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

Abstract

This invention provides deliverable immunostimulatory bacteria with enhanced antitumor activity, resulting in increased colony formation in tumors, the tumor microenvironment, and / or tumor-resident immune cells. [Solution] An immunostimulatory bacterium is provided, comprising a plasmid encoding a modified interferon gene-stimulating (STING) protein or a STING protein from a non-human species, wherein the STING protein has type I IFN signaling activity and attenuated NF-κB signaling activity compared to human STING.

Description

[Technical Field]

[0001] Related applications Applicant Actim Therapeutics, Inc. and inventors Christopher Di Thanos, Laura Hicks Glickman, Justin Scoble, Alexandre Charles Michel Ianello, and Haisin Keough claim priority under U.S. Provisional Application 62 / 962,140, ​​filed January 16, 2020, entitled "Immunostimulatory Bacteria Engineered To Colonize Tumors, Tumor-Resident Immune Cells, And The Tumor Microenvironment."

[0002] The applicant also claims the benefit of priority under U.S. Provisional Application 62 / 934,478, filed November 12, 2019, entitled "Immunostimulatory Bacteria Engineered To Colonize Tumors And The Tumor Microenvironment," filed by applicant Actim Therapeutics, Inc. and inventors Christopher Di Thanos, Laura Hicks Glickman, Justin Scoble, and Alexandre Charles Michel Ianello.

[0003] The applicant also claims the benefit of priority under U.S. Provisional Application 62 / 828,990, filed on April 3, 2019, entitled "Salmonella Strains Engineered To Colonize Tumors And The Tumor Microenvironment," filed by applicant Actim Therapeutics, Inc. and inventors Christopher Di Thanos, Laura Hicks Glickman, Justin Scoble and Alexandre Charles Michel Ianello.

[0004] The applicant also claims the benefit of priority under U.S. Provisional Application 62 / 811,521, filed on 27 February 2019, entitled "Tumor-Targeting Microorganisms that Promote Immuno-Stimulation of the Tumor Microenvironment," filed by applicant Actim Therapeutics, Inc. and inventors Christopher Di Thanos, Laura Hicks Glickman, Justin Scoble and Alexandre Charles Michel Ianello.

[0005] To the extent permitted, the subject matter of each of these applications is incorporated herein by reference in whole. Immunostimulatory bacteria provided in each of these applications may be modified as described herein, and such bacteria are incorporated herein by reference.

[0006] Import by referencing electronically submitted sequence listings. We hereby submit an electronic version of the sequence listing, and its contents are incorporated herein by reference in their entirety. The electronic file was created on February 27, 2020, is 603 kilobytes in size, and is named 1706SEQPC.txt. [Background technology]

[0007] background Tumors have developed a deeply immunosuppressive environment. They have invented multiple mechanisms to evade immune surveillance, reprogrammed anti-tumor immune cells to suppress immunosuppression, and continuously mutated their resistance to the latest cancer therapies (e.g., Mahoney et al. (2015) Nat. Rev. Drug Discov. 14(8):561-584). The field of cancer immunotherapy has made significant progress, as evidenced by the clinical success of anti-CTLA4, anti-PD-1, and anti-PD-L1 immune checkpoint antibodies (see, for example, Buchbinder et al. (2015) J. Clin. Invest. 125:3377-3383; Hodi et al. (2015) J. Clin. Invest. 125:3392-4000; and Chen et al. (2015) J. Clin. Invest. 125:3384-3391). Designing immunotherapies that overcome immune tolerance and evasion while limiting the autoimmune-related toxicity of current immunotherapies is a challenge in the field of cancer immunology. Therefore, supplementary and innovative immunotherapies and other treatments are needed. [Overview of the Initiative]

[0008] overview Offered herein are bacteria modified to be immunostimulant for anti-cancer therapy. The immunostimulant bacteria offered herein provide a multifaceted approach to antitumor therapy. The bacteria provide a platform that brings forth numerous pathways to elicit antitumor immunostimulant activity. Bacteria such as the Salmonella species offered herein are meticulously engineered to possess potent antitumor activity through enhanced ability to accumulate in or target tumors, tumor resident immune cells, and / or the tumor microenvironment (TME). This can be achieved, for example, through modifications that alter the type of cells they can infect (targeting), toxicity, ability to evade the immune system such as avoiding inactivation by complement, and / or the environment in which they can grow. Immunostimulant bacteria can also encode, for example, products that induce or enhance immune responses and other therapeutic / anti-cancer agents. The immunostimulant bacteria offered herein can be administered systemically by improving colonization of tumor / tumor microenvironment / tumor resident immune cells and their resistance to complement and other antibacterial immune responses.

[0009] Bacteria inherently stimulate the immune system; bacterial infections induce immune and inflammatory pathways and responses, some of which are desirable for antitumor treatment, while others are undesirable. Modification of bacteria by deletion or modification of genes and products that result in undesirable inflammatory responses, and by addition or modification of genes that induce desirable immunostimulatory antitumor responses, improves the antitumor activity of bacteria.

[0010] Bacteria can accumulate in tumor cells and tissues and replicate within them, thereby lysing the cells. Bacteria can migrate from the administration site and accumulate in other (e.g., distal / metastatic) tumors and tumor cells, providing a long-range effect. The bacteria provided here are modified to preferentially infect and accumulate tumor resident immune cells, tumors, and the tumor microenvironment, and to deliver plasmids and products encoding therapeutic anti-cancer proteins. Here, these properties of the bacteria are utilized to reliably produce immunostimulatory bacteria with multiple antitumor activities and properties that can act individually and synergistically.

[0011] The bacterial genomes provided herein are modified to increase accumulation in tumors and tumor-resident immune cells, as well as in the tumor microenvironment. This is achieved here by deleting or inactivating genes responsible for infection or invasion of non-tumor cells, such as epithelial cells, and / or reducing the cytopathogenicity of the bacteria, particularly to immune cells and tumor-resident immune cells.

[0012] Upon accumulation in tumor-resident immune cells, plasmid-encoded proteins are expressed and secreted to the tumor mesenteric cell (TME) under eukaryotic regulatory signaling. The immunostimulatory bacteria provided herein encode proteins that possess anticancer activity, such as through modification of the antitumor immune response. The bacteria provided herein encode proteins that result in the expression of type I interferon (IFN). Such proteins include STING (interferon gene stimulant) and other immunostimulatory proteins that are part of the cytosolic DNA / RNA sensor pathway that results in the expression of type I IFN, as well as variants of these proteins that increase type I IFN expression or result in constitutive expression of IFN. For example, immunostimulatory proteins include constitutively active variants of cytosolic DNA / RNA sensors, such as those with gain-of-function mutations.

[0013] Compositions, their use, and methods for modulating immune responses for the treatment of diseases, including cancer, are provided. The compositions comprise immunostimulatory bacteria provided herein. Treatment methods and the use of bacteria for treatment are also provided. Subjects to treatment include humans and other primates, pets such as dogs and cats, and other animals such as horses, cattle, and other agricultural and zoo animals.

[0014] Pharmaceutical compositions containing immunostimulatory bacteria, as well as methods and uses for treating proliferative disorders such as diseases and disorders, particularly tumors including solid tumors and hematological malignancies, are provided.

[0015] Methods are also provided for inhibiting the growth or reducing the volume of solid tumors by administering immunostimulatory bacteria or pharmaceutical compositions, or by using compositions for treatment. For example, a method is provided for administering or using a composition containing an effective amount of immunostimulatory bacteria, such as Salmonella species, in a single dose to a subject such as a human patient with solid tumor cancer.

[0016] Immunostimulatory bacteria are provided that encode constitutively active proteins that stimulate or induce the expression of type I IFNs. These immunostimulatory bacteria can also encode RNAi and other antitumor therapeutic agents such as cytokines and chemokines, and the other bacteria and plasmid modifications described herein can be combined in any desired combination.

[0017] Immunostimulating bacteria are provided in which colonization of tumors, the tumor microenvironment, and / or tumor-resident immune cells is enhanced, and antitumor activity is enhanced. The immunostimulating bacteria are modified by deletion of the flagellum-coding gene and / or modification of the gene so that functional flagella are not produced and / or modification of pagP so that pagP is deleted or inactive pagP products are produced. As a result, the immunostimulating bacteria are flagellum - (fuC - / fljB - ) and / or pagP - Separately or in addition to this, immunostimulatory bacteria are pagP - / msbB - It is possible.

[0018] Immunostimulatory bacteria are characterized by the disruption or deletion of all or part of the endogenous gene encoding aspartate semialdehyde dehydrogenase (ASD), etc. - (asd - ) may be the case, and as a result, endogenous ASD is not expressed. Immunostimulatory bacteria can be modified to encode aspartate semialdehyde dehydrogenase (ASD) in a plasmid under the control of a bacterial promoter for bacterial growth in vitro, so that bacterial replication is limited in vivo.

[0019] The immunostimulatory bacterium has, if desired, further genomic modifications such that the bacterium is an adenosine or purine auxotroph. The bacterium can be one or more of, if desired, asd - , purI - and msbB - . Immunostimulatory bacteria such as Salmonella species are modified to encode an immunostimulatory protein that confers antitumor activity in the tumor microenvironment and / or the bacterium preferentially infects immune cells in the tumor microenvironment or tumor resident immune cells and / or is modified to induce cell death of fewer immune cells than other cells. Methods of inhibiting the growth or reducing the volume of solid tumors by administration of immunostimulatory bacteria are also provided.

[0020] A method of increasing tumor colony formation of immunostimulatory bacteria such as Salmonella species by modifying the genome of the immunostimulatory bacterium to be flagellin - (fliC - / fljB - ) such that flagella are not produced and / or pagP - . In particular, the bacterium is a flagellin - ]>adenosine auxotroph and is also asd - . Bacteria that are flagellin - are derived from bacterial species that express flagella.

[0021] Bacteria also include plasmids encoding therapeutic products such as antitumor agents, proteins that increase the immune response of a target, and / or proteins that result in the constitutive expression or increased expression of immunostimulatory proteins such as type I interferons (IFNs), including interferon-β. This includes encoding proteins that stimulate the immune system as part of a pathway resulting in type I IFN expression, and in particular, those that make such proteins constitutively active. Plasmids may also encode immunostimulatory proteins such as cytokines that increase the antitumor immune response in a target. Bacteria also include plasmids encoding anticancer therapeutic agents such as interfering RNAs, including microRNAs, shRNAs, and siRNAs, designed to suppress, inhibit, interfere with, or otherwise silence immune checkpoint genes and products and other targets that play a role in immunosuppressive pathways. Bacteria may also encode tumor antigens in plasmids that stimulate an immune response against tumors. Encoding proteins are expressed under the control of promoters recognized by mammals and eukaryotes, such as animal or viral promoters. Bacteria may express one, two, or more therapeutic proteins / products, including combinations of gain-of-function immunostimulatory proteins and / or cytokines. These heterologous proteins are encoded on plasmids that are promoter-controlled, such as by the RNA polymerase II or III promoter, and are recognized by the eukaryotic host.

[0022] An immunostimulatory bacterium containing a plasmid encoding a product under the control of a eukaryotic promoter, wherein the genome of the immunostimulatory bacterium is modified, thereby enabling the bacterium to produce flagellin - (fuC - / fljB - ) and / or pagP - The bacteria provided are flagellin - (fuC - / fljB - ) and pagP - It may be one or both. These immunostimulatory bacteria exhibit increased tumor / tumor microenvironment and tumor-resident immune cell colony formation, and possess increased antitumor activity.

[0023] An immunostimulatory bacterium containing a plasmid encoding a therapeutic product under the control of a eukaryotic promoter, wherein the genome of the immunostimulatory bacterium is modified, thereby enabling the bacterium to use pagP - / msbB - Bacteria that are also provided are also increased. These bacteria also increase colonization in tumors, tumor resident immune cells, and the tumor microenvironment. The resulting changes in the bacterial membrane and structure modify the host immune response, such as complement activity, so that the bacteria are not eliminated by systemic administration. These bacteria are flagellin - (fuC - / fljB - ) may also include other modifications described herein, including modifications that alter cells to be able to infect and accumulate in the tumor microenvironment, tumor, and tumor-resident immune cells. Therefore, systemic administration of the immunostimulatory bacteria provided herein may result in high levels of tumor / tumor microenvironment and / or tumor-resident immune cell colonization. The immunostimulatory bacteria are purI - (purM - ), asd - and msbB - 1 or more of and flagellin - (fuC - / fljB - ) and pagP - It could be one or both.

[0024] Immunostimulating bacteria are purI - (purM - ), msbB - purD - Flagellin - (fuC - / fljB - ), pagP - adrA - , csgD - qseC - hilA - lppA - and lppB - and especially flagellin - (fuC - / fljB -) and / or pagP - and / or msbB - / pagP - It may be one or more of the following. For example, immunostimulatory bacteria may have genomic mutations such as deletions or destructions that reduce the toxicity or infectivity of non-immune cells in the host. For example, immunostimulatory bacteria may have pagP - It is possible. Another example is the immunostimulatory bacterium flagellin - (fuC - / fljB - ) and pagP - However, this is also possible. Bacteria can be modified to accumulate in tumor-resident immune cells and the tumor microenvironment (TME), express therapeutic products, thereby delivering immunotherapeutic antitumor products to an environment with beneficial activity, and avoiding harmful or toxic side effects from expression in other cells / environments. Nucleic acids encoding immunostimulatory proteins / therapeutic products can be manipulably ligated to nucleic acids encoding secretory signals for expression, thereby causing the immunostimulatory proteins / therapeutic products to be secreted into the tumor microenvironment in the host upon expression.

[0025] As described above, the genomes of immunostimulatory bacteria are also modified so that the bacteria preferentially infect immune cells such as tumor resident immune cells, and / or the genomes are modified so that the bacteria induce less cell death (reduced pyroptosis) of tumor resident immune cells than unmodified bacteria. As a result, immunostimulatory bacteria accumulate in tumors or the tumor microenvironment or tumor resident immune cells, or to a greater extent than unmodified bacteria, thereby delivering immunostimulatory proteins and their constitutively active variants and other therapeutic products to cells, stimulating or inducing the expression of type I interferon. Bacteria are involved in the expression of flagellin - (fuC - / fljB - ), pagP - and msbB - It can be one or more of the above and may include other modifications as listed here.

[0026] Immunostimulatory bacteria are characterized by the disruption or deletion of all or part of the endogenous gene encoding aspartate semialdehyde dehydrogenase (ASD), etc. - (asd - ) This is also possible, and as a result, endogenous ASD is not expressed. These immunostimulatory bacteria can be modified to encode aspartate semialdehyde dehydrogenase (ASD) in plasmids under the control of a bacterial promoter, so that the bacteria can be produced in vitro.

[0027] Immunostimulatory bacteria can be nutritionally demanding of specific nutrients that are abundant or accumulate in the tumor microenvironment, such as adenosine and adenine. They can also be modified to be nutritionally demanding of such nutrients in order to reduce or eliminate their replication capacity. Inactivated / deleted bacterial genomic genes can be compensated for by placing them under the control of promoters recognized by the host.

[0028] Furthermore, the genome of immunostimulatory bacteria is modified to preferentially infect tumor-resident immune cells. This is achieved by deleting or disrupting bacterial genes that play a role in bacterial invasiveness or infectivity and / or in inducing cell death. The bacteria are modified to preferentially infect tumor-resident immune cells and / or induce less cell death in such cells than unmodified bacteria or other cells that the bacteria can infect.

[0029] The immunostimulatory bacteria provided herein may include modifications to the bacterial genome that reduce the cell death of tumor-resident immune cells induced by the bacteria; and / or modifications to the bacterial genome that cause the bacteria to accumulate more efficiently in tumors, TMEs, or tumor-resident immune cells. These immunostimulatory bacteria can be modified so that the bacteria preferentially infect tumor-resident immune cells, and / or the genome of the immunostimulatory bacteria can be modified to reduce the induction of cell death of tumor-resident immune cells (reduced pyroptosis), thereby allowing the immunostimulatory bacteria to accumulate in tumors or the tumor microenvironment or tumor-resident immune cells, thereby delivering a constituently active immunostimulatory protein or other therapeutic product to the cells that stimulates or induces type I IFN expression.

[0030] Immunostimulatory bacteria may contain deletions or modifications of one or more genes or operons involved in SPI-1-dependent invasion (and / or SPI-2), thereby preventing them from invading or infecting epithelial cells. Examples of genes that may be deleted or inactivated include one or more of the following: avrA, hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP. Elimination of the ability to infect epithelial cells can be achieved by manipulating the immunostimulant bacteria described herein to include knockouts or deletions of genes encoding proteins involved in SPI-1 independent invasion, such as one or more genes selected from rck, pagN, hlyE, pefI, srgD, srgA, srgB, and srgC. Similarly, immunostimulant bacteria may include deletions of genes and / or operons in SPI-2, for example, to allow the bacteria to evade Salmonella-containing vacuoles (SCVs). These genes include, for example, sifA, sseJ, sseL, sopD2, pipB2, sseF, sseG, spvB, and steA.

[0031] For example, immunostimulatory bacteria can be modified to reduce pathogenicity compared to unmodified bacteria, thereby reducing infection of epithelial and / or other non-immune cells. These modifications include modifications of the type 3 secretion system (T3SS) or type 4 secretion system (T4SS), such as modifications of the Salmonella SPI-1 pathway or T3SS system described and illustrated herein. Bacteria can also be modified to induce less cell death, such as by deletion or disruption of the nucleic acid encoding PagP (lipid A palmitoyltransferase), which reduces bacterial pathogenicity.

[0032] The genomes of immunostimulatory bacteria provided herein may be modified to increase or promote infection of immune cells, such as phagocytes, particularly in the tumor microenvironment. This may include reduced infection of non-immune cells, such as epithelial cells, or increased infection of immune cells. Bacteria may also be modified to reduce pyroptosis in immune cells. Numerous modifications of bacterial genomes may result in either increased infection of immune cells or decreased pyroptosis, or both. Immunostimulatory bacteria provided herein may include, for example, deletion and / or disruption of genes involved in the SPI-1 T3SS pathway, such as the disruption or deletion of hilA, and / or disruption / deletion of genes encoding flagellin, rod-shaped protein (PrgJ), needle-shaped protein (PrgI), and QseC.

[0033] Therapeutic products encoded in plasmids for expression in eukaryotic hosts such as humans are under the control of eukaryotic regulatory sequences, including eukaryotic promoters such as those recognized by RNA polymerase II or III. These include viral and mammalian RNA polymerase II promoters.

[0034] Examples of viral promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the SV40 promoter, the Epstein-Barr virus (EBV) promoter, the herpesvirus promoter, the respiratory multinuclear virus (RSV) promoter, and the adenovirus promoter. Other RNA polymerase II promoters include, but are not limited to, the elongation factor-1 (EF-1) alpha promoter or the UbC promoter (lentivirus) or the PGK (3-phosphoglycerate kinase) promoter, the synthetic MND promoter, and the CAGG (or CAG) promoter. The synthetic CAG promoter includes the cytomegalovirus (CMV) initial enhancer element (C); the promoter, first exon and first intron of the chicken beta-actin gene (A); and the splice acceptor of the rabbit beta-globin gene (G). MND is a synthetic promoter containing the U3 region of a modified MoMuLV LTR and a myeloproliferative sarcoma virus enhancer (mouse leukemia virus-derived MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substitution; see, e.g., Li et al. (2010) J. Neurosci. Methods 189:56-64). Other strongly controllable or constitutive promoters may be used. Examples of promoters include the EF-1 alpha promoter, CMV, SV40, PGK, EIF4A1, CAG, and CD68 promoters. Regulatory sequences also include terminators, enhancers, secretory, and other transport signals.

[0035] Plasmids contained in immunostimulatory bacteria may exist in low or medium copy numbers, such as due to the selection of origins of replication that result in medium to low copy numbers, such as low copy number origins. It is shown here that the antitumor activity and other properties of bacteria are improved when the plasmid is present in low to medium copy numbers, where medium copy numbers are less than or about less than 150 and greater than or about 20 or 20 or 25 to 150 copies, and low copy numbers are less than or less than 25 or about 25 or less than 20 copies.

[0036] The immunostimulatory bacteria provided herein include any of the strains and bacteria described in U.S. Patent Application 16 / 033,187, which are further modified to express immunostimulatory proteins and / or to preferentially infect tumor microenvironment or tumor-resident immune cells and / or to be less toxic to immune cells within them, as described and illustrated herein.

[0037] Coding Therapeutic Proteins / Products Immunostimulatory bacteria encode therapeutic proteins or products on bacterial plasmids, under the control of a eukaryotic promoter, that, when expressed in mammalian subjects, confer or contribute to antitumor immunity in the tumor microenvironment.

