, or a reovirus.

160. The delivery vehicle of any of claims 136-159, wherein the nucleic acid encoding the immunostimulatory protein is expressed under control of a eukaryotic regulatory sequence.

161. The delivery vehicle of any of claims 136-160, wherein the nucleic acid encoding the immunostimulatory protein is expressed under control of a eukaryotic promoter.

162. The delivery vehicle of any of claims 136-161, wherein the immunostimulatory protein is a constitutively active STING protein.

163. The delivery vehicle of any of claims 136-162 that further comprises nucleic acids encoding an immunostimulatory protein, that, when expressed in a mammalian subject, confers or contributes to anti -turn or immunity in the tumor microenvironment.

164. The delivery vehicle of claim 163, wherein the immunostimulatory protein is a cytokine, a chemokine, a co-stimulatory protein or receptor, or a co stimulatory receptor with the cytoplasmic domain deleted.

165. The delivery vehicle of any of claims 136-164 that is a cell.

166. The delivery vehicle of claim 165, wherein the cell is a stem cell.

167. The delivery vehicle of claim 166, wherein the stem cell is a mesenchymal stem cell (MSC).

168. The delivery vehicle of claim 167, wherein the MSC is genetically modified to express a combination of immunomodulatory cytokines.

169. The delivery vehicle of claim 168, wherein the cytokines areInterleukin 12 (IL-12) and Interleukin 21 (IL-21).

170. The delivery vehicle, STING protein, immunostimulatory protein, or immunostimulatory bacterium of any of claims 1-169, wherein the type I interferon is interferon-a or interferon-b.

171. An isolated cell, comprising the delivery vehicle of any of claims 136- 164.

172. The cell of claim 171 that is an immune cell, a stem cell, a tumor cell, or a primary cell line.

173. The cell of claim 172 that is a hematopoietic cell.

174. The cell of claim 172 that is a T-cell.

175. The cell of any of claims 171-174 that is produced ex vivo by infecting the cell with the delivery vehicle.

176. An isolated cell, comprising an immunostimulatory bacterium, wherein:the immunostimulatory bacterium is modified so that it preferentially infects tumor-resident immune cells, and / or the genome of the immunostimulatory bacterium is modified so that it induces less cell death in tumor-resident immune cells; andthe cell is an immune cell, a stem cell, a cell from a primary cell line, or a tumor cell.

177. The cell of claim 176, wherein the immunostimulatory bacterium is a Salmonella species that is flagellin (fliC / fljB ).

178. An isolated cell or cultured cells, comprising the immunostimulatory bacterium of any of claims 30-135.

179. The cell of any of claims 176-178 that is a T-cell, or a hematopoietic cell.

180. The cell of any of claims 177-179 that is produced ex vivo by infecting the cell with the immunostimulatory bacterium.

181. A method of treatment of cancer, comprising administering the cell of any of claims 176-180 to a subject with a cancer that comprises a solid tumor or is a hematological malignancy.

182. The method of claim 181, wherein the cancer comprises a solid tumor.

183. The method of claim 181 or claim 182, wherein the cancer is metastatic.

184. The method of any of claims 181-183, wherein the cancer is selected from among cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix, liver, gastric cancer, lymphoma, and leukemia.

185. Use of the cell of any of claims 176-180 for the treatment of cancer.

186. The use of claim 185, wherein the cancer comprises a solid tumor or a hematological malignancy.

187. The use of claim 185 or claim 186, wherein the cancer is metastatic.

188. The use of any of claims 185-187, wherein the cancer is selected from among cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix, liver, gastric cancer, lymphoma, and leukemia.

189. A pharmaceutical composition, comprising the modified STING protein of any of claims 1-29, the immunostimulatory bacterium of any of claims SO US, the delivery vehicle of any of claims 136-170, or the cell of any of claims 171- 180, in a pharmaceutically acceptable vehicle.

190. An isolated cell, comprising the modified STING protein of any of claims 1-29, the immunostimulatory bacterium of any of claims 30-135, or the delivery vehicle of any of claims 136-170, wherein the cell is not a zygote of a fertilized human egg.

191. The cell of claim 190 that is an immune cell, a stem cell, a tumor cell, or a primary cell line.

192. The cell of claim 191 that is a hematopoietic cell, wherein the hematopoietic cell is a chimeric antigen myeloid cell that is a macrophage.

193. The cell of claim 191 that is a T-cell.

194. The cell of any of claims 190-193 that is produced ex vivo by infecting the cell with the modified STING protein, immunostimulatory bacterium or delivery vehicle.

195. A method of treatment of cancer, comprising administering the modified STING protein of any of claims 1-29, or the immunostimulatory bacteriumof any of claims 30-135, or the delivery vehicle of any of claims 136-170, or the cell of any of claims 171-180 and 190-194, or the pharmaceutical composition of claim 189, or the cell of any of claims 190-194, to a subject with a cancer that comprises a solid tumor or is a hematological malignancy.