[0038] Products encoded by immunostimulant bacteria include proteins and their variants that are part of the cytosolic DNA / RNA sensor pathway that leads to type I interferon (IFN) expression. These include variant proteins with increased activity and variant proteins that lead to constitutive expression of type I interferon. These also include proteins that, either naturally or through mutation, have reduced signaling activity in pathways that lead to undesirable immune responses, but possess type I interferon-stimulating activity and / or interferon-β-stimulating activity equivalent to or greater than that of native human proteins. In particular, immunostimulant bacteria encode gain-of-function (GOF) variants of immunostimulant proteins that, in their unmodified form, are part of the cytosolic DNA / RNA sensor pathway that leads to type I interferon (IFN) expression. An example is a gain-of-function, constitutively activated variant of an immunostimulant protein that promotes or induces interferonosis in humans, where the genome of the immunostimulant bacterium is modified to preferentially infect tumor resident immune cells and / or the genome of the immunostimulant bacterium is modified to induce less cell death (decreased pyroptosis) in tumor resident immune cells, thereby allowing the immunostimulant bacterium to accumulate in the tumor or tumor microenvironment or tumor resident immune cells, thereby delivering the constitutively activated immunostimulant protein to cells to stimulate or induce the expression of type I IFN. The variant may include mutations that eliminate phosphorylation sites in the immunostimulant protein, thereby reducing nuclear factor-kappa light chain enhancer (NF-κB) signaling in activated B cells. 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 their variants, such as those expressed in interferonial diseases and their constitutively active or increasedly active conservative variants.In one embodiment, these include type I IFN-inducing proteins such as STING, RIG-I, IRF-3, IRF-7, or MDA5, and variants thereof with increased or constitutive activity, where the immunostimulatory protein is STING, RIG-I, IRF-3, IRF-7, or MDA5.

[0039] Therefore, hereby provided are immunostimulatory bacteria comprising a plasmid containing heterologous nucleic acids encoding gain-of-function variants of immunostimulatory proteins, which are part of the cytosolic DNA / RNA sensor pathway resulting in the expression of type I interferon (IFN) in an unmodified form. These gain-of-function proteins are encoded on the plasmid under the control of a promoter and optionally other regulatory signals, eukaryotic regulatory signals including enhancers, poly(A) and transcriptional terminators. The nucleic acids encoding the protein / product on the plasmid may be multiplexed, thereby encoding multiple products. Strategies for polygene co-expression include the use of multiple promoters, fusion proteins, inter-gene protein cleavage sites, intrasequence ribosome entry sites (IRESs), and "self-cleavage" (ribosome skipping) 2A peptides in a single vector. The 2A peptide is an 18-22 amino acid (aa) long viral oligopeptide that mediates polypeptide "cleavage" during translation in eukaryotic cells. Therefore, plasmids encoding therapeutic products are provided under single-promoter control by incorporating 2A self-cleaving peptides such as T2A, P2A, F2A, and E2A between coding regions.

[0040] Unmodified immunostimulatory proteins are proteins that sense cytosolic DNA / RNA in signaling pathways. They include proteins modified with amino acid substitutions or deletions that increase and / or constitutively activate their activity. Provided are immunostimulatory bacteria containing plasmids encoding gain-of-function, constitutively active variants of immunostimulatory proteins. These gain-of-function proteins include proteins that promote or induce interferonism in humans and proteins that result in the constitutive expression of type I interferon in signaling pathways, including selected, gain-of-function variants modified to result in the constitutive expression of type I interferon. Unmodified immunostimulatory proteins sense or interact with cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides to induce type I interferon expression, either directly or indirectly as part of a signaling pathway, while variant proteins induce type I interferon expression in the absence of sensing or interaction with cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides (CDNs). This category includes gain-of-function variants that do not require cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides to express type I interferons. Examples of such proteins include 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.

[0041] In these immunostimulatory bacteria, the encoded variant gain-of-function protein may involve the elimination of a phosphorylation site in the immunostimulatory protein, thereby reducing nuclear factor-kappa light chain enhancer (NF-κB) signaling in activated B cells. Alternatively, the bacteria may contain one or more substitutions of the amino acid serine (S) or threonine (T) at the phosphorylation site to aspartate (D), resulting in increased or constitutive activity. Examples of proteins in the signaling pathway that leads to type I interferon expression include STING, RIG-I, IRF-3, IRF-7, and MDA5. Examples of mutations that result in gain-of-function activity for each of these proteins are described herein. The mutations involve the encoding immunostimulatory protein having variants STING, RIG-I, IRF-3, IRF-7, or MDA5, in which one or more serine (S) or threonine residues phosphorylated as a result of viral infection are replaced with aspartic acid (D), and the resulting variants are phosphorylation mimes that constitutively induce type I interferon. For example, an immunostimulatory bacterium is provided in which the immunostimulatory protein is IRF-3, having one or more substitutions at the residues at positions 385, 386, 396, 398, 402, 404, and 405, with the residues being replaced by aspartic acid residues; this includes IRF-3 with substitution 396D relative to SEQ ID NO: 312 and IRF-3 with mutations 396D / S398D / S402D / T404D / S405D relative to SEQ ID NO: 312.Other examples include immunostimulatory proteins selected from STING, MDA5, IRF-7, and RIG-I, with the following mutations in STING: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, relative to human STING of sequence numbers 305-309. , R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316 A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232 a) One or more selected from A / K236A / R238A, S358A, E360A, S366A, R238A, R375A and S324A / S326A in MDA5, relative to sequence number 310, T331I, T331R, A489T, R822Q, G821S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F and A452T One or more; c) In RIG-I, one or both of E373A and C268F based on SEQ ID NO: 311; and d) In IRF-7, one or more selected from S477D / S479D, S475D / S477D / S479D, S475D / S476D / S477D / S479D / S483D / S487D and Δ247-467 based on SEQ ID NO: 313. Any of these substitutions may be replaced by a conservative mutation according to the table of exemplary conserved amino acid substitutions below.

[0042] Delivery media are also provided, such as exosomes, liposomes, oncolytic viruses, nanoparticles, immunostimulatory bacteria, and other such media, containing nucleic acids encoding gain-of-function proteins and other therapeutic products as described above and elsewhere in this specification. For example, delivery media are provided, including nucleic acids, generally DNA, that encode a gain-of-function immunostimulatory protein, which is part of a signaling pathway that results in the expression of type I interferon. Gain-of-function variants may constitutively induce the expression of type I interferon. For example, these variants include any of those described herein, such as modified STING, where the modification in STING constitutively induces its activity and does not require cGAMP (or other ligand / CDN) for activity; modified STING is encoded by the modified TMEM173 gene; the modification includes amino acid insertions, deletions, or substitutions; and modified STING has enhanced immunostimulatory activity compared to unmodified STING. Based on sequence numbers 305-309, these amino acid substitutions in STING are 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, Includes one or more selected from 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.

[0043] The immunostimulatory bacteria provided herein may also include sequences of nucleotides encoding immunostimulatory proteins that, when expressed in mammalian subjects, confer or contribute to antitumor immunity in the tumor microenvironment; the immunostimulatory proteins are encoded on plasmids in bacteria under the control of eukaryotic promoters. Examples of promoters include, but are not limited to, the elongation factor-1 (EF1) alpha promoter, UbC promoter, PGK promoter, CAGG, or CAG promoter.

[0044] Immunostimulatory bacteria may also encode inhibitory RNA (RNAi) that, when expressed in mammalian subjects, confer or contribute to antitumor immunity. RNAi is encoded on plasmids in bacteria under the control of a eukaryotic promoter. The genome of immunostimulatory bacteria is modified to induce less cell death in tumor-resident immune cells and / or to accumulate in tumor-resident immune cells and the tumor microenvironment / tumor.

[0045] The immunostimulatory bacteria provided herein may also encode other immunostimulatory proteins. Immunostimulatory proteins can be cytokines, such as chemokines. Examples of immunostimulatory proteins include 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 involved in, affecting, or enhancing T cell recruitment / persistence, CD40, CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), B7-CD28 family members, and tumor necrosis factor receptor (TNFR) superfamily members. In one embodiment, these include, for example, IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-15, IL-23, IL-36 gamma, IL-2 with reduced binding to IL-2Ra, IL-15 / IL-15R alpha chain complex, IL-18, IL-2 modified not to bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, CCL3, CCL4, CCL5, proteins involved in, affecting, or enhancing T cell recruitment / persistence, CD40, CD40 ligand, OX40, OX40 ligand, 4-1BB, 4-1BB ligand, B7-CD28 family members, TGF-beta polypeptide antagonists, and tumor necrosis factor receptor (TNFR) superfamily members.

[0046] Immunostimulating bacteria may also contain sequences of nucleotides encoding inhibitory RNA (RNAi) that, if desired, inhibit, suppress, or interfere with the expression of immune checkpoints. RNAi can be encoded on bacterial plasmids. Immunostimulating proteins and, if desired, nucleotides encoding RNAi may be present in plasmids at low to medium copy numbers.

[0047] Immunostimulating bacteria can also encode therapeutic products such as RNAi or CRISPR cassettes that inhibit, suppress or interfere with the expression of immune checkpoints, or other targets that increase the antitumor immune response in the target; RNAi or CRISPR cassettes are encoded on bacterial plasmids. Other therapeutic products include antibodies that bind to immune checkpoints to inhibit their activity, such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies.

[0048] RNAi comprises 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 may be used interchangeably in the embodiments disclosed and described herein. Generally, RNAi is encoded on bacterial plasmids. Plasmids may contain other heterologous nucleic acids encoding products intended to modulate or add activity or products to bacteria, or other such products that can modulate the immune system of the target being treated with the bacteria. Bacterial genes may also be added, deleted, or disrupted. These genes may encode products for bacterial growth and replication, or products that also modulate the host's immune response to bacteria.

[0049] Bacterial species include, but are not limited to, strains of Salmonella, Shigella, Listeria, Escherichia coli, and Bifidobacteriae. For example, species include Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, and Salmonella enteritidis.

[0050] Species include, for example, Salmonella, Shigera, Escherichia coli, Bifidobacteria, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, and Chlamydia. a) A strain of Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, or Erysipelothrix, or an attenuated or modified strain of any of the aforementioned bacterial strains.

[0051] Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Cytrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix.For example, Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, and Citrobacter freundii. Chlamydia freundii, Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana Includes Agrobacterium quintana and Agrobacterium tumerfacium.

[0052] Examples of Salmonella strains, particularly Salmonella typhimurium strains, such as the strain named YS1646 (ATCC #202165) or the strain deposited as VNP20009 and ATCC #14028, or strains possessing all the identified characteristics of ATCC #14028, are illustrated hereby. Other strains include, for example, RE88, SL7207, χ8429, χ8431, and χ8468. Examples of Salmonella strains provided herein are the immunostimulatory bacterial 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-121, AST-122, and AST-123. These strains may be further modified to encode immunostimulatory proteins, which are gain-of-function variants of proteins in signaling pathways that result in the expression of type I interferons or other immunomodulatory proteins. Immunostimulatory bacteria may also encode immunostimulatory proteins that increase immune responses in the tumor microenvironment, such as cytokines. Immunostimulatory bacteria may also be modified to preferentially infect immune cells in the tumor microenvironment or tumor-resident immune cells and / or induce less cell death in such immune cells, as described herein. Their sequences and descriptions are provided in the detailed description, examples, and sequence listings. Immunostimulatory bacteria may be derived from attenuated bacterial strains or attenuated by modifications described herein, such as ASD deletion, which thereby limit replication to in vivo.

[0053] When bacterial genes are modified and referred to herein, it is understood that the nomenclature (name) is based on that of Salmonella species, which are examples of bacteria that can produce immunostimulatory bacteria. Those skilled in the art will recognize that other species may have corresponding proteins, but their nomenclature or designation may differ from that in Salmonella. However, the general descriptions herein may also apply to other bacterial species. For example, flagellin in Salmonella, as shown herein. - (fuC - / fljB -Deletion or inactivation of pagP and / or pagP increases tumor colonization. Similar genes in flagella or similar functions in infection may be modified in other bacterial species to achieve increased tumor colonization. Similarly, inactivation / deletion of bacterial products such as pagP and / or msbB products, 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 involved in the activation of complement pathways or other inflammatory pathways may be deleted, as exemplified here by Salmonella.

[0054] The immunostimulatory bacteria provided herein encode inhibitors of various genes that contribute to the reduction of the antitumor immune response and / or stimulate the immune system, thereby expressing immunostimulatory genes and / or gene products and / or products.

[0055] The immunostimulant bacteria provided herein possess properties that make them immunostimulant. Adenosine nutrient requirements are also immunostimulant. They may also encode therapeutic payloads in plasmids, such as gain-of-function / constitutively active STING variants and other immunostimulant proteins. The effect of this combination is enhanced by the strains provided herein, which have an adenosine nutrient requirement that provides preferential accumulation or recruitment to an adenosine-rich immunosuppressive tumor microenvironment (TME). Adenosine reduction in such TMEs further enhances the immunostimulant effect. Such combinations of traits of any known or manipulable bacterial strains for therapeutic administration provide similar immunostimulant effects.

[0056] The engineered immunostimulatory bacteria provided herein, such as Salmonella typhimurium immunostimulatory bacteria, include multiple synergistic modalities that induce immune reactivation in non-inflammatory tumors (cold tumors) to promote tumor antigen-specific immune responses while inhibiting immune checkpoint pathways that tumors utilize to overcome and evade persistent anti-tumor immunity. Included in these embodiments are adenosine nutrient requirements and enhanced vascular destruction. Improved tumor targeting and enhanced vascular destruction via adenosine nutrient requirements increase potency while localizing inflammation to limit systemic cytokine exposure and autoimmune toxicity observed with other immunotherapy modalities.

[0057] Heterogeneous proteins and RNAs, such as immunostimulant proteins and gain-of-function immunostimulant proteins, are expressed on plasmids under the control of promoters recognized by eukaryotic host cell transcription mechanisms, such as RNA polymerase II (RNAP II) and RNA polymerase III (RNAP III) promoters. The RNAP III promoter is generally constitutively expressed in eukaryotic hosts; the RNAP II promoter may be regulated. The therapeutic product / immunostimulant protein is supplied to a plasmid that is stably expressed by bacteria. Examples of such bacteria are attenuated Salmonella strains, generally those that are attenuated by passage or other methods or modifications described herein, such as adenosine nutrient requirement. An example of a Salmonella strain is a modified Salmonella typhimurium strain with a functional asd gene. These bacteria can be modified to include the carrying of a functional asd gene in the introduced plasmid; this maintains plasmid selection so that an antibiotic-based plasmid maintenance / selection system is not required. Asd-deficient strains that do not contain a functional asd gene in the plasmid autolyze in the host.

[0058] The promoter can be selected based on the tumor cell environment, such as a promoter expressed in the tumor microenvironment (TME), a promoter expressed under hypoxic conditions, or a promoter expressed under conditions where the pH is less than 7.

[0059] Plasmids in any of the bacteria listed above encode therapeutic products. Plasmids can exist in many or few copies. This can be controlled by the selection of factors such as the origin of replication. Low, high, and medium copy number plasmids and origins of replication are well known to those skilled in the art and can be selected. In the embodiments of immunostimulatory bacteria present herein, plasmids can exist in low to medium copy numbers, such as about 150 or fewer copies, or low copy numbers, such as less than about 25 or about 20 or 25 copies. Examples of origins of replication are those derived from pBR322, p15A, pSC101, pMB1, colE1, colE2, pPS10, R6K, R1, RK2, and pUC.

[0060] Plasmids encode therapeutic polypeptides, such as type I interferon-inducing polypeptides, including those expressed in interferonopathy, and / or therapeutic proteins described herein and / or known to those skilled in the art for their use in cancer treatment. Plasmids may also contain nucleic acid sequences encoding a listeriolisin O (LLO) protein lacking a signal sequence (cytoLLO), a CpG motif, a DNA nuclear targeting sequence (DTS), and a retinoic acid-inducible gene-I (RIG-I) binding element. Immunostimulatory bacteria containing nucleic acids may contain a CpG motif recognized by Toll-like receptor 9 (TLR9). The CpG motif may be encoded on a plasmid. The CpG motif may be included in or part of a bacterial gene encoded on a plasmid. For example, a gene containing CpG may be an asd gene encoded on a plasmid. The immunostimulatory bacteria provided herein may contain one or more of the CpG motif, an asd gene selectable marker for plasmid maintenance, and a DNA nuclear targeting sequence.

[0061] Immunostimulatory bacteria may be flagellin-deficient, such as due to a deletion or disruption of a flagellum-coding gene. For example, a deletion in the gene encoding one or both of the flagellin subunits fliC and fljB may result in a flagellum-deficient bacterium, where wild-type bacteria express flagella. Immunostimulatory bacteria may also have a deletion or modification in the gene encoding endonuclease I (endA), thereby inhibiting or eliminating endA activity.

[0062] The immunostimulatory bacteria provided here can grow in DAP-supplemented medium but, when administered to the target, have aspartate semialdehyde dehydrogenase that restricts their replication in vivo. - (asd - ) may be such. Such bacteria are self-limiting and may be advantageous for treatment. Bacteria may have aspartate semialdehyde dehydrogenase (asd) due to the destruction or deletion of all or part of the endogenous gene encoding aspartate semialdehyde dehydrogenase (asd). - This may be the case, and as a result, endogenous ASD is not expressed. In other embodiments, the gene encoding aspartate semialdehyde dehydrogenase may be included in a plasmid for in vivo expression.

[0063] Any immunostimulant bacteria provided herein may generally contain nucleic acids on the plasmid that include a CpG motif or CpG island, where the CpG motif is recognized by Toll-like receptor 9 (TLR9). Nucleic acids encoding CpG motifs or islands are abundant in prokaryotes; therefore, CpG motifs may be included in or part of bacterial genes encoded on plasmids. For example, the bacterial gene asd contains immunostimulant CpG.

[0064] The immunostimulatory bacteria provided herein may be nutrient-requiring for adenosine or for both adenosine and adenine. Any of the bacteria here may be nutrient-requiring for adenosine, which can favorably increase antitumor activity because adenosine accumulates in many tumors and is immunosuppressive.

[0065] The immunostimulatory bacteria provided herein may be flagellin-deficient, where wild-type bacteria possess flagella. Flagellin deficiency can be caused by the disruption or deletion of all or part of one or more genes encoding flagella. For example, immunostimulatory bacteria are provided that have a deletion in one or both of the genes encoding flagellin subunits FliC and FljB, thereby causing the bacteria to be flagellal-deficient.

[0066] The immunostimulatory bacteria provided herein may contain nucleic acids encoding cytoLLO, a listeriolisin O(LLO) protein that lacks a peripheral secretory signaling sequence and accumulates in the cytosol. This mutation is asd - It combines favorably with bacteria. LLO is a cholesterol-dependent pore-forming hemolysin from Listeria monocytogenes that mediates bacterial phagosome evasion. When an autolyzing strain is introduced into a tumor-bearing host such as a human, the bacteria are taken up by phagocytic immune cells and enter vacuoles. In this environment, DAP deletion prevents bacterial replication and leads to bacterial autolysis in the vacuole. The lysis then releases a plasmid, and the accumulated LLO forms pores in the cholesterol-containing vacuolar membrane, allowing the plasmid to be delivered to the host cell cytosol. Here, the therapeutic product can be expressed using host cell mechanisms and released into the tumor microenvironment to perform antitumor therapy.

[0067] Immunostimulating bacteria may contain DNA nuclear targeting sequences (DTSs) encoded on plasmids, such as the SV40 DTS.

[0068] Immunostimulatory bacteria may have deletions or modifications in the gene encoding endonuclease-1 (endA), thereby inhibiting or eliminating endA activity. Examples of these include immunostimulatory bacteria containing one or more CpG motifs, asd gene selectable markers for plasmid maintenance, and DNA nuclear targeting sequences.

[0069] An immunostimulatory bacterium can contain, on a plasmid, a nucleic acid encoding two or more different RNA molecules that inhibit, suppress or interfere with the expression of immune checkpoints or an RNA molecule encoding an inhibitor of a metabolite that is immunosuppressive or in an immunosuppressive pathway.

[0070] A nucleic acid encoding RNAi such as shRNA or miRNA or siRNA can include a transcription terminator after the RNA-encoding nucleic acid. In all embodiments, the RNAi encoded on the plasmid in the immunostimulatory bacterium can be short hairpin-type RNA (shRNA) or microRNA (miRNA).

[0071] The plasmid in any of the immunostimulatory bacteria can also encode an agonist of retinoic acid-inducible gene I (RIG-I) or a sequence of nucleotides that is a RIG-I binding element.

[0072] The immunostimulatory bacterium is purI - (purM - )、msbB - 、purD - 、flagellin - (fliC - / fljB - )、pagP - 、adrA - 、csgD - and hilA - can include one or more deletions of one or more genes, such as one or more of. The immunostimulatory bacterium can be msbB - . For example, the immunostimulatory bacterium can include one or more of a purI deletion, an msbB deletion, an asd deletion and an adrA deletion and optionally a csgD deletion. Examples of bacterial gene deletions / modifications are any of the following. One or more mutations in a gene that modifies the biosynthesis of lipopolysaccharide selected from one or more of rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA and lpxT; and / or A suicide gene is introduced, and one or more mutations selected from one or more of sacB, nuk, hok, gef, kil or phlA are introduced; and / or Introducing a bacterial lysis gene and one or more mutations selected from either hly or cly; and / or Mutations in one or more pathogenic factors selected from IsyA, pag, prg, iscA, virG, plc, and act; and / or One or more mutations that modify the stress response selected from recA, htrA, htpR, hsp, and groEL; and / or Mutations at min that disrupt the cell cycle; and / or One or more mutations that interfere with or inactivate a regulatory function selected from cya, crp, phoP / phoQ, and ompR.

[0073] Immunostimulating bacteria may be strains of Salmonella, Sigella, Escherichia coli, Bifidobacteria, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Cytrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, or Erysipelothrix, or attenuated or modified strains of any of the aforementioned bacterial strains.

[0074] Examples of immunostimulatory bacteria include plasmids containing one or more nucleic acid sequences encoding a listeriolisin O (LLO) protein (cytoLLO) lacking a signal sequence, a CpG motif, a DNA nuclear targeting sequence (DTS), and a retinoic acid-inducible gene-I (RIG-I) binding element.

[0075] When a plasmid contains two or more therapeutic products under the control of separate promoters, each is separated by at least approximately 75 nucleotides or from at least 75 nucleotides to approximately 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 approximately 1600 or 1600 nucleotides (or base pairs), or 75 to 1500 or 1600 nucleotides (or base pairs).

[0076] Other examples of immunostimulant bacteria include those that are nutrient-dependent to adenosine and contain one or more deletions in the flagellum-coding gene; a deletion in endA; a plasmid encoding CytoLLO; a nuclear localization sequence; and an asd plasmid complement, which, in their unmodified form, encode therapeutic products containing gain-of-function variants of immunostimulant proteins that are part of the cytosolic DNA / RNA sensor pathway resulting in type I interferon (IFN) expression, as described herein.

[0077] Such immunostimulatory bacteria include Salmonella strains, such as wild-type Salmonella typhimurice strains, including strains deposited under ATCC deposit number 14028 or strains possessing all the identified characteristics of strains deposited under ATCC deposit number 14028. Other strains include, for example, strains named AST-100, VNP20009, or strain YS1646 (ATCC #202165), and attenuated Salmonella typhimurice strains selected from RE88, SL7207, χ8429, χ8431, and χ8468.

[0078] Immunostimulating bacteria may contain one or more purI deletions, msbB deletions, asd deletions, and adrA deletions, in addition to modifications that increase accumulation in tumor cells and / or reduce cell death, and may encode the immunostimulating proteins described herein. Immunostimulating bacteria also One or more mutations in a gene that modifies the biosynthesis of lipopolysaccharide selected from one or more of rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; and / or Introduction of a suicide gene and one or more mutations selected from one or more of sacB, nuk, hok, gef, kil, and phlA; and / or Introduction of a bacteriolytic gene and one or more mutations selected from one or both of hly and cly; and / or Mutations in one or more virulence factors selected from IsyA, pag, prg, iscA, virG, plc, and act; and / or One or more mutations that modify the stress response selected from recA, htrA, htpR, hsp, and groEL; and / or Mutations in min that interfere with the cell cycle; and / or One or more mutations that interfere with or inactivate a regulatory function selected from cya, crp, phoP / phoQ, and ompR May also be included.

[0079] The strain is msbB - 、asd - 、hilA - And / or flagellin - (fliC - / fljB - ) And / or pagP -It may be one or more of the following. In the unmodified form, therapeutic products such as immunostimulant proteins, including gain-of-function variants of immunostimulant proteins that are part of the cytosolic DNA / RNA sensor pathway leading to type I interferon (IFN) expression, RNAi, and chemokines / cytokines, are expressed under the control of a host-recognized promoter such as the RNAP III promoter or RNAP II promoter, as described herein. Immunostimulant bacteria may be strains of Salmonella, Sigella, Escherichia coli, Bifidobacteria, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Cytrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, or Erysipelothrix, or attenuated or modified strains of any of the aforementioned bacterial strains. Generally, a strain is one that is attenuated in a host, either as an attenuated strain or through modifications that alter its properties, and includes the ability to infect cells and replicate in some or all cells. Salmonella strains such as Salmonella typhimurium are an example of bacteria. Examples of strains include strains named AST-100, VNP20009, or strain YS1646 (ATCC #202165), RE88, SL7207, χ8429, χ8431, χ8468, and Salmonella typhimurium strains derived from the wild-type strain ATCC #14028.

[0080] Compositions comprising immunostimulatory bacteria are provided. Such compositions comprise bacteria and pharmaceutically acceptable excipients or media. Immunostimulatory bacteria include any described herein or in patents / applications incorporated herein or known to those skilled in the art. Such bacteria are modified to encode variants of immunostimulatory proteins that are part of a signaling pathway resulting in the expression of type I interferons. Proteins such as STING proteins are modified to result in increased activity and / or constitutive expression of type I interferons such as interferon-α or interferon-β. Bacteria may also encode immunostimulatory proteins, such as cytokines, that increase antitumor activity in the tumor microenvironment or tumors. The genome of bacteria may be modified to increase the infectivity of immune cells and / or decrease the infectivity of non-immune cells and / or reduce the ability of immune cells to induce cell death. Therefore, bacteria are modified as described herein for the delivery of immunostimulatory proteins that promote accumulation and / or antitumor activity in tumors or the tumor microenvironment or tumor-resident immune cells. Immunostimulating bacteria may further contain plasmids encoding RNAi or CRISPR cassettes, such as miRNA or shRNA, that target immune checkpoints or otherwise enhance the bacteria's antitumor activity.

[0081] A single dose is therapeutically effective in treating diseases or disorders in which immune stimulation results in treatment. Examples of such stimulation include, but are not limited to, specific and nonspecific immune responses, specific and nonspecific immune responses, innate responses, primary immune responses, adaptive immunity, secondary immune responses, memory immune responses, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression.

[0082] Immunostimulatory bacteria that are cGAS agonists are provided. Examples of such bacteria are Salmonella species such as Salmonella typhimurium, which are either cGAS agonists and interferon gene-stimulating agent (STING) agonists, or both. These may be administered in use or method, for example, in radiotherapy and chemotherapy, where cytosolic DNA is produced or accumulated. STING activates innate immunity in a nucleic acid-sensing response in the cytosol. Downstream signaling is activated via the binding of cyclic dinucleotides (CDNs), which are synthesized by the bacterial or host enzyme cGAS in response to binding to cytosolic double-stranded DNA (dsDNA). The phosphate cross-linking structures of bacterial and host-produced CDNs differ, resulting in different abilities to activate STING. CDNs are synthesized by the bacterial or host enzyme cGAS in response to binding to cytosolic dsDNA. IFN-β is the signature cytokine of activated STING.

[0083] Also provided are modified non-human STING proteins and STING protein chimeras, as well as delivery media, proteins and / or pharmaceutical compositions comprising any of the above, including bacteria, liposomes, exosomes, minicells, nanoparticles, vectors, for example, oncolytic viruses, cells encoding or comprising these STING proteins and / or delivery media, and uses and methods for treating the cancer of the same. Modified non-human STING proteins and STING protein chimeras, as well as delivery media, cells, immunostimulatory bacteria, uses, methods and pharmaceutical compositions include, but are not limited to, the following:

[0084] 1. Modified non-human STING proteins, where the non-human STING proteins have lower NF-κB activation than human STING proteins and, optionally, higher type I interferon activation / signaling activity compared to wild-type (WT) human STING proteins. These non-human STING proteins are modified to include one or more mutations to increase activity or to act constitutively in the absence of cytosolic nucleic acid signaling. The mutations are amino acid mutations that generally occur in humans in interferonial diseases, as described above for human STING. The corresponding mutations are introduced into non-human species STING proteins, where the corresponding amino acid residues are identified by alignment (see, for example, Figures 1-13). In one embodiment, the TRAF6 binding site in the C-terminal tail (CTT) of the STING protein is deleted, reducing NF-κB signaling activity.

[0085] 2. Modified STING proteins, particularly human STING proteins, are chimeras in which the CTT (C-terminal tail) region of a STING protein from a certain species, such as humans, is replaced with a CTT from another non-human STING protein that has lower NF-κB signaling activity and / or higher type I IFN signaling activity than human STING. In addition, the TRAF6 binding site is optionally deleted from the CTT in these chimeras.

[0086] 3. Modified STING protein 2, including mutation 1.

[0087] 4. A delivery medium such as an immunostimulatory bacterium provided herein or known to those skilled in the art, which encodes any modified STING protein of any of 1-3, including exosomes, minicells, liposomes, nanoparticles, oncolytic viruses, and other viral vectors.

[0088] 5. Delivery media, such as exosomes, minicells, liposomes, nanoparticles, oncolytic viruses, and other viral vectors, including any immunostimulatory bacteria provided herein or known to those skilled in the art, encoding unmodified STING from non-human species, wherein the STING protein has reduced NF-κB signaling activity compared to human STING and, optionally, increased type I interferon-stimulated / signaling activity.

[0089] 6. Cells used in cell therapy, such as T cells and stem cells, and cells used to produce any of the proteins 1-3 above (non-zygotes if human).

[0090] 7. A pharmaceutical composition comprising STING proteins 1-3, delivery media 4 and 5, or cells 6.

[0091] 8. Uses and methods for treating cancer by administering immunostimulatory bacteria as described herein, in any of the manners described in 1-7.

[0092] 9. Also provided are immunostimulant bacteria encoding non-human STING proteins, particularly those having lower NF-κB activity (signaling activity) and similar or higher type I interferon-stimulating activity or interferon-β stimulating activity compared to human STING.

[0093] Assays and methods for evaluating the NF-κB activity (signaling activity) and type I interferon-stimulating or interferon-β stimulating activity of STING are described herein and are known to those skilled in the art. These methods include, for example, those described in de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175, which, in particular, describes the interferon-β and NF-κB signaling activity of STING proteins from various species, including humans, thereby identifying STING proteins from various species that have lower NF-κB activity than human STING and also have interferon-β activity equivalent to or higher than human STING. de Oliveira Mann et al. (2019) describes species alignments and identifies domains of STING in various species, including the CTT domain (see also Supplemental Information for de Oliveira Mann et al. (2019)).

[0094] Non-human STING proteins may be from, but are not limited to, the following species: Tasmanian devil (Sarcophilus harrisii; SEQ ID NO: 331), marmoset (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 NO: 345-346), wild boar (Sus scrofa; SEQ ID NO: 347), bat (Rousettus aegyptiacus; SEQ ID NO: 348), manatee (Trichechus manatus latirostris; SEQ ID NO: 349), elephant shark (Callorhinchus milii; SEQ ID NO: 350), and 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, demonstrating that the molecular mechanism of STING signaling is shared among vertebrates (see de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175).

[0095] Pharmaceutical compositions comprising any immunostimulatory bacteria and other delivery media are also provided. The same applies to their use for the treatment of cancer and methods for treating cancer. Methods and uses include the treatment of a subject having cancer, comprising administering immunostimulatory bacteria or a pharmaceutical composition to a subject such as a human. Methods for treating a subject having cancer, comprising administering immunostimulatory bacteria, are also provided.

[0096] The methods and uses include combination therapy in which a second anticancer agent or treatment is applied. The second anticancer agent may be a chemotherapeutic agent that delivers cytosolic DNA or radiotherapy or an immune checkpoint inhibitor such as an anti-PD-1 or anti-PD-L1 or anti-CTLA-4 antibody or other therapeutic cells such as CAR-T cells or stem cells, TIL cells and modified cells for cancer treatment.

[0097] Administration may be by any suitable route, such as non-enteral, and may include further agents that can facilitate or enhance delivery. Administration may be oral, rectal, or pulmonary aerosol, or intratumor, intravenous, intramuscular, or subcutaneous. Administration may be by any suitable route, such as non-enteral, systemic, topical, or external, including oral, rectal, or pulmonary aerosol, intratumor, intravenous, intramuscular, or subcutaneous.

[0098] Cancer includes solid tumors and hematological malignancies, such as lymphoma, leukemia, gastric cancer, and cancers of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectal, ovaries, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix, and liver.

[0099] Immunostimulating bacteria can be formulated into administration compositions, such as suspensions. They can be dried and stored as powders. Combinations of immunostimulating bacteria with other anticancer agents are also provided.

[0100] Combination therapies are offered for the treatment of cancer and malignant tumors. Immunostimulating bacteria may be administered intermittently or concurrently with other cancer treatments, including radiotherapy, chemotherapy, particularly genotoxic chemotherapy that delivers cytosolic DNA, and immunotherapy such as checkpoint inhibitor antibodies including anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and other such immunotherapies.

[0101] Also provided are isolated cells containing immunostimulatory bacteria or any other delivery medium such as exosomes, liposomes, and other such media containing nucleic acids encoding gain-of-function variant proteins and other therapeutic agents as described herein. The cells include, but are not limited to, immune cells, stem cells, tumor cells, primary cell lines, and other cells used in cell therapy. Examples of cells include, for example, hematopoietic cells such as T cells and hematopoietic stem cells. Hematopoietic cells may be chimeric antigen myeloid cells such as macrophages. The delivery medium and immunostimulatory bacteria can be introduced into cells ex vivo. Therefore, for example, isolated cells containing immunostimulatory bacteria are provided, where the immunostimulatory bacteria are modified to preferentially infect tumor resident immune cells and / or the genome of the immunostimulatory bacteria is modified to induce less cell death in tumor resident immune cells; and the cells are immune cells, stem cells, cells from primary cell lines, or tumor cells. The cells are used in methods of cell therapy, such as for the treatment of cancer. The cells may be allogeneic or autologous to the target of treatment.

[0102] Methods are also provided for increasing tumor / tumor microenvironment colonization by immunostimulatory bacteria. One such method involves, for example, using bacterial flagellin - (fuC - / fljB - ) and / or pagP - This includes modifying it to be so.

[0103] The terms and expressions used are not intended to be descriptive or limiting, nor are they intended to exclude any equivalent or part of any nature of what is indicated and described; rather, it is recognized that various modifications are intended. [Brief explanation of the drawing]

[0104] [Figure 1] The alignment of wild-type human and Tasmanian devil STING proteins is described.

[0105] [Figure 2]Alignment of wild-type human and marmoset STING proteins is described.

[0106] [Figure 3] Alignment of wild-type human and bovine STING proteins is described.

[0107] [Figure 4] Alignment of wild-type human and feline STING proteins is described.

[0108] [Figure 5] Alignment of wild-type human and ostrich STING proteins is described.

[0109] [Figure 6] Alignment of wild-type human and crested ibis STING proteins is described.

[0110] [Figure 7] Alignment of wild-type human and coelacanth (SEQ ID NO: 345) STING proteins is described.

[0111] [Figure 8] Alignment of wild-type human and zebrafish STING proteins is described.

[0112] [Figure 9] Alignment of wild-type human and wild boar STING proteins is described.

[0113] [Figure 10] Alignment of wild-type human and bat STING proteins is described.

[0114] [Figure 11] The alignment of wild-type human and manatee STING proteins is described.

[0115] [Figure 12] Alignment of wild-type human and elephant shark STING proteins is described.

[0116] [Figure 13] The alignment of wild-type human and mouse STING proteins is described. [Modes for carrying out the invention]

[0117] Detailed description overview A. Definition B. Overview of immunostimulant bacteria C. Cancer immunotherapy 1. Immunotherapy 2. Adoptive immunotherapy 3. Cancer vaccines and oncolytic viruses D. Bacterial cancer immunotherapy 1. Bacterial treatment 2. Comparison of immune responses to bacteria and viruses 3. Salmonella treatment a. Tumor-tropic bacteria b. Salmonella enterica serovar typhimurium (a type of fungus that causes typhimosis in mice) c. Bacterial attenuation i. msbB - mutant ii. purI - mutant iii. Combinations of attenuating mutations iv. VNP20009 and other attenuated Salmonella typhimurium strains v. Salmonella typhimurium engineered to deliver macromolecules 4. Enhancement of immunostimulatory bacteria for increased therapeutic index and expression in tumor-resident immune cells a. ASD gene deletion b. Adenosine nutritional requirements c. Flagellin-deficient strains d. Gene deletion in the LPS biosynthesis pathway e. Deletion of genes necessary for biofilm formation f. Salmonella manipulated to avoid Salmonella-containing vacuoles (SCVs) g. Deletion of SPI-1 and SPI-2 genes and / or other genes to eliminate the ability of bacteria to infect epithelial cells, including the deletion of flagella. i. Salmonella pathogenic island 1 (SPI-1) SPI-1-dependent host cell invasion SPI-1-independent host cell invasion ii. Salmonella pathogenic island 2 (SPI-2) h. Endonuclease-1 (endA) mutations to increase plasmid delivery i. RIG-I binding sequence j. DNase II inhibition k. RNase H2 inhibition l. Stabilin-1 / CLEVER-1 inhibition m. CpG motif and CpG Island 5. Modifications that increase the uptake of Gram-negative bacteria such as Salmonella by immune cells and reduce immune cell death. a. Bacterial uptake by immune cells b. Macrophage pyroptosis i. Flagellin ii. SPI-1 protein Rod-shaped protein (PrgJ) Needle-shaped protein (PrgI) iii. QseC 6. Bacterial culture conditions 7. Increased tumor colony formation E. Gain-of-function mutations in non-human STING proteins and proteins for stimulating immune responses in the tumor microenvironment. 1. Type I interferon and pathway 2. Type I interferonosis and gain-of-function variants 3. STING-mediated immune activation 4. TMEM173 allergen 5. Constitutive STING expression and gain-of-function mutations 6. Non-human STING proteins and variants thereof with increased or constitutive activity, STING chimeras, and variants thereof with increased or constitutive activity 7. Other gene products and constitutive variants that act as cytosolic DNA / RNA sensors a. Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) b. MDA5 / IFIH1 c. RIG-I d. IRF-3 and IRF-7 8. Other type I IFN regulatory proteins 9. Other therapeutic products F. Construction of immunostimulatory bacteria encoding proteins and exemplary plasmids and delivery vehicles 1. Replication origin and plasmid copy number 2. Plasmid maintenance / selection components 3. RNA polymerase promoter 4. DNA nuclear targeting sequence 5. CRISPR G. Other delivery vehicles encoding gain-of-function modified proteins that constitutively induce non-human STING proteins, type I interferons, and other therapeutic products 1. Exosomes, extracellular vesicles, and other vesicular delivery vehicles 2. Oncolytic viruses a. Adenovirus b. Herpes simplex virus c. Poxvirus d. Measles virus e. Reovirus f. Vesicular stomatitis virus (VSV) g. Newcastle disease virus h. Parvovirus i. Coxsackievirus j. Seneca Valley virus[[ID=?]] H. Pharmaceuticals, compositions, and formulations 1. Manufacture a. Cell bank manufacture b. API manufacture c. Pharmaceutical manufacturing 2. Composition 3. Formulation a. Liquids, injections, emulsions b. Dried heat-stable preparations 4. Compositions for other routes of administration 5. Dosage / Dosage 6. Packaging and Products I. Treatment method and use 1. Tumor 2. Administration 3. Monitoring J. Examples

[0118] A. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. All patents, patent applications, published applications and publications, GenBank arrays, databases, websites and other publications cited throughout this disclosure are incorporated herein by reference in their entirety unless otherwise noted. If a term has multiple definitions, the one in this section shall prevail. When URLs or other such identifiers or addresses are referenced, it is understood that such identifiers may change, and certain information on the Internet may appear and disappear, but equivalent information may be found through an Internet search. Such references demonstrate that such information is available and publicly distributed.

[0119] The therapeutic bacteria used here are those that, when administered to subjects such as humans, produce therapeutic effects such as cancer or anti-tumor therapy.

[0120] The immunostimulant bacteria used herein are therapeutic bacteria that, when introduced into a target, accumulate and replicate in immune-privileged tissues and cells, such as tumors, and / or express immunostimulant or immunostimulant products. For example, immunostimulant bacteria are attenuated in the host by reducing toxicity or pathogenicity and / or encoding products that reduce toxicity or pathogenicity, so that, primarily outside of immune-specific environments, the immunostimulant bacteria cannot replicate and / or express their products (or replication / product expression is reduced). The immunostimulant bacteria provided herein are modified to encode or exhibit one or more products that constitute immunostimulant activity. Such products, properties, and traits include, but are not limited to, at least one immunostimulatory protein such as cytokines or costimulatory molecules; DNA / RNA sensors or their gain-of-function variants (e.g., STING, MDA5, RIG-I); RNAi such as siRNA (shRNA and microRNA) that target, interfere with, or inhibit checkpoint genes such as TREX1 and / or PD-L1; CRISPR; or an immune checkpoint inhibitor such as an anti-immune checkpoint antibody. Immunostimulatory bacteria may also include modifications that make the bacteria nutritionally dependent on metabolites that are immunosuppressive or in immunosuppressive pathways, such as adenosine.

[0121] The strain designations VNP20009 (see, for example, International PCT Application Publication WO99 / 13053, and also U.S. Patent 6,863,894) and YS1646 and 41.2.9 used herein are interchangeable and refer to strains deposited with the American Type Culture Collection (ATCC) and assigned deposit number ATCC202165, respectively. VNP20009 refers to a modified attenuated strain of Salmonella typhimurium produced from the wild-type strain ATCC 14028, having deletions in msbB and purI.

[0122] The strain designations YS1456 and 8.7 used herein are interchangeable and refer to strains deposited with ATCC, each assigned deposit number ATCC202164 (see U.S. Patents 6,863,894).

[0123] Interferonosis as used herein refers to disorders associated with the upregulation of interferon due to mutations in gene products involved in pathways that control or induce interferon expression. The activity of the product is usually regulated by mediators such as cytosolic DNA or RNA or nucleotides; when mutated, the activity is constitutive. Type I interferonosis includes a range of conditions, including the severe form of Eicardi-Goutier syndrome (AGS) and mild familial Chilbran lupus (FCL). Nucleic acid molecules encoding mutant products with these properties can be produced in vitro, for example, by comparing the product of an allele with normal activity to result in gain of function in the product, or by selecting mutations that further result in gain of function compared to disease-associated gain-of-function mutants described herein.