196. The method of claim 195, wherein the cancer comprises a solid tumor.

197. The method of claim 195 or claim 196, wherein the cancer is metastatic.

198. The method of any of claims 195-197, wherein the cancer is selected from among lymphoma, leukemia, gastric cancer, and cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix, and liver.

199. Use of the modified STING protein of any of claims 1-29, or the immunostimulatory bacterium of any of claims 30-135, or the delivery vehicle of any of claims 136-170, or the cell of any of claims 171-180 and 190-194, or the pharmaceutical composition of claim 189, or the cell of any of claims 190-194 for the treatment of cancer.

200. The use of claim 199, wherein the cancer comprises a solid tumor or a hematological malignancy.

201. The use of claim 199 or claim 200, wherein the cancer is metastatic.

202. The use of any of claims 199-201, wherein the cancer is selected from among lymphoma, leukemia, gastric cancer, and cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix, and liver.

203. The modified STING protein, delivery vehicle, immunostimulatory bacterium, pharmaceutical composition, cell, method or use of any of claims 1-202, wherein the non-human STING protein has the sequence of amino acids set forth in any of SEQ ID NOs: 331, 338 or 341-351, or is an allelic variant of the STING protein of each species, having at least 98% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOs: 331, 338 or 341-351.

204. The modified STING protein, delivery vehicle, immunostimulatory bacterium, pharmaceutical composition, cell, method or use of any of claims 1-202, wherein the non-human STING protein or chimera has the sequence of amino acids set forth in any of SEQ ID NOs: 332-337, 339, or 340.

205. A pharmaceutical composition, comprising the immunostimulatory bacterium of any of claims 30-135 in a pharmaceutically acceptable vehicle.

206. The pharmaceutical composition of claim 189 or claim 205 that is formulated for administration without dilution.

207. The pharmaceutical composition of any of claims 189, 205 and 206 that is formulated as a single dose.

208. The pharmaceutical composition of any of claims 189 and 205-207 that is formulated for parenteral administration.

209. A method of treatment of cancer that comprises a solid tumor or a hematological malignancy in a subject, comprising administering the pharmaceutical composition of any of claims 189 and 205-208.

210. Use of the pharmaceutical composition of any of claims 189 and 205- 208 for treatment of a cancer that comprises a solid tumor or a hematological malignancy in a subject.

211. The immunostimulatory bacterium of any of claims 30-135 for use for treatment of a cancer that comprises a solid tumor or a hematological malignancy in a subject.

212. The method or use or immunostimulatory bacterium of any of claims 195-202 and 209-211, wherein the subject is a human.

213. The method or use or immunostimulatory bacterium of any of claims 195-202 and 209-212, wherein the treatment comprises combination therapy in which a second anti-cancer agent or treatment is administered.

214. The method or use or immunostimulatory bacterium of claim 213, wherein the second anti-cancer agent or treatment is administered before,concomitantly with, after, or intermittently with, the immunostimulatory bacterium.

215. The method or use or immunostimulatory bacterium of claim 214, wherein the second anti-cancer agent or treatment is an immunotherapy.

216. The method or use or immunostimulatory bacterium of any of claims 195-202 and 209-215, wherein administration of the protein, cell, delivery vehicle, pharmaceutical composition, or immunostimulatory bacterium is parenteral.

217. The method or use or immunostimulatory bacterium of any of claims 195-202 and 209-216, wherein administration is oral or rectal or by aerosol into the lung or intratumoral.

218. The method or use or immunostimulatory bacterium of any of claims 195-202 and 209-216, wherein administration is intravenously, intramuscularly, or subcutaneously.

219. The method or use or immunostimulatory bacterium of any of claims195-202 and 209-218, wherein the cancer is selected from among leukemia, lymphoma, gastric cancer, and cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix, and liver.

220. The method or use or immunostimulatory bacterium of claim 215, wherein the immunotherapy comprises administration of an anti-PD-1, or anti-PD-Ll, or anti-CTLA-4 antibody.

221. A method of increasing the ability of a therapeutic bacterium to colonize a tumor, comprising modifying the genome, whereby the bacterium is flagellin (fliC / fljR ) and / or pagF .

222. The method of claim 220, wherein the therapeutic bacterium is an immunostimulatory bacterium.

223. The method of claim 221 or claim 222, wherein the bacterium is a Salmonella species.

224. A method of producing an immunostimulatory bacterium or oncolytic virus for treating cancer, comprising:identifying a mutated gain-of-function, constitutively active immuno stimulatory protein that promotes interferonopathies in human patients; andintroducing nucleic acid encoding the identified protein into a tumor-targeting bacterium or virus, whereby the resulting bacterium or virus, when introduced into a subject, promotes immunostimulation of the tumor microenvironment.

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