[0124] The gain-of-function mutations used here refer to proteins whose activity is increased compared to the same protein without the mutation. For example, if the protein is a receptor, its affinity for the ligand is increased; if it is an enzyme, there is an increase in activity, including constitutive activity.

[0125] The origin of replication used here is the DNA sequence from which replication begins in a chromosome, plasmid, or virus. For small DNA, including bacterial plasmids and small viruses, a single origin is sufficient.

[0126] The origin of replication determines the vector copy number, depending on the selected origin. For example, if the expression vector is derived from the low-copy-number plasmid pBR322, the copy number is approximately 25-50 copies / cell, while if it is derived from the high-copy-number plasmid pUC, it may be 150-200 copies / cell.

[0127] The medium copy number of the plasmid in the cells used herein is about 150 or 150 or less than 150, and the low copy number is 15 - 30, such as 20 or less than 20. The low to medium copy number is less than 150. The high copy number is more than 150 copies / cell.

[0128] The 2A peptide used herein is a viral oligopeptide 18 - 22 amino acids (aa) long that mediates the cleavage of polypeptides during translation in eukaryotic cells. The name "2A" refers to a specific region of the viral genome, and different viral 2As are generally named according to the virus they are derived from. These examples are F2A (foot-and-mouth disease virus 2A), E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (Thosea asigna virus 2A) (for example, for the coding sequences, see Liu et al. (2017) Scientific Reports 7:2193, Figure 1; also see SEQ ID NOs: 367 - 370).

[0129] The CpG motif used herein refers to a pattern of bases that includes an unmethylated central CpG (where "p" refers to the nucleotide phosphodiester bond between consecutive C and G) surrounded by at least one base adjacent to the central CpG (both 3' and 5' sides). CpG oligodeoxynucleotides are at least about 10 nucleotides long and are oligodeoxynucleotides that contain unmethylated CpG. The C of at least 5'CG3' is unmethylated.

[0130] The RIG-I binding sequence used herein refers to a sequence synthesized by RNA pol III from a poly(dA-dT) sequence that can directly activate type I IFN through the RIG-I pathway by virtue of the 5'-triphosphate (5'ppp) structure or through interaction with RIG-I. The RNA contains at least 4 A ribonucleotides (A - A - A - A); this can include 4, 5, 6, 7, 8, 9, 10 or more. The RIG-I binding sequence is introduced into a plasmid in bacteria for the transcription of poly A.

[0131] The term "cytokine" used here is broad and broadly refers to a category of small proteins (approximately 5-20 kDa) that are important for cell signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines are cell signaling molecules that facilitate intercellular communication in immune responses and stimulate the migration of cells toward sites of inflammation, infection, and trauma.

[0132] The term "chemokine" as used herein refers to chemotactic cytokines that bind to chemokine receptors, and includes proteins isolated from natural sources and those synthesized by recombinant means or chemical synthesis. Examples of chemokines include IL-8, IL-10, GCP-2, GRO-α, GRO-β, GRO-γ, ENA-78, PBP, and CTAP. This includes, but is not limited to, III, NAP-2, LAPF-4, MIG(CXCL9), CXCL10, CXCL11, PF4, IP-10, SDF-1α, SDF-1β, SDF-2, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1α(CCL3), MIP-1β(CCL4), MIP-1γ, MIP-2, MIP-2α, MIP-3α, MIP-3β, MIP-4, MIP-5, MDC, HCC-1, ALP, Langkaine, Tim-1, Eotaxin-1, Eotaxin-2, I-309, SCYA17, TRAC, RANTES(CCL5), DC-CK-1, Lymphotactin, Fractalkine, and others known to those skilled in the art. Chemokines are involved in the migration of immune cells to inflammatory sites, as well as the maturation of immune cells and the production of adaptive immune responses.

[0133] As used herein, "immunostimulatory proteins" refer to proteins that exhibit or promote an anti-tumor immune response in the tumor microenvironment. Examples of such proteins include, but are not limited to, GM-CSF, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-36 gamma, IFNα, IFNβ, 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), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), B7-CD28 family members, and TNFR superfamily members, as well as cytokines, chemokines, and costimulatory molecules.

[0134] The cytosolic DNA / RNA sensor pathway used herein is initiated by the presence of DNA, RNA, nucleotides, dinucleotides, cyclic nucleotides and / or cyclic dinucleotides or other nucleic acid molecules that result in type I interferon production. These nucleic acid molecules in the cytosol arise from viruses or bacteria or radiation or other such exposures, leading to the activation of an immune response in the host.

[0135] The immunostimulatory proteins used here that induce innate immune responses, such as type I interferon induction, are proteins that are part of the cytosolic DNA / RNA sensor pathway that leads to the expression of immune response mediators such as type I interferons. For example, as described herein and known to those skilled in the art, cytosolic DNA is sensed by cGAS, leading to cGAMP production and subsequent STING (interferon gene stimulant) / TBK1 (tank-binding kinase 1) / IRF3 (interferon regulator) signaling and type I IFN production. Bacterial cyclic dinucleotides (CDNs such as bacterial cyclic di-AMP) also activate STING. Therefore, STING is an immunostimulatory protein that induces type I interferons. 5'-triphosphate RNA and double-stranded RNA are sensed by RIG-I and MDA-5 alone or MDA-5 / LGP2. This leads to polymerization of mitochondrial MAVS (mitochondrial antiviral signaling protein), and also activates TBK1 and IRF3. Proteins in these pathways are immunostimulatory proteins that result in the expression of innate immune response mediators, such as type I interferons. Immunostimulatory proteins in DNA / RNA sensor pathways can be modified to act in the absence of cytosolic nucleic acids, either by increased activity or constitutively, to elicit an immune response, such as the expression of type I interferons.

[0136] The "carboxy-terminal tail" or "C-terminal tail" (CTT) of the innate immune protein STING used herein refers to the C-terminal portion of the STING protein, which, in the wild-type STING protein, is anchored to the cGAMP-binding domain by a flexible linker region. The CTT contains an IRF3-binding site, a TBK1-binding site, and a TRAF6-binding site. STING promotes the induction of interferon-beta (IFN-β) production via phosphorylation of the STING protein C-terminal tail (CTT) by TANK-binding kinase 1 (TBK1). The interaction between STING and TBK1 is mediated by the stretching of eight evolutionarily conserved amino acid residues in the carboxy-terminal tail (CTT) of STING. TRAF6 catalyzes the formation of the K63-linked ubiquitin chain of STING, resulting in the activation of the transcription factor NF-κB and the induction of an alternative STING-dependent gene expression program. Deletion of the TRAF6-binding site in the CTT may reduce the activation of NF-κB signaling. Substitution of human CTT (or a portion thereof) from certain STINGs in CTT (or its corresponding portion) may reduce NF-κB activation by human STINGs. STING CTT is an unstructured stretch of approximately 40 amino acids containing sequence motifs necessary for STING phosphorylation and IRF3 recruitment (see de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175). Human STING residue 366 has been identified as a primary TBK1 phosphorylation site, which is part of the LxIS motif shared by innate immune adapter 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 including residue L374, which is necessary for TBK1 binding; the LxIS and PxPLR sequences are conserved in the vertebrate STING allele (see de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175). Examples of STING CTT sequences and IRF3, TBK1, and TRAF6 binding sites are shown in the table below. [Table 1]

[0137] The term "bacteria modified to induce less cell death in tumor-resident immune cells" as used herein refers to bacteria that are less virulent than unmodified bacteria or have reduced pathogenicity compared to unmodified bacteria. Examples of such modifications include those that eliminate pyroptosis and those that alter the bacterial LPS profile. These modifications include disruption or deletion of one or more components of the SPI-1 pathway, such as the flagellin gene, hilA, rod-shaped protein, needle-shaped protein, QseC, and pagP.

[0138] The bacteria described here as "modified to preferentially infect tumor-resident immune cells" have genome modifications that reduce their ability to infect non-immune cells. Examples of such modifications include those that disrupt the type 3 or type 4 secretory system or other genes or systems that affect the bacteria's ability to invade non-immune cells. For example, Salmonella may have disrupted / deleted the SPI-1 component, which is necessary for the infection of cells such as epithelial cells but does not affect the infection of immune cells such as phagocytic cells.

[0139] The term "modification" as used herein relates to the modification of the amino acid sequence of a polypeptide or the nucleotide sequence of a nucleic acid molecule, and includes the deletion, insertion, and substitution of amino acids or nucleotides, respectively. Methods for modifying polypeptides are commonplace to those skilled in the art, such as by using recombinant DNA methods.

[0140] The modifications of bacterial genomes, plasmids, or genes used herein include deletions, substitutions, and insertions of nucleic acids.

[0141] RNA interference (RNAi), as used here, is a biological process in which an RNA molecule inhibits gene expression or translation by neutralizing a targeted mRNA molecule in a way that inhibits the translation and, consequently, the expression of a targeted gene.

[0142] The RNA molecules used here, which act via RNAi, are referred to as inhibitory because they silence the expression of the targeted gene. Expression silencing means that the expression of the targeted gene is reduced, suppressed, or inhibited.

[0143] The RNAi-mediated gene silencing used here can be described as inhibiting, suppressing, disrupting, or silencing the expression of a targeted gene. The targeted gene contains a nucleotide sequence that corresponds to the sequence of the inhibitory RNA, thereby silencing mRNA expression. Small interference RNA (siRNA) is a small piece of double-stranded (ds) RNA, usually about 21 nucleotides long, with 3' overhangs (2 nucleotides) at each end that are used to "interfere" with protein translation by binding to messenger RNA (mRNA) at a specific sequence and promoting its degradation. In this process, the siRNA blocks the production of a specific protein based on the nucleotide sequence of the corresponding mRNA. The process is called RNA interference (RNAi), and is also referred to as siRNA silencing or siRNA knockdown. Short hairpin RNA or small hairpin RNA (shRNA) is an artificial RNA molecule with tight hairpin turns that can be used to silence targeted gene expression via RNA interference (RNAi). ShRNA expression in cells is generally achieved via plasmid delivery or viral or bacterial vectors.

[0144] The inhibition, suppression, disruption, or silencing of a targeted gene as used herein refers to a process that modifies the expression of the targeted gene, such as translation, thereby reducing the activity or expression of the product encoded by the targeted gene. Reduction includes complete knockout or partial knockout, thereby performing the treatment in relation to the immunostimulant bacteria provided herein and the administration herein.

[0145] The tumor microenvironment (TME) as used herein is the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). The conditions present include, but are not limited to, increased angiogenesis, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure, and metabolites or metabolic modifications such as high adenosine levels, which are signs of tumority.

[0146] The human type I interferons (IFNs) used here are a subgroup of interferon proteins that regulate the activity of the immune system. All type I IFNs bind to specific cell surface receptor complexes, such as the IFN-α receptor. Type I interferons include, among others, IFN-α and IFN-β. IFN-β proteins are produced by fibroblasts and have antiviral activity, primarily involved in the innate immune response. The two types of IFN-β are IFN-β1 (IFNB1) and IFN-β3 (IFNB3).

[0147] The statement that a nucleic acid or encoding RNA used herein targets a gene means that it inhibits, represses, or silences gene expression by some mechanism. Generally, such nucleic acids contain at least a portion complementary to the targeted gene, where the portion is sufficient to form a hybrid with the complementary portion.

[0148] As used herein, "deletion" refers to the removal of one or more nucleotides or amino acids when compared to a nucleic acid or polypeptide sequence, or to a target polynucleotide or polypeptide sequence, or to a natural or wild-type sequence.

[0149] As used herein, "insertion" refers to the addition or inclusion of one or more nucleotides or amino acids into a target, natural, wild-type, or other relevant sequence when comparing a nucleic acid or amino acid sequence. Therefore, a nucleic acid molecule containing one or more insertions compared to a wild-type sequence contains one or more added nucleotides in the linear length of the sequence.

[0150] As used herein, "addition" to nucleic acid and amino acid sequences refers to the addition of a nucleotide or amino acid to any end of a sequence compared to other sequences.

[0151] As used herein, "substitution" or "replacement" refers to the replacement of one or more nucleotides or amino acids with alternative nucleotides or amino acids in a natural, target, wild-type, or other nucleic acid or polypeptide sequence without changing the length of the molecule (expressed in terms of the number of residues). Therefore, one or more substitutions in a molecule do not change the number of amino acid residues or nucleotides in the molecule. Amino acid substitutions compared to a specific polypeptide may be expressed in terms of the number of amino acid residues along the length of the polypeptide sequence.

[0152] The descriptions of the "corresponding positions" of sequences and their corresponding nucleotide or amino acid positions, as shown in the sequence listings used herein, refer to nucleotide or amino acid positions identified by alignment with the disclosed sequences to maximize identity using a standard alignment algorithm such as the GAP algorithm. By aligning the sequences, those skilled in the art can identify the corresponding residues, for example, by using conserved and identical amino acid residues as guides. Generally, amino acid sequences are arranged to obtain top-level matches in order to identify corresponding positions (see, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, 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).

[0153] As used herein, sequence alignment refers to the use of homology for the alignment of two or more nucleotide sequences or amino acids. Generally, two or more related sequences are aligned by 50% or more identity. An aligned sequence set refers to two or more sequences aligned at corresponding positions, and includes the alignment of RNA-derived sequences such as ESTs and other cDNAs aligned with genomic DNA sequences. Related or variant polypeptides or nucleic acid molecules can be aligned in any way known to those skilled in the art. Such methods generally include maximizing the match and using manual alignment and the use of one of the many available alignment programs (e.g., BLASTP) and others known to those skilled in the art. By aligning polypeptide or nucleic acid sequences, those skilled in the art can identify similar regions or positions using conserved and identical amino acid residues as guides. Furthermore, those skilled in the art can also find corresponding amino acid or nucleotide residues between human and non-human sequences using conserved amino acid or nucleotide residues as guides. Corresponding positions can also be based on structural alignment, for example, by using alignment obtained by computer simulation of protein structures. In other examples, corresponding regions can be identified. Those skilled in the art can also use conserved amino acid residues as guides to discover corresponding amino acid residues between human and non-human sequences.

[0154] The "properties" of polypeptides such as antibodies used herein refer to all properties exhibited by the polypeptide, including, but not limited to, binding specificity, structural arrangement or three-dimensional structure, protein stability, resistance to proteolysis, steric stability, thermal tolerance, and tolerance to pH conditions. Changes in properties can alter the "activity" of the polypeptide. For example, changes in the binding specificity of an antibody polypeptide can alter the polypeptide's ability to bind to an antigen and / or various binding activities, such as affinity, avidity, or in vivo activity.

[0155] The “activity” or “functional activity” of polypeptides such as antibodies used herein refers to any activity exhibited by the polypeptide. Such activity can be determined empirically. Examples of activity include, but are not limited to, the ability to interact with biomolecules via antigen binding, DNA binding, ligand binding or dimerization, or enzymatic activity, such as kinase activity or proteolytic activity. For antibodies (including antibody fragments), activity includes, but is not limited to, the ability to specifically bind to a particular antigen, antigen binding affinity (e.g., high or low affinity), antigen binding avidity (e.g., high or low avidity), binding rate, dissociation rate, effector functions such as the ability to promote antigen neutralization or elimination, viral neutralization, and in vivo activity such as the ability to prevent or eliminate infection or invasion by pathogens or to promote penetration into specific tissues, fluids or cells in the body. Activity can be evaluated in vitro or in vivo using recognized assays such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays to measure binding or dissociation rates, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry and binding assays (e.g., panning assays).

[0156] The terms "bonding," "bonded," or their grammatical variations used herein refer to the participation of molecules in some attractive interaction with another molecule, resulting in a stable association where two molecules are in close proximity to each other. Bonding includes, but is not limited to, non-covalent bonds and covalent bonds (e.g., reversible and irreversible covalent bonds), and includes, but is not limited to, interactions between molecules such as small molecules found in proteins, nucleic acids, carbohydrates, lipids, and chemical compounds in drugs.

[0157] As used herein, "antibody" refers to an immunoglobulin fragment, including a fragment of an immunoglobulin molecule that contains at least a portion of the variable heavy and light chain regions of an immunoglobulin molecule, whether naturally occurring or partially or completely synthesized by recombinant production, etc., sufficient for the formation of an immunoglobulin and an antigen-binding site, and which, when assembled, specifically binds to an antigen. Therefore, an antibody includes any protein having a binding domain that is homologous or substantially homologous to the immunoglobulin antigen-binding domain (antibody-binding site). For example, an antibody refers to an antibody comprising two heavy chains (which may be denoted as H and H') and two light chains (which may be denoted as L and L'), where each heavy chain may be a full-length immunoglobulin heavy chain or a portion thereof sufficient for the formation of an antigen-binding site (e.g., the heavy chains include, but are not limited to, the VH chain, VH-CH1 chain, and VH-CH1-CH2-CH3 chain), and each light chain may be a full-length light chain or a portion thereof sufficient for the formation of an antigen-binding site (e.g., the light chains include, but are not limited to, the VL chain and VL-CL chain). Each heavy chain (H and H') pairs with one light chain (each L and L'). Generally, an antibody contains at least all or part of the variable heavy (VH) chain and / or variable light (VL) chains. The antibody may also contain all or part of the constant region.

[0158] For the purposes herein, the term antibody includes full-length antibodies and some of them, including antibody fragments such as anti-EGFR antibody fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), bispecific antibodies, anti-idiotype (anti-Id) antibodies, or any of the antigen-binding fragments described above. Antibodies also include synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies. The antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or sub-subclasses (e.g., IgG2a and IgG2b).

[0159] As used herein, “nucleic acid” generally refers to at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), linked by phosphodiester bonds. The term “nucleic acid” also includes analogs of nucleic acids, such as peptide nucleic acids (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acids also include DNA and RNA derivatives that include nucleotide analogs or “backbone” bonds other than, for example, phosphodiester bonds, such as phosphotriester bonds, phosphoramidate bonds, phosphorothioate bonds, thioester bonds, or peptide bonds (peptide nucleic acids). The term also includes, as equivalents, variants and analogs of RNA or DNA produced from derivatives, nucleotide analogs, single-stranded (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For RNA, the uracil base is uridine.

[0160] The term "isolated nucleic acid molecule" as used herein refers to a molecule isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of culture medium when produced by other cellular material or recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Examples of isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding resistant antibodies or antigen-binding fragments.

[0161] With respect to nucleic acid sequences, regions, elements, or domains used herein, "operably linked" means that the nucleic acid regions are functionally related to one another. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, thereby allowing the nucleic acid to be transcribed and translated to express a functional fusion protein, where the leader peptide causes the secretion of the fusion polypeptide. In some examples, a nucleic acid encoding a primary polypeptide (e.g., a leader peptide) can be operably linked to a nucleic acid encoding a secondary polypeptide, where the nucleic acid is transcribed as a single mRNA transcript, but the translation of the mRNA transcript may result in the expression of one of the two polypeptides. For example, when an amber stop codon is introduced into a partial amber suppressor cell, it may be positioned between the nucleic acid encoding the primary polypeptide and the nucleic acid encoding the secondary polypeptide so that the resulting single mRNA transcript can be translated to produce a fusion protein containing both the primary and secondary polypeptides, or to produce only the primary polypeptide. In other examples, a promoter can be operably linked to a nucleic acid encoding a polypeptide, thereby allowing the promoter to control or mediate the transcription of the nucleic acid.

[0162] For example, with respect to synthetic nucleic acid molecules, synthetic genes, or synthetic peptides, the term "synthetic" as used herein refers to nucleic acid molecules or polypeptide molecules produced by recombinant and / or chemical synthesis methods.

[0163] The naturally occurring α-amino acid residues used here are 20 naturally occurring α-amino acid residues that are incorporated into proteins in humans through the specific recognition of the accompanying tRNA molecule by homologous mRNA codons.

[0164] As used here, "polypeptide" refers to two or more amino acids linked by a covalent bond. The terms "polypeptide" and "protein" are interchangeable in this context.

[0165] The term "peptide" used here refers to polypeptides that are 2 to approximately 40 or exactly 40 amino acids long.

[0166] The term "amino acid" as used herein refers to an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For the purposes described herein, the amino acids contained in the antibodies provided include 20 naturally occurring amino acids (see table below), unnatural amino acids, and amino acid analogs (e.g., amino acids with an α-carbon side chain). The amino acids used herein, present in the various amino acid sequences of the polypeptides described herein, are identified by well-known three-letter or one-letter abbreviations (see table below). Nucleotides present in various nucleic acid molecules and fragments are designated by standard one-letter nomenclature commonly used in this field.

[0167] The term "amino acid residue" used herein refers to an amino acid formed by the chemical digestion (hydrolysis) of a polypeptide via peptide bonds. The amino acid residues described herein are generally "L" isomers. "D" isomers can be substituted with any L-amino acid residue, provided that the desired functional properties are maintained by the polypeptide. NH2 refers to the free amino group at the amino terminus of the polypeptide. COOH refers to the free carboxyl group at the carboxyl terminus of the polypeptide. The abbreviations for amino acid residues are shown in the table below, following the standard polypeptide nomenclature described in J. Biol. Chem., 243:3557-59 (1968) and approved in 37 CFR §§ 1.821-1.822. [Table 2]

[0168] The sequences of all amino acid residues shown herein by formula have a left-to-right orientation in the conventional direction from the amino terminus to the carboxyl terminus. The term "amino acid residue" is defined to include the amino acids, modified, unnatural, and unusual amino acids listed in the correspondence table above. The dotted lines at the start or end of the amino acid residue sequence indicate a further sequence of one or more amino acid residues or a peptide bond to an amino-terminal group such as NH2 or a carboxyl-terminal group such as COOH.

[0169] In peptides or proteins, appropriate conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0170] Such substitutions, such as those in gain-of-function mutations described and provided herein, can be performed according to the example substitution sets shown in the table below. [Table 3]

[0171] Other substitutions are also permissible and may be determined empirically or according to other known conservative or non-conservative substitutions.

[0172] The term "naturally occurring amino acids" used here refers to the 20 L-amino acids found in polypeptides.

[0173] The term "non-natural amino acid" as used herein refers to organic compounds that have a structure similar to natural amino acids but are structurally modified to mimic the structure and reactivity of natural amino acids. Therefore, amino acids that do not exist in nature include, for example, 20 amino acids other than those found in nature, or analogs of amino acids, and include, but are not limited to, D-stereoisomers of amino acids. Examples of non-natural amino acids are known to those skilled in the art, including 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β-alanine / β-aminopropionic acid (Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid / piperidine acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), N -Includes, but is not limited to, ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), allo-hydroxylysine (Ahyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), allo-isoleucine (Aile), N-methylglycine, sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle), and ornithine (Orn).

[0174] The DNA constructs used herein are single-stranded or double-stranded, linear or circular DNA molecules containing DNA segments combined and parallelized in ways not found in nature. These DNA constructs exist as a result of human manipulation and include clones and other copies of the manipulated molecule.

[0175] The DNA segments used here are parts of larger DNA molecules that possess specific attributes. For example, a DNA segment encoding a particular polypeptide is part of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from 5' to 3', encodes the amino acid sequence of a specific polypeptide.

[0176] As used herein, the term polynucleotide refers to 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 origin, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is expressed here in terms of nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The term nucleotide is used for single-stranded and double-stranded molecules whenever possible. When this term is applied to a double-stranded molecule, it is used to mean the overall length and is understood to be equivalent to the term base pair. It is recognized by those skilled in the art that the chain lengths of double-stranded polynucleotides may vary slightly and their ends may be mismatched; therefore, not all nucleotides in a double-stranded polynucleotide molecule can be paired. Such unpaired ends generally do not exceed 20 nucleotides in length.

[0177] The recombinant production method used here refers to the use of well-known molecular biology methods for the expression of proteins encoded by cloned DNA.

[0178] As used herein, “heterogeneous nucleic acids” are nucleic acids that encode products not normally produced in vivo by the cells in which they are expressed (i.e., RNA and / or proteins), or nucleic acids that are located at or in loci that do not normally exist, or that mediate or encode mediators that alter the expression of endogenous nucleic acids such as DNA by affecting transcription, translation, or other controllable biochemical processes. Heterogeneous nucleic acids such as DNA are also called exogenous nucleic acids. Any nucleic acid such as DNA that a person skilled in the art would recognize or consider to be heterogeneous or exogenous to the cells in which it is expressed is encompassed herein by heterogeneous nucleic acids; heterogeneous nucleic acids include exogenously added nucleic acids that are also expressed endogenously. Heterogeneous nucleic acids are generally not endogenous to the cells in which they are introduced, but are obtained from other cells, prepared by synthesis, introduced to genomic loci that do not occur naturally, or their expression is under the control of one or more regulatory sequences that differ from the natural regulatory sequences or sequences.

[0179] Examples of heterogeneous nucleic acids here include, but are not limited to, nucleic acids encoding immunostimulatory proteins such as proteins or their gain-of-function variants in DNA / RNA sensor pathways, or cytokines that confer or contribute to antitumor immunity in the tumor microenvironment. In immunostimulatory bacteria, heterogeneous nucleic acids are generally encoded in introduced plasmids, but can also be introduced into the bacterial genome, such as promoters that alter the expression of bacterial products. Heterogeneous nucleic acids, such as DNA, include nucleic acids that can, in some way, intervene in the expression of DNA encoding a therapeutic product, or can encode products such as peptides or RNA that, in some way, directly or indirectly intervene in the expression of a therapeutic product.

[0180] The cell therapy used herein involves the delivery of cells to a target for treating a disease or condition. Allogeneic or autologous cells are ex vivo modified, such as by infecting them with immunostimulatory bacteria provided herein, so that they deliver or express products when introduced into the target.

[0181] The gene therapy used herein involves the introduction of heterologous nucleic acids, such as DNA, into certain cells, particularly human target cells, in mammals with the disorder or condition for which such therapy is being explored. The nucleic acids, such as DNA, are introduced into selected target cells in such a manner that the heterologous nucleic acids are expressed and the therapeutic product encoded by them is produced. Gene therapy may also be used to deliver nucleic acids encoding gene products that complement gene products produced by the mammal or cell replicating or introducing the deficient gene. The introduced nucleic acids may encode therapeutic compounds that are not normally produced in the mammalian host, or not produced in therapeutically effective amounts or for therapeutically useful durations, such as growth factors or their inhibitors, or tumor necrosis factor or its inhibitors, such as their receptors. The heterologous nucleic acids, such as DNA, encoding the therapeutic product may be modified before introduction into the cells of the affected host to enhance or alter the product or its expression. Gene therapy may also involve the delivery of gene expression inhibitors or repressors or other modulators.

[0182] As used herein, "expression" refers to the process by which polypeptides are produced through the transcription and translation of polynucleotides. Polypeptide expression levels can be assessed using any method known in this art, including, for example, a method for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantitative analysis of polypeptides in cell solubles by ELISA, Coomassie blue staining after gel electrophoresis, the Lowry protein assay, and the Bradford protein assay.

[0183] The term "host cell" as used here refers to a cell that receives, maintains, regenerates, and / or amplifies the vector. The host cell may also 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 acid.

[0184] As used herein, a “vector” is a nucleic acid capable of replicating one or more heterologous proteins that can be expressed when the vector is transformed into a suitable host cell. The description of a vector generally includes vectors into which nucleic acids or fragments thereof encoding polypeptides can be introduced by restriction digestion and ligation. The description of a vector also includes vectors containing nucleic acids encoding polypeptides, such as modified anti-EGFR antibodies. A vector is used to introduce nucleic acids encoding polypeptides into host cells for nucleic acid amplification or expression / presentation of the polypeptide encoded by the nucleic acid. Vectors generally remain in the episome, but may be designed to integrate a gene or a portion of its genome into a chromosome. Also considered are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such media are well known to those skilled in the art. The term “vector” also includes “viral vectors” or “viral vectors.” A viral vector is an engineered virus manipulably linked to a foreign gene for introducing a foreign gene into a cell (as a medium or shuttle).

[0185] The term "expression vector" as used herein includes a vector capable of expressing DNA that is manipulably linked to a control sequence, such as a promoter region, the control sequence enabling the expression of such DNA fragments. Such additional segments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally originate from plasmids or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that, upon introduction into a suitable host cell, results in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and / or prokaryotic cells and that remain in the episome or integrate into the host cell genome.

[0186] As used here, "primary sequence" refers to the sequence of amino acid residues in a polypeptide or the nucleotide sequence of a nucleic acid molecule.

[0187] As used herein, “sequence identity” refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test polypeptide or polynucleotide and a control polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For the purposes herein, sequence identity is generally determined by alignment to identify identical residues. Alignment may be local or comprehensive. Matches, mismatches, and gaps can be identified between comparison sequences. A gap is a null amino acid or nucleotide inserted between residues of an aligned sequence so that identical or similar properties are aligned. In general, internal and terminal gaps may exist. When using a gap penalty, sequence identity can be determined without penalty for terminal gaps (e.g., terminal gaps are not penalized). Alternatively, sequence identity can be determined without considering gaps as: number of identical positions / total length of aligned sequence × 100.

[0188] The "inclusive alignment" used here is an alignment that aligns two sequences from beginning to end, aligning each letter of each sequence only once. The alignment is created regardless of whether the sequences are similar or identical. For example, 50% sequence identity based on "inclusive alignment" means that 50% of the residues are identical in the alignment of the complete sequences of two 100-nucleotide lengths of comparison sequences. It is understood that inclusive alignment can also be used to determine sequence identity even if the aligned sequences are not the same length. Differences at the ends of the sequences are considered in determining sequence identity unless "no penalty for terminal gaps" is selected. In general, inclusive alignment is used for sequences that share considerable similarity over most of their length. An example of an algorithm for performing inclusive alignment is the Needleman-Wunsch algorithm (including Needleman et al. (1970) J. Mol. Biol. 48: 443). Examples of programs for performing comprehensive alignment are publicly available, including the Global Sequence Alignment Tool available on the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ) and programs available at deepc2.psi.iastate.edu / aat / align / align.html.

[0189] The "local alignment" used here aligns two sequences, but only aligns the portion of the sequences that share similarity or identity. Therefore, local alignment determines whether a subsegment of one sequence exists in the other sequence. If there is no similarity, the alignment is not answered. Local alignment algorithms include BLAST or the Smith-Waterman algorithm (Adv. Appl. Math. 2: 482 (1981)). For example, 50% sequence identity based on "local alignment" means that in the alignment of the complete sequences of two comparison sequences of some length, a 100-nucleotide similarity or identity region has 50% of the residues that are identical within that similarity or identity region.

[0190] For the purposes of this invention, sequence identity can be determined by a standard alignment algorithm program using default gap penalties established by each supplier. Default parameters for the GAP program may include: (1) a unary comparison matrix (including values ​​of 1 for agreement and 0 for mismatch) and a weighted comparison matrix according to Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, as described in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol within each gap; and (3) no penalty for terminal gaps. Whether the nucleotide sequences of any two nucleic acid molecules or the amino acid sequences of any two polypeptides are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical" or have other similar differences using percentage identity can be determined using known computer algorithms based on local or comprehensive alignment (see, for example, wikipedia.org / wiki / Sequence_alignment_software, which provides links to numerous known and publicly available alignment databases and programs).Generally, for the purposes presented here, sequence identity is determined using computer algorithms based on comprehensive alignment, such as the Needleman-Wunsch comprehensive sequence alignment tool available at NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (William Pearson (Adv. Appl. Math. (1991) 12:337-357) implementing the Huang and Miller algorithm); and Xiaoqui Huang's program available at deepc2.psi.iastate.edu / aat / align / align.html. Generally, the full-length sequences of each comparison polypeptide or nucleotide are aligned across the full length of each sequence in the comprehensive alignment. Local alignment may also be used when the sequences being compared are substantially the same length.

[0191] Therefore, the term “identity” as used herein refers to the comparison or alignment between a test polypeptide or polynucleotide and a control polypeptide or polynucleotide. In some non-restrictive examples, “at least 90% identical” means a 90–100% agreement with respect to the control polypeptide or polynucleotide. A level of identity of 90% or higher is, for illustrative purposes, an indicator of the fact that, assuming a comparison of 100 amino acid or nucleotide lengths of the test polypeptide or polynucleotide and the control polypeptide or polynucleotide, no more than 10% (i.e., 10 out of 100) of the test polypeptide or polynucleotide differs from the control polypeptide. Similar comparisons can be performed between test polynucleotides and control polynucleotides. Such differences may represent point mutations randomly distributed across the entire length of the amino acid sequence, or one or more positions of various lengths up to an acceptable maximum, e.g., 10 / 100 amino acid differences, may cluster (approximately 90% identity). Differences may also be due to deletions or shortenings of amino acid residues. Differences may be defined as nucleic acids or amino acid substitutions, insertions, or deletions. Depending on the length of the comparison sequences, results can be independent of the program and gap parameter set, with homology or agreement levels exceeding approximately 85-90%; such high levels of identity can often be easily assessed without relying on software.

[0192] As used herein, “disease or disorder” refers to a pathological condition in an organism that is caused by an infection, acquired condition, or genetic condition, and is characterized by identifiable symptoms.

[0193] As used herein, "treatment" of a subject with a disease or condition means that, after treatment, the subject's symptoms are partially or completely relieved or remain at rest.

[0194] The term "treatment" as used herein refers to any effect that improves the symptoms of a disease or disorder. Treatments include prevention, therapy, and / or cure. Treatments also encompass any pharmaceutical use of any immunostimulant bacteria or composition provided herein.

[0195] The term "prevention" used here refers to the prevention of the possibility of disease and / or the prevention of symptom exacerbation or disease progression.

[0196] As used herein, "prevention" or "prevention" and its grammatically equivalent forms refer to methods of reducing the risk or likelihood of developing a disease or condition.

[0197] The term "pharmaceutically effective agent" as used herein includes, but is not limited to, any conventional therapeutic agent, including, for example, anesthetics, vasoconstrictors, dispersants, and small molecule drugs and therapeutic proteins.

[0198] As used herein, "therapeutic effect" refers to the effect resulting from the treatment in question, which alters the symptoms of a disease or condition, generally improves or enhances them, or cures the disease or condition.

[0199] The term "therapeutic dose" as used herein refers to the amount of a drug, compound, substance, or composition containing a compound that, after administration to a subject, is sufficient to produce at least a therapeutic effect. Therefore, it is the amount necessary for the prevention, cure, improvement, suppression, or partial suppression of the symptoms of a disease or disorder.

[0200] As used herein, "therapeutic efficacy" refers to the ability of a drug, compound, substance, or composition containing a compound to produce a therapeutic effect in a subject to which the drug, compound, substance, or composition containing a compound is administered.

[0201] The terms "effective prophylactic dose" or "effective prophylactic dose" as used herein refer to the amount of a drug, compound, substance, or composition containing a compound that, when administered to a subject, produces the intended prophylactic effect, such as prevention or delay of the onset or recurrence of a disease or symptom, reduction of the likelihood of the onset or recurrence of a disease or symptom, or reduction of the incidence of viral infection. A single dose may not necessarily produce a complete prophylactic effect, and may only occur after a series of doses. Therefore, an effective prophylactic dose may be administered in one or more doses.

[0202] The improvement of symptoms of a particular disease or disorder by treatment such as the administration of the pharmaceutical compositions or other therapeutic agents used herein means any reduction of the symptoms, whether permanent or transient, which may be caused by or related to the administration of the composition or treatment.

[0203] As used herein, "anticancer agents" refers to any drugs that are destructive or toxic to malignant cells and tissues. For example, anticancer agents include drugs that kill cancer cells or otherwise inhibit or impair the growth of tumors or cancer cells. An example of an anticancer agent is a chemotherapeutic agent.

[0204] The term "therapeutic activity" used here refers to the in vivo activity of a therapeutic polypeptide. Generally, therapeutic activity is the activity associated with treating a disease or condition.

[0205] The term "subject" used here refers to animals, including mammals such as humans.

[0206] The term "patient" used here refers to a human subject.

[0207] The animals used herein include, but are not limited to, all animals such as humans, primates including gorillas and monkeys; rodents such as mice and rats; poultry such as chickens; ruminants such as goats, cattle, deer and sheep; and pigs and other animals. Non-human animals exclude humans from being considered. The polypeptides provided herein are of all origins, derived from animals, plants, prokaryotes and fungi. The majority of polypeptides are of animal origin, including mammalian origin.

[0208] As used herein, "composition" refers to any mixture. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0209] As used here, "combination" refers to any connection between two or more items. A combination can be two or more separate items, such as two compositions or two collections; a mixture of such items, such as a single mixture; or any variation thereof. The elements of a combination are generally functionally linked or related.

[0210] The combination therapy used herein refers to the administration of two or more different therapeutic agents. The different therapeutic agents may be provided and administered separately, sequentially, intermittently, or as a single composition.

[0211] The kits used herein refer to packaged combinations that include, optionally, additional reagents and other elements such as instructions for use of the combination or its components, for purposes including, but not limited to, activation, administration, diagnosis, and evaluation of biological activity or properties.

[0212] As used herein, "unit dosage form" refers to a physically distinct unit that is suitable for human and animal use and is individually packaged, as is well known in this field.

[0213] The term "single-dose preparation" used here refers to a preparation intended for direct administration.

[0214] The multi-dose formulations used herein refer to formulations that contain multiple doses of a therapeutic agent and can be administered directly to provide several single doses of the therapeutic agent. Multiple doses may be administered over minutes, hours, weeks, days, or months. Multi-dose formulations may allow for dose adjustment, dose accumulation, and / or dose splitting. Because multi-dose formulations are used over time, they generally contain one or more preservatives to inhibit microbial growth.

[0215] As used herein, “product” refers to a manufactured and sold product. To the extent used throughout this specification, the term is intended to encompass any of the compositions provided herein that are contained in a package.

[0216] As used herein, "fluid" refers to any composition that can flow. Fluids therefore include compositions that are semi-solid, paste, solution, aqueous mixture, gel, lotion, cream, and other such compositions.

[0217] The isolated or purified polypeptide or protein (e.g., isolated antibody or antigen-bound fragment thereof) or its biologically active portion (e.g., isolated antigen-bound fragment) used herein is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein originates, or substantially free of chemical precursors or other chemicals when chemically synthesized. A preparation can be determined to be substantially free of other impurities if it appears to be free of impurities readily detectable by standard analytical methods used by those skilled in the art for assessing such purity, such as thin-layer chromatography (TLC), gel electrophoresis, and high-performance liquid chromatography (HPLC), or if it is pure enough that further purification would not detectably alter the physical and chemical properties of the substance, such as its enzymatic and biological activity. Methods for purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, substantially chemically pure compounds can be mixtures of stereoisomers. In such cases, further purification increases the specific activity of the compound. As used herein, “cell extract” or “lysate” means a preparation or fraction produced from lysed or disrupted cells.

[0218] As used herein, "control" refers to a sample that is substantially identical to the test sample, except that it is not treated with the test parameters, or, if it is a plasma sample, may be derived from a normal volunteer without the condition in question. The control may also be an internal control.

[0219] The singular expressions used herein include multiple references unless otherwise clearly indicated. Therefore, for example, a description of a polypeptide containing an "immunoglobulin domain" includes polypeptides having one or more immunoglobulin domains.

[0220] The term "or" as used herein is used to mean "and / or" unless it is explicitly stated that the alternatives are mutually exclusive or that the alternatives are mutually exclusive.

[0221] The ranges and quantities used here can be expressed as "approximately" a specific value or range. "Approximately" can also indicate an exact quantity. Therefore, "approximately 5 amino acids" means "approximately 5 amino acids" and also "5 amino acids."

[0222] As used herein, “optional” or “at will” means whether or not the event or situation described thereafter occurs, and this statement includes both cases in which such event or situation occurs and cases in which it does not. For example, “at will” variant portion means that the portion is either variant or non-variant.

[0223] Unless otherwise specified, any abbreviations used herein for protecting groups, amino acids, and other compounds follow their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 11(9):1726–1732).

[0224] To clarify disclosure and not for limiting purposes, detailed descriptions are further subdivided into smaller categories.

[0225] B. Overview of immunostimulant bacteria Modified bacteria, hereafter referred to as immunostimulatory bacteria, are provided, which encode inhibitory RNAs such as designed shRNAs and designed microRNAs that accumulate and / or replicate in tumors and exert an effective tumor therapeutic effect by inhibiting, suppressing, or silencing the expression of RNA in the target of treatment. The bacterial strains for modification may be any suitable for therapeutic use. The modified immunostimulatory bacteria provided herein are for use and methods for treating cancer. The bacteria are modified for such use and methods.

[0226] The immunostimulatory bacteria provided herein are modified by deletion or modification of bacterial genes to attenuate the inflammatory response and to enhance the antitumor immune response in the host treated with the bacteria. For example, in the host, plasmids encoding therapeutic products such as antitumor products are incorporated into the bacteria, and the bacteria may be nutrient-dependent on adenosine. Attenuation of the inflammatory response to the bacteria can be achieved by deletion of the msbB gene and / or flagellin gene knockout, which reduces TNF-alpha in the host. Bacteria are modified to stimulate host antitumor activity, for example, by the addition of plasmids encoding immunostimulatory proteins, STING proteins, variant STING proteins, and proteins targeting host immune checkpoints, and by the addition of CpG to nucleic acids.

[0227] Bacterial strains may be attenuated strains or strains that have been attenuated by standard methods or by modifications provided herein, thereby limiting their colony-forming ability primarily to immune-privileged tissues and organs, particularly immune and tumor cells, including solid tumors. For the purposes herein, bacteria do not need to be attenuated in themselves, but rather by modifications such as genomic modifications that limit or alter the cells that can be infected by the bacteria. Bacteria include, but are not limited to, strains of Salmonella, Shigella, Listeria, Escherichia coli, and Bifidobacteria. For example, species include Shigella sonnei, Shigella flexneri, Shigella shigai, Listeria monocytogenes, Salmonella typhi, Salmonella mucinosa, Salmonella trityphi, and Bacillus gertner's. Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Cytrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix. For example, this includes Rickettsia japonica, Rickettsia typhus, Rickettsia scrub typhus, Rickettsia sibirica, Bacillus subtilis bronchoseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas euclenophila, Aeromonas salmonicida, Bacillus tularensis, Mycobacterium pseudotuberculosis, Cytrobacter freundii, Chlamydia pneumoniae, Haemophilus somnus, Mycobacterium bovine malformation, Mycobacterium intracellulare, Bacillus regalis, Rhodococcus equii, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelas swine, Yersinia enterocolitica, Rosaliamea quintana, and Agrobacterium tumelfasium.

[0228] Bacteria accumulate in environments containing replicating cells that provide an environment for bacterial invasion and replication, by one or more properties including diffusion, migration, and chemotaxis into immune-privileged tissues or organs or the environment, providing an environment for bacterial invasion and replication. Immunostimulating bacteria and species exhibiting such therapeutic effects include Salmonella species, Listeria, and Escherichia coli. Bacteria contain plasmids encoding one or more therapeutic products under the control of a eukaryotic promoter, such as the RNA polymerase (RNAP) II or III promoter. Generally, the RNAPIII (also known as POLIII) promoter is constitutive, while RNAPII (also known as POLII) can be regulated. When multiple products are encoded, each expression may be under the control of a different promoter.

[0229] Among the bacteria presented here are those modified to be adenosine-dependent. 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-dependent bacteria. Adenosine is immunosuppressive and accumulates in high concentrations in tumors; adenosine-dependent bacteria improve the antitumor activity of bacteria because they selectively replicate in adenosine-rich tissues.

[0230] Bacteria modified to lack the ASD gene are also provided. For in vivo use, these bacteria are modified to include the transport of a functional ASD gene in the introduced plasmid; this maintains plasmid selection so as not to require an antibiotic-based plasmid maintenance / selection system. Also provided is the use of ASD-deficient strains that do not contain a functional ASD gene in the plasmid and are therefore engineered to autolyze in the host.

[0231] Bacteria modified to be incapable of flagellar production are also provided. This can be achieved by modifying the bacteria by deleting the gene encoding the flagellin subunit. Flagellin-deficient modified bacteria are less inflammatory, and therefore more tolerable, and induce a more potent antitumor response.

[0232] Bacteria modified to produce listeriolisin O, which improves plasmid delivery in phagocytic cells, are also provided.

[0233] Bacteria modified to support low-copy, CpG-containing plasmids are also provided. The plasmids may contain further modifications.

[0234] Bacteria can also be modified to grow in a way that minimizes the expression of the toxic SPI-1 (Salmonella pathogenicity island-1) gene, if they are Salmonella species. In Salmonella, the genes responsible for pathogenicity, invasion, survival, and extraintestinal spread are located in the Salmonella pathogenicity island (SPI).

[0235] Bacteria can be further modified for other desirable traits, including plasmid maintenance selection, particularly antibiotic-free selection, and strain preparation. Immunostimulatory bacteria may optionally encode therapeutic polypeptides and drugs, including antitumor therapeutic polypeptides.

[0236] The immunostimulatory bacteria provided herein are also examples of Salmonella species. Examples of bacteria for modification described herein are engineered strains of Salmonella typhimurii, such as strain YS1646 (ATCC Catalog # 202165; also see International PCT Application Publication WO99 / 13053, also known as VNP20009), which has been plasmid-engineered to complement asd gene knockout and antibiotic-free plasmid maintenance.

[0237] Nutritionally modified immunostimulatory bacterial strains that are resistant to adenosine are provided herein as pharmaceutical compositions containing such strains, formulated for administration to subjects such as humans for use in methods of treating tumors and cancer.

[0238] The modified immunostimulatory bacteria provided herein encompass multiple synergistic modalities that induce immune reactivation in non-inflammatory tumors and promote tumor antigen-specific immune responses while inhibiting immune checkpoint pathways that tumors utilize to overcome and evade persistent anti-tumor immunity. Improved tumor targeting, mediated through enhanced adenosine nutrient requirements and vasode breakdown, exhibits improved efficacy while localizing inflammation to limit systemic cytokine exposure and autoimmune toxicity observed with other immunotherapy modalities. An example of such modified bacteria is strain YS1646, particularly asd. - This is a Salmonella typhimurium strain that includes such modifications to the strain and the wild-type strain.

[0239] C. Cancer immunotherapy The immunosuppressive environment found within the tumor microenvironment (TME) is a driving mechanism for tumor initiation and progression. Cancer emerges after the immune system fails to control and contain the tumor. Multiple tumor-specific mechanisms create a tumor environment in which the immune system is forced to tolerate the tumor and its cells rather than eliminate them. The goal of cancer immunotherapy is to reclaim the immune system's innate ability to eliminate tumors.

[0240] 1. Immunotherapy Several clinical cancer immunotherapies explore disrupting the balance of immunosuppression against antitumor immunity. Strategies involving immune stimulation via direct administration of cytokines such as IL-2 and IFN-α elicit moderate clinical responses in a small number of patients but induce severe systemic inflammation-related toxicity (Sharma et al. (2011) Nat. Rev. Cancer 11:805-812). The immune system has evolved several checks and balances that regulate autoimmunity, including the upregulation of its binding to programmed cell death protein 1 (PD-1) on T cells and its homologous ligand, programmed cell 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 is linked to CD8 + It interferes with the T cell signaling pathway and CD8 +They impair T cell proliferation and effector function, inducing T cell tolerance. PD-1 and PD-L1 are two examples of numerous inhibitory "immune checkpoints" that function by downregulating the immune response. Other inhibitory immune checkpoints include cytotoxic T lymphocyte-associated protein 4 (CTLA-4), signal regulatory protein α (SIRPα), V-domain Ig suppressor of T cell activation (VISTA), programmed death-ligand 2 (PD-L2), indoleamine 2,3-dioxygenase (IDO) 1 and 2, lymphocyte-activating gene 3 (LAG3), Ga lectin-9, T cell immune receptor with Ig and ITIM domains (TIGIT), T cell immunoglobulins, and mucin-domain-containing It includes -3 (TIM-3, also known as hepatitis A virus cell receptor 2 (HAVCR2)), herpesvirus entry mediator (HVEM), CD39, CD73, B7-H3 (also known as CD276), B7-H4, CD47, CD48, CD80 (B7-1), CD86 (B7-2), CD155, CD160, CD244 (2B4), B- and T lymphocyte attenuators (BTLA or CD272), and carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1 or CD66a).

[0241] Antibodies designed to block immune checkpoints such as anti-PD-1 (e.g., pembrolizumab, nivolumab) and anti-PD-L1 (e.g., atezolizumab, avelumab, durvalumab) have successfully prevented persistent T-cell anergy and disrupted immune tolerance. Only a small fraction of patients who receive these treatments achieve clinical efficacy, and those who do often exhibit autoimmune-related toxicity (see, e.g., Ribas (2015) N. Engl. J. Med. 373:1490-1492; Topalian et al. (2012) N. Engl. J. Med. 366:2443-2454). This further demonstrates the need for a more effective and less toxic treatment, which is offered here.

[0242] Another checkpoint blocking strategy involves inhibiting CTLA-4 on T cells. CTLA-4 binds to and inhibits costimulatory receptors on APCs such as CD80 or CD86, and also defeats costimulatory differentiation antigen group 28 (CD28), which binds to the same receptors, but with lower affinity. This blocks stimulatory signals from CD28 while transmitting inhibitory signals from CTLA-4 and preventing T cell activation (see Phan et al. (2003) Proc. Natl. Acad. Sci. USA 100:8372-8377). Anti-CTLA-4 therapy (e.g., ipilimumab) shows clinical success and persistence in some patients, but also exhibits a greater incidence of severe immune-related adverse events (see, e.g., Hodi et al. (2010) N. Engl. J. Med. 363:711-723; Schadendorf et al. (2015) J. Clin. Oncol. 33:1889-1894). Tumors have been shown to acquire resistance to anti-immune checkpoint antibodies, highlighting the need for more sustained anti-cancer therapies, such as those provided here.

[0243] 2. Adoptive immunotherapy In the pursuit of reactivation to make non-inflammatory tumors more immunogenic, a group of immunotherapies known as adoptive cell therapy (ACT) encompasses diverse strategies that utilize immune cells and reprogram them to possess antitumor activity (Zielinski et al. (2011) Immunol. Rev. 240:40-51). Dendritic cell-based therapies introduce genetically modified dendritic cells (DCs) with more immunostimulatory properties. These therapies have been unsuccessful because they fail to disrupt immune tolerance to cancer (see, e.g., Rosenberg et al. (2004) Nat. Med. 12:1279). Methods using whole irradiated tumor cells containing endogenous tumor antigens and granulocyte-macrophage colony-stimulating factor (GM-CSF) to stimulate DC recruitment, known as GVAX, have similarly failed clinically because they lack the ability to disrupt tumor tolerance (Copier et al. (2010) Curr. Opin. Mol. Ther. 12:14-20). Another autologous cell-based therapy, cyproisel T (Provenge), was approved by the FDA in 2010 for castration-resistant prostate cancer. It utilizes APCs (artificial prostate cells) harvested from patients, rearmed to express prostatic acid phosphatase (PAP) antigen to stimulate a T-cell response, and then reintroduced after lymphodesection. Unfortunately, its broad application is limited due to the low observed objective response rate and high cost, and its use is restricted to the early stages of prostate cancer (Anassi et al. (2011) P T. 36(4):197-202). Similarly, autologous T-cell therapy (ATC) involves harvesting the patient's own T cells, reactivating them ex vivo to overcome tumor resistance, and then introducing them into the patient after lymphodesection. Clinical success of ATC has been limited, and is limited to melanoma, while simultaneously presenting with serious safety and feasibility issues that limit its usefulness (Yee et al. (2013) Clin. Cancer Res. 19:4550-4552).

[0244] Chimeric antigen receptor T cell (CAR-T) therapy involves patient-derived T cells that have been reprogrammed to express a fusion protein of the T cell receptor and the variable extracellular domain of the antibody Ig. This confers the antigen-recognizing properties of the antibody and 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 fatal immune-related adverse events (Jackson et al. (2016) Nat. Rev. Clin. Oncol. 13:370-383). Tumors can also mutate to evade recognition by target antigens, including CD19 (Ruella et al., (2016) Comput Struct Biotechnol J. 14: 357-362) and EGFRvIII (O'Rourke et al. (2017) Sci Transl Med. Jul. 19; 9(399):eaaa0984), thereby promoting immune evasion. While CAR-T therapy is approved in the context of hematological malignancies, its feasibility for treating solid tumors faces a significant obstacle: overcoming the highly immunosuppressive nature of the solid tumor microenvironment. Numerous additional modifications to existing CAR-T therapies are needed to potentially provide feasibility for solid tumors (Kakarla, et al. (2014) Cancer J. Mar-Apr; 20(2):151-155).

[0245] 3. Cancer vaccines and oncolytic viruses Non-inflammatory tumors lack T-cell and dendritic cell (DC) infiltration and are non-T-cell inflammatory (Sharma et al. (2017) Cell 9;168(4):707-723). In the pursuit of reactivation to make non-inflammatory tumors more immunogenic, other classes of immunotherapy utilize microorganisms that accumulate in tumors naturally or through manipulation. These include viruses designed to express tumor antigens and stimulate the immune system to activate and reprogram the immune system to eliminate the tumor. Viral-based cancer vaccines have clinically failed due to numerous factors, including pre-existing or acquired immunity to the viral vector itself and insufficient immunogenicity for expression at tumor antigens (Larocca et al. (2011) Cancer J. 17(5):359-371). The lack of adequate adjuvant activation of APCs also interferes with other non-viral vector cancer vaccines, such as DNA vaccines. Oncolytic viruses preferentially replicate in dividing tumor cells rather than healthy tissue, leading to subsequent tumor cell lysis, immunogenic tumor cell death, and further viral transmission. Tarimodine laherparepvec (T-VEC), an oncolytic virus using a combination of modified herpes simplex virus and the DC mobilization cytokine GM-CSF, is FDA approved for metastatic melanoma (Bastin et al. (2016) Biomedicines 4(3):21). It has shown clinical benefit in some melanoma patients and is less immunotoxic than other immunotherapies, but its efficacy is limited; it has no distal tumor efficacy and lacks broad applicability to other tumor types. Among many others, other oncolytic virus (OV)-based vaccines, such as those utilizing paramyxoviruses, reoviruses, and picornaviruses, have encountered similar limitations in inducing systemic antitumor immunity (Chiocca et al. (2014) Cancer Immunol. Res. 2(4):295-300). Systemic administration of oncolytic viruses presents unique challenges. IV administration easily dilutes the virus, thus requiring high titers that can lead to hepatotoxicity.If pre-existing immunity is present, the virus is rapidly neutralized in the bloodstream, and subsequent acquired immunity limits repeated administration (Maroun et al. (2017) Future Virol. 12(4):193-213).

[0246] Among the limitations of virus-based vaccine vectors and oncolytic viruses, the greatest limitation may be the virus itself. Viral antigens have a remarkably high affinity for human T cell receptors (TCRs) compared to tumor antigens (Aleksic et al. (2012) Eur J Immunol. 42(12):3174-3179). Tumor antigens presented on the surface of rigidly activated APCs along with viral vector antigens by MHC-1 are defeated in binding to TCRs, resulting in very poor antigen-specific anti-tumor immunity. Tumor-targeting immunostimulatory vectors offered here, which do not themselves provide high-affinity T cell epitopes, may be able to circumvent these limitations.

[0247] D. Bacterial cancer immunotherapy Provided herein are immunostimulatory bacteria modified to accumulate on tumor-resident immune cells and prevent infection of epithelial or other cells. These immunostimulatory bacteria contain plasmids that encode and express therapeutic products, such as immunostimulatory proteins, which are part of a cytosolic DNA / RNA sensor pathway resulting in the expression of type I IFNs under the control of a host recognition promoter. Therefore, immunostimulatory bacteria represent a cancer therapy that delivers immunotherapy directly to the tumor microenvironment through modification of the bacterial genome and the encoding of therapeutic products. The bacteria, methods, and uses provided herein address the challenges encountered in other cancer immunotherapies. Immunostimulatory proteins, which are part of a cytosolic DNA / RNA sensor pathway resulting in the expression of type I IFNs, are encoded or delivered by other delivery media such as exosomes, liposomes, oncolytic viruses, and gene therapy vectors, in addition to expression in the immunostimulatory bacteria provided herein.

[0248] 1. Bacterial treatment Acute inflammation associated with microbial infections has been linked to spontaneous tumor shedding based on centuries of observation. The recognition that bacteria possess anti-cancer activity dates back to the 1800s when tumor regression in patients infected with Streptococcus pyogenes was observed by several physicians. William Coley initiated the first research into the use of bacteria for the treatment of terminal cancer and developed a vaccine consisting of Streptococcus pyogenes and Serratia marcescens. This vaccine has been successful in treating a variety of cancers, including sarcomas, carcinomas, lymphomas, and melanomas. Since then, numerous bacteria, including Clostridium, Mycobacterium, Bifidobacterium, Listeria, and species such as Listeria monocytogenes and Escherichia, have been studied as sources of anti-cancer vaccines (see, for example, published international PCT applications WO1999 / 013053 and WO2001 / 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).

[0249] Bacteria can infect animal and human cells, and some possess the innate ability to deliver DNA into the cell's cytosol. Bacteria are also suitable as therapeutic agents because they can be administered orally, readily reproduce in vitro and in vivo, and can be stored and transported in a lyophilized state. Bacterial genes are easy to manipulate, and the complete genomes of many strains are well-characterized (Felgner et al. (2016) mbio 7(5):e01220-16). As a result, bacteria are used for the delivery and expression of a diverse range of genes, including those encoding cytokines, angiogenesis inhibitors, toxins, and prodrug-converting enzymes. For example, Salmonella has been used to express immunostimulatory 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 tumor treatment. Bacterial vectors are also cheaper and easier to manufacture than viral vectors, and bacterial delivery is preferable to viral delivery because, if necessary, they can be rapidly eliminated with antibiotics, making them a safer alternative.

[0250] However, for use, the strain must be non-pathogenic, or non-pathogenic after modification for use as a therapeutic agent. For example, in the treatment of cancer, the therapeutic bacterial strain should be attenuated or sufficiently non-toxic to avoid causing systemic disease and / or septic shock, while still maintaining a certain level of infectivity to effectively colonize tumors. Genetically modified bacteria used as antitumor agents to induce direct tumor-killing effects and / or deliver tumor-killing molecules have been described (Clairmont, et al. (2000) J. Infect. Dis. 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). These include strains of Salmonella typhimurium created through bioengineering. These bacteria preferentially accumulate in tumors >1,000 times more abundantly than in normal tissue and are uniformly distributed within tumor tissue (Pawelek, J. et al. (1997) Cancer Res. 57:4537-4544; Low, KB et al. (1999) Nat. Biotechnol. 17:37-41). This preferential replication allows the bacteria to produce and deliver diverse anticancer agents directly within the tumor at high concentrations while minimizing toxicity to normal tissue.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 one viable attenuated Salmonella strain has been shown to be well tolerated after oral administration in human clinical trials (Chatfield, SN et al. (1992) Biotechnology 10:888-892; DiPetrillo, MD et al. (1999) Vaccine 18:449-459; Hohmann, EL et al. (1996) J. Infect. Dis. 173:1408-1414; Sirard, JC et al. (1999) Immunol. Rev. 171:5-26). The Salmonella typhimurium phoP / phoQ operon is a typical bacterial two-component regulatory system consisting of membrane-associated sensor kinase (PhoQ) and a cytosolic transcription regulator (PhoP: Miller, SI et al. (1989) Proc Natl Acad Sci USA 86:5054-5058; Groisman, EA et al. (1989) Proc Natl Acad Sci USA 86: 7077-7081).PhoP / phoQ is necessary for pathogenicity, and its deletion leads to insufficient survival of this bacterium in macrophages and significant attenuation in mice and humans (Miller, SI et al. (1989) Proc Natl Acad Sci USA 86:5054-5058; Groisman, EA et al. (1989) Proc Natl Acad Sci USA 86: 7077-7081; Galan, JE and Curtiss, R. III. (1989) Microb Pathog 6:433-443; Fields, PI et al. (1986) Proc Natl Acad Sci USA 83:51). PhoP / phoQ deletion strains have been used as effective vaccine delivery vehicles (Galan, JE and Curtiss, R. III. (1989) Microb Pathog 6:433-443; Fields, PI et al. (1986) Proc Natl Acad Sci USA 83:5189-5193; Angelakopoulos, H. and Hohmann, EL (2000) Infect Immun 68:2135-2141). Attenuated Salmonella strains have been used for targeted delivery of tumor-killing proteins (Bermudes, D. et al. (2002) Curr Opin Drug Discov Devel 5:194-199; Tjuvajev J. et al. (2001) J Control Release 74:313-315).

[0251] The clinical benefits of bacterial-based cancer therapies have been shown to be limited. Clostridium novyi (Dang et al. (2001) Proc. Natl. Acad. Sci. USA 98(26):15155-15160; U.S. Patent Publication 2017 / 0020931 and 2015 / 0147315; and U.S. Patents 7,344,710 and 3,936,354), Mycobacterium bovis (U.S. Patent Publication 2015 / 0224151 and 2015 / 0071873), Bifidobacterium bifidum (Kimura et al. (1980) Cancer Res. 40:2061-2068), Lactobacillus casei (Yasutake et al. A variety of bacterial species, including Listeria monocytogenes (Le et al. (2012) Clin. Cancer Res. 18(3):858-868; Starks et al. (2004) J. Immunol. 173:420-427; U.S. Patent Publication 2006 / 0051380) and Escherichia coli (U.S. Patent 9,320,787), are being studied as potential agents for anti-cancer treatment.

[0252] For example, the Bacillus calmette-Guérin (BCG) strain is approved for the treatment of bladder cancer in humans, is more effective than intravesical chemotherapy, and is often used as a first-line treatment (Gardlik et al. (2011) Gene therapy 18:425-431). Another approach involves potent CD8 cytosis induced in response to tumor antigens expressed in mice. +Listeria monocytogenes is a surviving, attenuated intracellular bacterium capable of inducing T cells (Le et al. (2012) Clin. Cancer Res. 18(3):858-868). In a clinical trial of a Listeria-based vaccine incorporating the tumor antigen mesothelin, a prime-boost approach combined with an allogeneic pancreatic cancer-based GVAX vaccine demonstrated a median survival of 6.1 months in patients with advanced pancreatic cancer, compared to 3.9 months in patients treated with GVAX vaccine alone (Le et al. (2015) J. Clin. Oncol. 33(12):1325-1333). These results were not replicated in large-scale phase 2b trials, likely indicating the difficulty of inducing immunity to low-affinity autoantigens such as mesothelin.

[0253] Bacterial strains may be modified as described herein. Strains can be attenuated or modified by standard methods and / or gene deletions or modifications, and by gene alterations or introductions that enable bacteria to grow in vivo, primarily in immune-privileged environments such as TMEs, tumor cells, tumor resident immune cells, and solid tumors. Starting strains for the modifications described herein may be selected from, for example, Shigella, Listeria, Escherichia coli, Bifidobacteria, and Salmonella. For example, Shigella Sonne's, Shigella flexner's, Shigella shiga, Listeria monocytogenes, Salmonella typhi, Salmonella murine, Salmonella trityphi, and Salmonella gertner's. Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Cytrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix. For example, Rickettsia japonica, Rickettsia typhus, Rickettsia scrub typhus, Rickettsia sibirica, Bacillus subtilis, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas euclenophila, Aeromonas salmonicida, Bacillus tularemia, Mycobacterium pseudotuberculosis, Cytrobacter freundii, Chlamydia pneumoniae, Haemophilus somnus, Mycobacterium bovine, Mycobacterium intracellulare, Bacillus reticulata, Rhodococcus equii, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelas swine, Yersinia enterocolitica, Rosaliamea quintana, and Agrobacterium tumelfasium. Bacteria known for any therapeutic use, including immunostimulant effects, may be modified as described herein.

[0254] 2. Comparison of immune responses to bacteria and viruses Viruses and other bacteria possess the advantage of being naturally immunostimulant. 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 induces a downstream signaling cascade, which leads to the induction of cytokines and chemokines and the initiation of an immune response that results in pathogen elimination (Iwasaki and Medzhitov (2010) Science 327(5963):291-295). How the innate immune system binds to PAMPs and what type of infectious agent it originates from determines the appropriate adaptive immune response to fight invading pathogens.

[0255] The PRR class, known as Toll-like receptors (TLRs), recognizes PAMPs of bacterial and viral origin and is located in various compartments within cells. TLRs bind to a range of ligands, including lipopolysaccharides (TLR4), lipoproteins (TLR2), flagellin (TLR5), unmethylated CpG motifs in DNA (TLR9), double-stranded RNA (TLR3), and single-stranded RNA (TLR7 and TLR8) (Akira et al. (2001) Nat. Immunol. 2(8):675-680; Kawai and Akira (2005) Curr. Opin. Immunol. 17(4):338-344). For example, host surveillance in Salmonella typhimurium is largely mediated via TLR2, TLR4, and TLR5 (Arpaia et al. (2011) Cell 144(5):675-688). These TLRs signal via MyD88 and TRIF adapter molecules and mediate the induction of NF-κB-dependent pro-inflammatory cytokines such as TNF-α, IL-6, and IFN-γ (Pandey et. al. (2015) Cold Spring Harb Perspect Biol 7(1):a016246).

[0256] Another category of PRRs is the nod-like receptor (NLR) family. These receptors are present in the cytosol of host cells and recognize intracellular PAMPs. For example, Salmonella typhimurium flagellin has been shown to activate the NLRC4 / NAIP5 inflammasome pathway, which leads to caspase-1 cleavage and induction of pro-inflammatory cytokines IL-1β and IL-18, resulting in pyroptotic cell death of infected macrophages (Fink et al. (2007) Cell Microbiol. 9(11):2562-2570).

[0257] The involvement of TLR2, TLR4, TLR5, and inflammasomes mediates pro-inflammatory cytokines that induce bacterial elimination, but DCs and CD8 are necessary for antitumor immunity. + Not T cells, but mainly neutrophils, macrophages, and CD4 + It activates the NF-κB-driven signaling cascade, which leads to T cell recruitment and activation (Liu et al. (2017) Signal Transduct Target Ther. 2:e17023). CD8 + To activate T cell-mediated anti-tumor immunity, IRF3 / IRF7-dependent type I interferon signaling is used by CD8 +It is important for DC activation for T cell priming and for tumor antigen cross-presentation (Diamond et al. (2011) J. Exp. Med. 208(10):1989-2003; Fuertes et al. (2011) J. Exp. Med. 208(10):2005-2016). Type I interferons (IFN-α, IFN-β) are signature cytokines induced by two distinct TLR-dependent and TLR-independent signaling pathways. The TLR-dependent pathway for inducing IFN-β arises after pathogen endocytosis, thereby prompting TLR3, 7, 8, and 9 to detect pathogen-derived DNA and RNA elements within endosomes. TLRs 7 and 8 recognize viral nucleosides and nucleotides, and their synthetic agonists, such as reximod and imiquimod, have been clinically evaluated (Chi et al. (2017) Frontiers in Pharmacology 8:304). Synthetic dsRNAs such as poly(I:C) and poly(ICLC), an analog formulated with poly(I:C) and poly(L)-lysine to withstand RNase digestion, are agonists of the TLR3 and MDA5 pathways and potent inducers of IFN-β (Caskey et al. (2011) J. Exp. Med. 208(12):2357-66). TLR9 detection of endosomal CpG motifs present in viral and bacterial DNA also induces IFN-β via IRF3. Furthermore, TLR4 has been shown to induce IFN-β via MyD88-independent TRIF activation of IRF3 (Owen et al. (2016) mBio.7:1 e02051-15). Subsequently, TLR4 activation in DCs was shown to be type I IFN-independent, and therefore, the ability of TLR4 to activate DCs via type I IFN is unlikely to be biologically relevant (Hu et al. (2015) Proc. Natl. Acad. Sci. USA 112(45):13994-13999). In addition, TLR4 signaling is CD8 + It has not been shown to directly recruit or activate T cells.

[0258] One of the TLR-independent type I IFN pathways is cytosolic host recognition mediated by single-stranded (ss) and double-stranded (ds) RNAs. These are sensed by RNA helicases including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), and via phosphorylation of the IFN-β promoter-stimulating factor 1 (IPS-1; also known as mitochondrial antiviral signaling protein or MAVS) adapter protein of the IRF-3 transcription factor, leading to IFN-β induction (Ireton and Gale (2011) Viruses 3(6):906-919). The synthetic RIG-I binding element has also been unintentionally found in common lentiviral shRNA vectors in the form of the AA dinucleotide sequence at the U6 promoter transcription start site. Subsequent deletion in the plasmid prevented disruption of off-target type I IFN activation (Pebernard et al. (2004) Differentiation. 72:103-111).

[0259] The second type of TLR-independent type I interferon induction pathway is mediated via interferon gene stimulator (STING), a cytosolic ER-resident adapter protein that is now recognized as a central mediator for sensing cytosolic dsDNA from infectious pathogens or abnormal host cell injury (Barber (2011) Immunol. Rev 243(1):99-108). STING signaling activates the TANK-binding kinase (TBK1) / IRF3 axis and the NF-κB signaling axis, leading to the induction of IFN-β and other pro-inflammatory cytokines and chemokines that potently activate innate and adaptive immunity (Burdette et al. (2011) Nature 478(7370):515-518). STING-mediated sensing of cytosolic dsDNA requires cyclic GMP-AMP synthase (cGAS), a host cell nucleotidyltransferase that directly binds to dsDNA. In response, it synthesizes cyclic GMP-AMP (cGAMP), a cyclic dinucleotide (CDN) second messenger that binds to and activates STING (Sun et al. (2013) Science 339(6121):786-791; Wu et al. (2013) Science 339(6121):826-830). Bacterial CDNs such as c-di-AMP produced from intracellular Listeria monocytogenes can directly bind to mouse STING, but can only bind to three of the five human STING alleles. Unlike bacterial-produced CDNs, in which two purine nucleosides are linked by a 3'-3' phosphate crosslink, the internucleotide phosphate crosslinks in cGAMP synthesized by mammalian cGAS are linked by a non-standard 2'-3' bond.These 2'-3' molecules bind to STING with 300 times better affinity than bacterial 3'-3' CDNs, and are therefore more potent physiological ligands for human STING (see, for example, 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):pl1; Ablasser et al. (2013) Nature 503(7477):530-534).

[0260] The cGAS / STING signaling pathway in humans has evolved to preferentially respond to viral pathogens over bacterial pathogens, and this is because the aforementioned bacterial vaccine carrying host tumor antigens in humans is CD8 + This may explain why it was insufficient as a T cell priming vector. CD8 signaling via STING-dependent type I IFN signaling from conventional DCs. + TLR-independent activation of T cells is the primary mechanism for virus detection and, along with TLR-dependent type I IFN production by plasmacytoid DCs, is only activated when the STING pathway is virally inactivated (Hervas-Stubbs et al. (2014) J. Immunol. 193:1151-1161). Furthermore, while IFN-β induction via TLR4 is possible in bacteria such as Salmonella typhimurium, CD8 + T cells do not induce or are not required to induce elimination or protective immunity (Lee et al. (2012) Immunol Lett. 148(2): 138-143). CD8 is physiologically associated with many bacterial strains, including Salmonella typhimurium. + The absence of the T epitope hindered bacterial vaccine development and subsequent protective immunity against infection, even from the same gene strain (Lo et al. (1999) J. Immunol. 162:5398-5406). Bacterial-based cancer immunotherapy requires type I IFNs to cross-present tumor antigens and promote sustained anti-tumor immunity, as well as CD8 +Its ability to induce T cell recruitment and activation is biologically limited. However, the immunostimulatory bacteria provided herein have been engineered to overcome this challenge. The immunostimulatory bacteria provided herein preferentially utilize CD8 rather than TLR-dependent bacterial immune signaling. + This includes virus-like TLR-independent type I IFN signaling that induces T cell-mediated antitumor immunity.

[0261] STING activates innate immunity in response to nucleic acid sensing in the cytosol. Downstream signaling is activated via the binding of CDN, which is synthesized by bacterial or host enzyme cGAS in response to binding to cytosolic dsDNA. The phosphate crosslinking structure of bacterial and host-produced CDN differs, resulting in different abilities to activate STING. IFN-β is the signature cytokine of activated STING; it is a virally induced type I IFN, not a bacterially induced IFN, and is effective against CD8 + It is necessary for T cell-mediated antitumor immunity. The immunostimulatory bacteria provided here include those that are STING agonists and those that express STING.

[0262] 3. Salmonella treatment Salmonella is an example of a bacterial genus that can be used as a cancer treatment agent. The Salmonella species exemplified herein are attenuated species or modified as described herein to reduce toxicity for use as a cancer treatment agent.

[0263] a. Tumor-tropic bacteria Numerous bacterial species exhibit preferential replication within solid tumors when injected distally. These include, but are not limited to, Salmonella species, Bifodobacterium, Clostridium, and Escherichia. The combination of the bacteria's innate tumor-homing nature and the host's innate immune response to bacterial infection is thought to mediate the antitumor response. This tumor tropism has been shown to reduce tumor size to varying degrees. A contributing factor to the tumor tropism of these bacterial species is their ability to replicate in anaerobic or hypoxic environments. Numerous of these naturally tumor-targeting bacteria have been further engineered to enhance their antitumor response efficacy (reviewed in Zu et al. (2014) Crit Rev Microbiol. 40(3):225-235; and Felgner et al. (2017) Microbial Biotechnology 10(5):1074-1078).

[0264] b. Salmonella typhimurium Salmonella typhimurium is an example of a bacterial species used as an anti-cancer agent. The approach of using bacteria to stimulate the host immune response against cancer involves Salmonella typhimurium, a Gram-negative facultative anaerobic organism that preferentially accumulates in hypoxic and necrotic areas of the body, including the tumor microenvironment. Salmonella typhimurium accumulates in these environments due to the increased availability of nutrients from tissue necrosis, the leaky tumor vascular structure, and the immune-evading tumor microenvironment (Baban et al. (2010) Bioengineered Bugs 1(6):385-394). Salmonella typhimurium can survive under both aerobic and anaerobic conditions; therefore, it can colonize small tumors that are not too hypoxic and large tumors that are more hypoxic.

[0265] Salmonella typhimurium is a Gram-negative, facultative pathogen transmitted via the fecal-oral route. It can cause localized gastrointestinal infections, and after oral ingestion, it enters the bloodstream and lymphatic system, infecting systemic tissues such as the liver, spleen, and lungs. Systemic administration of wild-type Salmonella typhimurium overstimulates TNF-α induction, leading to a potentially fatal cytokine cascade and septic shock if left untreated. Consequently, pathogenic bacterial strains, including Salmonella typhimurium, must be attenuated to prevent systemic infection without completely suppressing their ability to efficiently colonize tumor tissue. Attenuation is often achieved by mutations in cellular structures that can induce immune responses, such as the bacterial outer membrane, or by limiting their replication capacity in the absence of supplemental nutrients.

[0266] Salmonella typhimurium is an intracellular pathogen that can be rapidly taken up by bone marrow cells such as macrophages, or induce its own uptake by non-phagocytic cells such as epithelial cells. Once inside a cell, it can replicate within the Salmonella-containing vacuole (SCV) and escape to the cytosol of some epithelial cells. Many of the molecular determinants of Salmonella typhimurium pathogenicity have been identified, and the genes are clustered into Salmonella pathogenic islands (SPIs). The two most characteristic pathogenic islands are SPI-1, which is responsible for mediating bacterial invasion by non-phagocytic cells, and SPI-2, which is necessary for replication within SCVs (Agbor and McCormick (2011) Cell Microbiol. 13(12):1858-1869). Both of these pathogenic islands encode a macromolecular structure called the type 3 secretory system (T3SS), which can translocate effector proteins across the host membrane (Galan and Wolf-Watz (2006) Nature 444:567-573).

[0267] c. Bacterial attenuation Therapeutic bacteria intended for administration as cancer treatment agents must be modified to avoid causing disease. Various methods for achieving this are known in this field. For example, nutrient requirement mutations and deletions / mutations in genes such as aro, pur, gua, thy, nad, and asd (US Patent Publication 2012 / 0009153), which make it impossible for bacteria to synthesize essential nutrients, are widely used. The nutrients produced by the involvement of these genes are often unavailable to host cells, thus making bacterial survival challenging. For example, attenuation of Salmonella and other species can be achieved by deletion of the aroA gene, which is part of the shikimic acid pathway that leads to glycolysis into aromatic amino acid biosynthesis (Felgner et al. (2016) mbio 7(5):e01220-16). The deletion of aroA therefore results in bacterial nutrient requirements for aromatic amino acids and subsequent attenuation (US Patent Publications 2003 / 0170276, 2003 / 0175297, 2012 / 0009153 and 2016 / 0369282; International Patent Publications WO2015 / 032165 and WO2016 / 025582). Similarly, other enzymes in the biosynthetic pathway of aromatic amino acids, including aroC and aroD, are deleted to achieve attenuation (US Patent Publication 2016 / 0369282; International Patent Publication WO2016 / 025582). For example, Salmonella typhimurium strain L7207 is an aromatic amino acid nutrient requirement strain (aroA - (Mutant); strains A1 and A1-R are leucine-arginine nutrient-requiring strains. VNP20009 is a purine nutrient-requiring strain (purI - This is a mutant. As shown here, it also has nutritional requirements for immunosuppressive nucleoside adenosine.

[0268] Mutations that weaken bacteria include mutations in genes that alter lipopolysaccharide biosynthesis, such as rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; mutations that introduce suicide genes, such as sacB, nuk, hok, gef, kill, or phlA; mutations that introduce bacterial lysis genes, such as hly and cly; and disease-causing genes, such as isyA, pag, prg, iscA, virG, plc, and act. Mutations in virulence factors; mutations that modify stress responses such as recA, htrA, htpR, hsp, and groEL; mutations that disrupt the cell cycle such as min; and mutations that disrupt or inactivate regulatory functions such as cya, crp, phoP / phoQ, and ompR, are included but not limited to these (U.S. Patent Publications 2012 / 0009153, 2003 / 0170276, 2007 / 0298012; U.S. Patent 6,190,657; International Patent Application Publication WO2015 / 032165; Felgner et al. (2016) Gut Microbes 7(2):171-177; Broadway et al. (2014) J. Biotechnology 192:177-178; Frahm et al. (2015) mBio 6(2):e00254-15; Kong et al. (2011) Infection and Immunity 79(12):5027-5038; Kong et al. (2012) Proc. Natl. Acad. Sci. USA 109(47):19414-19419). Ideally, genetic attenuation should involve gene deletions rather than point mutations to prevent spontaneous compensatory mutations that would lead to a return to the pathogenic phenotype.

[0269] i. msbB - mutant In Salmonella typhimurium, the enzyme myristoyltransferase, encoded by the msbB gene, catalyzes the addition of a terminal myristyl group to the lipid A domain of lipopolysaccharide (LPS) (Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Therefore, deletion of msbB alters the acyl composition of the lipid A domain of LPS, a major component of the outer membrane of Gram-negative bacteria. This modification significantly reduces LPS's ability to induce septic shock, attenuates bacterial strains, reduces potentially harmful TNFα production, and thus reduces systemic toxicity. The Salmonella typhimurium msbB mutant retains its ability to preferentially colonize tumors over other tissues in mice, thus preserving its antitumor activity and thereby enhancing the therapeutic index of salmonella-based immunotherapy (see, for example, U.S. Patent Publications 2003 / 0170276, 2003 / 0109026, 2004 / 0229338, 2005 / 0255088 and 2007 / 0298012).

[0270] For example, deletion of msbB in the Salmonella typhimurium strain VNP20009 results in the dominant production of pentaacylated LPS, which is less toxic than naturally occurring hexaacylated LPS, enabling systemic delivery without inducing toxic shock (Lee et al. (2000) International Journal of Toxicology 19:19-25). Other LPS mutations that dramatically reduce pathogenicity, provide low toxicity, and enable high-dose administration can be introduced into bacterial strains provided herein, including Salmonella strains.

[0271] ii. purI - mutant Immunostimulant bacteria that can be attenuated by making them nutrient-requiring for one or more essential nutrients such as purines (e.g., adenine), nucleosides (e.g., adenosine), or amino acids (e.g., arginine and leucine) are used. In particular, in embodiments of immunostimulant bacteria provided herein, such as Salmonella typhimurium, the bacteria are nutrient-requiring for adenosine, which preferentially accumulates in the tumor microenvironment. Therefore, the immunostimulant bacterial strains described herein are attenuated because they require adenosine for growth and preferentially colonize adenosine-rich TMEs, as described below.

[0272] The enzyme phosphoribosylaminoimidazole synthetase, encoded by the purI gene (synonymous with the purM gene), is involved in the purine biosynthesis pathway. Therefore, disruption of the purI gene makes bacteria nutrient-dependent on purines. In addition to attenuation, purI - The mutant is concentrated in the tumor environment and possesses considerable antitumor activity (Pawelek et al. (1997) Cancer Research 57:4537-454). This colonization has been previously described as being brought about by the high concentration of purines present in the tumor's interstitial fluid as a result of rapid cell turnover. -It was thought that because the bacteria could not synthesize purines, they required an external source of adenine, which led to their growth being limited to a purine-enriched tumor microenvironment (Rosenberg et al. (2002) J. Immunotherapy 25(3):218-225). The VNP20009 strain was initially reported to contain a deletion of the purI gene (Low et al. (2003) Methods in Molecular Medicine Vol. 90, Suicide Gene Therapy:47-59), but subsequent analysis of the entire VNP20009 genome showed that the purI gene was not deleted but disrupted by a chromosomal inversion (Broadway et al. (2014) Journal of Biotechnology 192:177-178). The entire gene is contained within two adjacent parts of the VNP20009 chromosome, separated by an insertion sequence (one of which contains an active transposase).

[0273] Here, the purI mutant Salmonella typhimurium strain is shown to be nutrient-dependent for the highly abundant nucleoside adenosine in the tumor microenvironment. Therefore, when using VNP20009, no further modifications are needed to achieve adenosine nutrient dependence. For other strains and bacteria, the purI gene may be disrupted, as in VNP20009, or all or part of the purI gene may be deleted to prevent reversion to the wild-type gene.

[0274] iii. Combinations of attenuating mutations Bacteria with multiple genetic attenuations due to gene deletions in different chromosomal regions are desirable for bacterial immunotherapy because they can increase attenuation while reducing the likelihood of reversion to the pathogenic phenotype through homologous recombination with wild-type genetic material. Restoration of pathogenicity through homologous recombination requires two separate recombination events in the same organism. Ideally, a combination of attenuating mutations selected for use in immunotherapeutic agents would increase tolerability without reducing potency, thereby increasing the therapeutic index.

[0275] For example, disruption of the msbB and purI genes in the Salmonella typhimurium strain VNP20009 has been used for tumor targeting and growth suppression, and has induced low toxicity in animal models (Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002; Bermudes et al. (2000) Cancer Gene Therapy: Past Achievements and Future Challenges, edited by Habib Kluwer Academic / Plenum Publishers, New York, pp. 57-63; Low et al. (2003) Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy:47-59; Lee et al. (2000) International Journal of Toxicology 19:19-25; Rosenberg et al. (2002) J. Immunotherapy 25(3):218-225; Broadway et al. (2014) J. Biotechnology 192:177-178; Loeffler et al. (2007) Proc. Natl. Acad. Sci. USA 104(31):12879-12883; Luo et al. (2002) Oncology Research 12:501-508). However, VNP20009 does not exhibit the same tumor accumulation and antitumor activity in human clinical trials. Therefore, the high doses required to exhibit any antitumor activity are not feasible due to toxicity. Immunostimulating bacteria provided herein, including combinations of gene modifications that improve the therapeutic index with desirable properties, such as reducing pathogenicity, increasing tolerability, reducing or eliminating bacterial infection of epithelial (and other non-immune) cells, increasing accumulation in tumor resident immune cells, and reducing cell death of tumor resident immune cells, address this challenge.

[0276] iv. VNP20009 and other attenuated Salmonella typhimurium strains An example of a therapeutic bacterium that can be modified as described here is the strain named VNP20009 (ATCC # 202165, YS1646). The clinical candidate, VNP20009 (ATCC # 202165, YS1646), has been attenuated by at least 50,000 times for safety by deleting 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:47-59; Lee et al. (2000) International Journal of Toxicology 19:19-25). Similar attenuated Salmonella strains should also be considered. As described above, deletion of msbB alters the composition of the lipid A domain of lipopolysaccharide, the main component of the outer membrane of Gram-negative bacteria (Low et al. (1999) Nat. Biotechnol. 17(1):37-41). This reduces potentially harmful TNFα production, prevents lipopolysaccharide-induced septic shock, attenuates bacterial strains, and reduces systemic toxicity (Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Deletion of the purI gene makes bacteria purine-dependent, which further attenuates bacteria and concentrates them in the tumor microenvironment (Pawelek et al. (1997) Cancer Res. 57:4537-4544; Broadway et al. (2014) J. Biotechnology 192:177-178).

[0277] The accumulation of VNP20009 in tumors is attributed to a combination of factors including the inherent invasiveness of the parent strain, ATCC #14028, its ability to replicate in hypoxic environments, and its demand for high concentrations of purines present in the tumor's interstitial fluid. Here, we also demonstrate that VNP20009 accumulates at pathologically high levels in the tumor microenvironment, contributing to an immunosuppressive tumor microenvironment, and that it is also nutritionally required for nucleoside adenosine (Peter Vaupel and Arnulf Mayer Oxygen Transport to Tissue XXXVII, Advances in Experimental Medicine and Biology 876 chapter 22, pp. 177-183). When VNP20009 was administered to mice carrying syngeneic or human xenograft tumors, the bacteria preferentially accumulated in the extracellular components of the tumors at a ratio exceeding 300–1000:1, reducing TNFα induction and demonstrating tumor growth inhibition and long-term survival compared to control mice (Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002). However, results from a phase 1 clinical trial in humans showed that while VNP20009 was relatively safe and well-tolerated, insufficient accumulation in human melanoma tumors was observed, and little antitumor activity was demonstrated (Toso et al. (2002) J. Clin. Oncol. 20(1):142-152). High doses that would likely be required to exert any antitumor activity were not feasible due to toxicity correlated with high levels of pro-inflammatory cytokines.

[0278] For example, leucine-arginine nutrient-requiring strain A-1 (Zhao et al. (2005) Proc. Natl. Acad. Sci. USA 102(3):755-760; Yu et al. (2012) Scientific Reports 2:436; US Patent 8,822,194; US Patent Publication 2014 / 0178341) and its derivative AR-1 (Yu et al. (2012) Scientific Reports 2:436; Kawaguchi 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 - The mutant Salmonella typhimurium strain L7207 (Guo et al. (2011) Gene therapy 18:95-105; US Patent Publication 2012 / 0009153, 2016 / 0369282 and 2016 / 0184456) and its obligate anaerobic derivative YB1 (International Patent Application Publication WO2015 / 032165; Yu et al. (2012) Scientific Reports 2:436; Leschner et al. (2009) PLoS ONE 4(8):e6692); strain aroA, a derivative of SL1344 (wild type). - / aroD - Mutant Salmonella typhimurium strain BRD509 (Yoon et al. (2017) European J. of Cancer 70:48-61); asd - / cya - / crp -mutant Salmonella typhimurium strain χ4550 (Sorenson et al. (2010) Biologics: Targets & Therapy 4:61-73) and phoP - / phoQ - Other strains of Salmonella typhimurium, such as Salmonella typhimurium strain LH430 (International Patent Publication WO2008 / 091375), may be used for tumor-targeted delivery and therapy.

[0279] Although strain VNP20009 failed to demonstrate clinical benefit in clinical trials involving patients with advanced melanoma, the treatment was safely administered to patients with advanced cancer. A maximum tolerated dose (MTD) was established. Therefore, this strain and other similarly modified bacterial strains can be used as starting materials for tumor-targeting therapeutic delivery vehicles. The modifications provided herein offer strategies for increasing efficacy by increasing the antitumor efficiency and / or safety and tolerability of therapeutic agents.

[0280] v. Salmonella typhimurium engineered to deliver macromolecules Salmonella typhimurium has also been modified to deliver tumor-associated antigen (TAA) survivin (SVN) to APCs to induce adaptive immunity (US Patent Publication 2014 / 0186401; Xu et al. (2014) Cancer Res. 74(21):6260-6270). SVN is an inhibitor of apoptotic proteins (IAPs) that prolong cell survival, regulate the cell cycle, and are overexpressed in all solid tumors and poorly expressed in normal tissues. This technique uses Salmonella pathogenic island 2 (SPI-2) and its type III secretion system (T3SS) to deliver TAA to the cytosol of APCs, which then triggers TAA-specific CD8 + It is activated to induce T cells and anti-tumor immunity (Xu et al. (2014) Cancer Res. 74(21): 6260-6270). Similar to Listeria-based TAA vaccines, this approach has shown promise in mouse models, but effective tumor antigen-specific T cell priming in humans has not yet been demonstrated.

[0281] In addition to gene delivery, Salmonella typhimurium has also been used to deliver small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) for cancer treatment. For example, attenuated Salmonella typhimurium has been modified to express certain shRNAs that target STAT3 and IDO1 (International Patent Publication WO2008 / 091375; and U.S. Patent 9,453,227). VNP20009 transformed with an shRNA plasmid targeting the immunosuppressive gene indoleamine deoxygenase (IDO) successfully silenced IDO expression in a mouse melanoma model, resulting in tumor cell death and significant tumor invasion by neutrophils (Blache et al. (2012) Cancer Res. 72(24):6447-6456). The combination of this vector and PEGPH20 (an enzyme that depletes extracellular hyaluronan) has shown promising results in the treatment of pancreatic ductal adenocarcinoma tumors (Manuel et al. (2015) Cancer Immunol. Res. 3(9):1096-1107; U.S. Patent Publication 2016 / 0184456). In other studies, a strain of Salmonella typhimurium attenuated by phoP / phoQ deletion and expressing signaling transcription factor 3 (STAT3)-specific shRNA was found to inhibit tumor growth, reduce the number of metastatic organs, and extend lifespan in C57BL6 mice (Zhang et al. (2007) Cancer Res. 67(12):5859-5864). In other examples, Salmonella typhimurium strain L7207 has been used to deliver shRNA targeting CTNNB1, a gene encoding β-catenin (Guo et al. (2011) Gene therapy 18:95-105; US Patent Publication 2009 / 0123426, 2016 / 0369282), while Salmonella typhimurium strain VNP20009 has been used to deliver shRNA targeting STAT3 (Manuel et al. (2011) Cancer Res. 71(12):4183-4191; US ​​Patent Publication 2009 / 0208534, 2014 / 0186401 and 2016 / 0184456; International Patent Application Publication WO2008 / 091375 and WO2012 / 149364).siRNAs targeting the autophagy genes Atg5 and Becrin 1 have been delivered to tumor cells using Salmonella typhimurium strains A1-R and VNP20009 (Liu et al. (2016) Oncotarget 7(16):22873-22882). Improvements to such strains are needed to more efficiently stimulate the immune response and possess other advantageous properties, such as the immunostimulatory bacteria provided herein. Further and alternative modifications of various bacteria are described in the published international PCT application Publication WO2019 / 014398 and the US publication 2019 / 0017050A1. The bacteria described in each of these publications, and also described herein, may be modified as described herein to further improve their immunostimulatory and tumor-targeting properties.

[0282] Bacteria can be modified as described herein to reduce inflammatory effects and therefore to be less toxic. As a result, they can be administered in higher doses, for example. These Salmonella strains, as well as any other bacterial species known to those skilled in the art and / or listed above and herein, can be modified as described herein, such as by introducing an adenosine nutrient requirement. An example is the Salmonella typhimurium species described herein.

[0283] The bacterial strains provided herein are engineered to deliver therapeutic molecules / products. The strains here deliver immunostimulatory proteins, including modified gain-of-function variants of cytosolic DNA / RNA sensors, which constitutively induce / induce immunostimulatory proteins such as type I IFN expression and other cytokines, thereby promoting an anti-tumor immune response in the tumor microenvironment. The strains may also include genomic modifications that reduce phagocytic pyroptosis, thereby providing a stronger immune response and / or reducing or eliminating the ability to infect / invade epithelial cells, while retaining the ability to infect / invade phagocytic cells to accumulate more efficiently in tumors and tumor-resident immune cells. The bacterial strains encode therapeutic products. Accumulation in tumor-resident immune cells leads to the expression and secretion of the encoded therapeutic product into the tumor microenvironment, increasing therapeutic efficacy.

[0284] 4. Enhancement of immunostimulatory bacteria for increased therapeutic index and expression in tumor-resident immune cells Provided herein are enhancements of immunostimulant bacteria that reduce toxicity and improve antitumor activity. Examples of such enhancements are given below; they are described in relation to Salmonella, particularly Salmonella typhimurium; it will be understood by those skilled in the art that similar enhancements can be carried out with other bacterial species and other Salmonella strains.

[0285] a. ASD gene deletion The bacterial ASD gene encodes aspartate semialdehyde dehydrogenase. (Example: ASD gene of Salmonella typhimurium) - Mutants exhibit an obligate requirement for diaminopimelic acid (DAP), which is necessary for cell wall synthesis, and undergo thawing in environments lacking DAP. This DAP nutrient requirement can be used in vivo for plasmid selection and maintenance of plasmid stability without antibiotics when the asd gene is complemented trans in a plasmid. Antibiotic-free plasmid selection systems are advantageous because they allow for 1) the use of antibiotics as a rapid elimination mechanism in case of adverse effects and 2) antibiotic-free scale-up of production where such use is generally avoided. The asd gene complementation system is provided for such selection (Galan et al. (1990) Gene 94(1):29-35). The use of the asd gene complementation system to maintain plasmids in the tumor microenvironment is expected to increase the potency of Salmonella typhimurium engineered to deliver plasmid-encoding genes and therapeutic products / proteins, such as STING protein and other immunostimulant proteins, as described here.

[0286] Another use of the asd mutant of Salmonella typhimurium is for the utilization of DAP trophotropy, producing autolyzing (or suicide) strains for the delivery of macromolecules that infect cells but lack the ability to persistently colonize host tumors. Deletion of the asd gene provides an asd-deleted strain that, when grown in vitro or in vivo, makes the bacterium trophotropic to DAP (see, e.g., Example 3), and contains a plasmid that is trophotropic to DAP and encodes a therapeutic product but does not contain the asd complement gene, resulting in a strain that lacks replication in vivo. This strain grows in vitro in the presence of DAP, proliferates normally, and is then administered as an immunotherapy agent to a mammalian host lacking DAP. The suicide strain can invade host cells but cannot replicate because DAP is absent in the mammalian tissue, and it autoly thaws, delivering its cytosolic contents (e.g., plasmid or protein) to the cytosol.

[0287] In the example provided herein, the asd gene deletion strain of VNP20009 is further modified to express the LLO protein (cytoLLO) lacking the endogenous peripheral secretory signaling sequence, causing it to accumulate in the Salmonella cytosol. LLO is a cholesterol-dependent pore-forming hemolysin from Listeria monocytogenes that mediates bacterial phagosome evasion. When the autolyzing strain is introduced into tumor-bearing mice, the bacteria are taken up by phagocytic immune cells and enter the Salmonella-containing vacuole (SCV). In this environment, the DAP deletion prevents bacterial replication and leads to bacterial autolysis in the SCV. Subsequently, the lysis of the suicide strain releases the plasmid and accumulated LLO, forming a pore in the cholesterol-containing SVC membrane, enabling plasmid delivery to the host cell cytosol. Here, the gene product encoded on the plasmid, under the control of a eukaryotic promoter, can be expressed by host cell mechanisms.

[0288] b. Adenosine nutritional requirements Tryptophan and metabolites derived from the ATP / adenosine pathway are major driving mechanisms in the formation of an immunosuppressive environment within tumors. Adenosine, present in both intracellular and extracellular release forms, is an effector of immune function. Adenosine reduces T cell receptor-induced activation of NF-κB and inhibits IL-2, IL-4, and IFN-γ. Adenosine reduces T cell cytotoxicity, increases T cell anergy, and promotes Foxp3 + Or Lag-3 + Adenosine increases T cell differentiation into regulatory T cells (T-regs). In NK cells, it reduces adenosine IFN-γ production and suppresses NK cell cytotoxicity. Adenosine blocks neutrophil adhesion and spillage, reduces phagocytosis, and attenuates superoxide and nitric oxide levels. Adenosine also reduces the expression of TNF-α, IL-12, and MIP-1α (CCL3) in macrophages, attenuates MHC class II expression, and increases IL-10 and IL-6 levels. Adenosine immunomodulatory activity occurs after the release of tumor cells into the extracellular space and activation of adenosine receptors (ADRs) on the surface of target immune cells, cancer cells, or endothelial cells. High adenosine levels in the tumor microenvironment result in local immunosuppression, limiting the immune system's ability to eliminate cancer cells.

[0289] Extracellular adenosine is produced by the secondary activity of membrane-attached ectoenzymes, CD39 and CD73, expressed in tumor stromal cells, along with adenosine production via phosphohydrolysis of ATP or ADP produced from dead or dying cells. CD39 converts extracellular ATP (or ADP) to 5'AMP, which is then converted to adenosine by CD73. The expression of CD39 and CD73 in endothelial cells increases under hypoxic conditions in the tumor microenvironment, thereby increasing adenosine levels. Tumor hypoxia can result from inadequate blood supply and disordered tumor vascular structure, which impair oxygen delivery (Carroll and Ashcroft (2005) Expert. Rev. Mol. Med. 7(6):1-16). Hypoxic conditions in the tumor microenvironment also inhibit adenylate kinase (AK), which converts adenosine to AMP, leading to extremely high extracellular adenosine concentrations. Extracellular adenosine concentrations in the hypoxic tumor microenvironment were measured at 10–100 μM, which is up to approximately 100–1000 times higher than the typical extracellular adenosine concentration of about 0.1 μM (Vaupel et al. (2016) Adv Exp Med Biol. 876:177-183; Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Because the hypoxic areas of the tumor are distal to the microvessels, the local concentration of adenine in the tumor area may be higher than elsewhere.

[0290] Because it directs the effects of inhibiting the immune system, adenosine can also control cancer cell proliferation and propagation by acting on cancer cell growth, apoptosis, and angiogenesis. For example, adenosine is primarily A 2A and A 2B Receptor stimulation can promote angiogenesis. Stimulation of endothelial cell receptors regulates the expression of intercellular adhesion molecule 1 (ICAM-1) and E-sectin in endothelial cells, maintaining vascular integrity and promoting vascular proliferation (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Adenosine stimulates various cells 2A , A 2BAlternatively, activation of A3 at level 1 or higher can stimulate the production of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), or angiopoietin 2 (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857).

[0291] Adenosine can also directly regulate tumor cell proliferation, apoptosis, and metastasis through its interaction with receptors on cancer cells. For example, in a certain breast cancer cell line, A1 and A 2A Receptor activation stimulates tumor cell proliferation, and in colon cancer cells, A 2B Some studies have shown that receptor activation has cancer growth-promoting properties (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Adenosine can also induce apoptosis in cancer cells, and various studies have shown that this activity is linked to the exogenous apoptotic pathway mediated by A3 or A3. 2A and A 2B This links to the activation of the endogenous apoptotic pathway via (Antonioli et al. (2013)). Adenosine can promote tumor cell migration and metastasis by increasing cell motility, adhesion to the extracellular matrix, and the expression of cell adhesion proteins and receptors that promote cell migration and motility.

[0292] Extracellular release of adenosine triphosphate (ATP) occurs from stimulated immune cells and damaged, dying, or stressed cells. When stimulated by this extracellular release of ATP, the NLR family pyrin domain-containing inflammasome 3 (NLRP3) activates caspase-1, leading to the secretion of cytokines IL-1β and IL-18, which subsequently activate innate and adaptive immune responses (Stagg and Smyth (2010) Oncogene 29:5346-5358). ATP is catabolized to adenosine by the enzymes CD39 and CD73. Activated adenosine acts as a highly immunosuppressive metabolite via a negative feedback mechanism, exhibiting pleiotropic effects on multiple immune cell types in the hypoxic tumor microenvironment (Stagg and Smyth (2010) Oncogene 29:5346-5358). Adenosine receptor A 2A and A 2B Adenosine is expressed in a variety of immune cells and is stimulated by adenosine to promote cAMP-mediated signaling alterations, resulting in immunosuppressive phenotypes in T cells, B cells, NK cells, dendritic cells, mast cells, macrophages, neutrophils, and NKT cells. As a result, adenosine levels can accumulate more than 100 times normal levels in pathological tissues such as solid tumors, where ectonucleotidases such as CD73 are shown to be overexpressed. Adenosine has also been shown to promote tumor angiogenesis and progression. Engineered bacteria that are nutrient-dependent on adenosine therefore exhibit enhanced tumor targeting and colonization.

[0293] Immunostimulatory bacteria such as Salmonella typhi can be made adenosine-dependent by deletion of the tsx gene (Bucarey et al. (2005) Infection and Immunity 73(10):6210-6219) or purD (Husseiny (2005) Infection and Immunity 73(3):1598-1605). In the Gram-negative bacterium Xanthomonas oryzae, a purD gene knockout strain has been shown to be adenosine-dependent (Park et al. (2007) FEMS Microbiol Lett 276:55-59). As illustrated here, Salmonella typhi strain VNP20009 is adenosine-dependent due to purI deletion, and therefore further modification to make it adenosine-dependent is unnecessary. Therefore, the embodiment of the immunostimulatory bacterial strain provided herein is adenosine-nutrient-requiring. Such nutrient-requiring bacteria selectively replicate in the tumor microenvironment, further increasing the accumulation and replication of the administered bacteria in the tumor, reducing adenosine levels within and around the tumor, thereby reducing or eliminating immunosuppression caused by adenosine accumulation. An example of such bacteria provided herein is purI for providing adenosine nutrient requirement. - / msbB - This is a modified strain of Salmonella typhimurium containing mutations. Other genomic mutations may also be incorporated to confer other advantageous properties to the bacterium, as described here.

[0294] c. Flagellin-deficient strains Flagella are organelles on the surface of bacteria consisting of long fibers connected via hooks to a rotating motor that can rotate clockwise or counterclockwise to provide a means of locomotion. In Salmonella typhimurium, flagella are important for establishing infection via the oral route due to their ability to mediate chemotaxis and motility across the mucous layer of the digestive tract. Flagella are necessary for chemotaxis and colonization of tumorigenic casts 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), but flagella are not necessary for tumor colonization in animals when the bacteria are administered intravenously (Stritzker et al. (2010) International Journal of Medical Microbiology 300:449-456). Each flagellar filament consists of tens of thousands of flagellin subunits. The Salmonella typhimurium chromosome contains two genes, fliC and fljB, which encode antigenically distinct flagellin monomers. Mutants lacking both fliC and fljB are non-motile and non-pathogenic when administered orally, but remain pathogenic when administered non-enterally.

[0295] Flagellin is a major pro-inflammatory determinant in Salmonella (Zeng et al. (2003) J. Immunol. 171:3668-3674) and is directly recognized by TLR5 on the cell surface and NLRC4 in the cytosol (Lightfield et al. (2008) Nat Immunol. 9(10):1171-1178). Both pathways lead to a pro-inflammatory response that results in the secretion of cytokines including IL-1β, IL-18, TNF-α, and IL-6. Attempts have been made to create more potent salmonella-based cancer immunotherapies by increasing the pro-inflammatory response to flagellin by manipulating bacteria to secrete Vibrio vulnificus flagellin B, which induces stronger inflammation than flagellin encoded by fliC and fljB (Zheng et al. (2017) Sci. Transl. Med. 9(376):eaak9537).

[0296] To reduce pro-inflammatory signaling, immunostimulatory bacteria such as Salmonella typhimurium are provided that are engineered to lack both flagellin subunits fliC and fljB. For example, as shown here, a Salmonella strain lacking msbB, which results in TNF-alpha-induced reduction, is combined with fliC and fljB knockout. The resulting Salmonella strain has a combination of TNF-alpha-induced reduction and TLR5 recognition reduction. These modifications, msbB - ,fliC - and fljB - This can be optionally combined with a bacterial plasmid containing a CpG and / or cDNA expression cassette to provide eukaryotic promoter-controlled expression of heterologous proteins, such as STING pathway gain-of-function protein variants, immunostimulatory cytokines, and / or similarly inhibitory RNAi molecules. The resulting bacteria have reduced pro-inflammatory signaling and strong antitumor activity.

[0297] Elimination of flagella confers an additional advantageous property that increases the therapeutic index of bacteria. For example, as shown here (see Example 6, for example), elimination of flagella (i.e., in Salmonella, fliC) - / fljB - This reduces pyroptosis in mouse macrophages and human monocytes, prevents infection in epithelial cells, and restricts bacterial uptake to tumor-resident immune / bone marrow cells.

[0298] As described below and elsewhere in this specification, flagellar loss is, for example, asd - msbB - purI - , pagP - , csgD - adrA - and / or other modifications described herein may be combined with one or more other genomic modifications that confer advantageous properties that improve the therapeutic index of the bacteria. Such modified bacteria may be transformed with plasmids encoding therapeutic products that increase the antitumor immune response in a target, including, for example, cytosolic DNA / RNA sensors and their gain-of-function variants, as well as immunostimulatory proteins such as cytokines.

[0299] For example, as provided here, fliC - and fljB - A double mutant was constructed using an asd-deficient strain of Salmonella typhimurium strain VNP20009. VNP20009, attenuated in terms of pathogenicity by disrupting PurI / purM, was also modified to include an msbB deletion, which results in the production of a lipid A subunit of LPS that is less toxin-producing than wild-type lipid A. This leads to reduced TNF-α production in a mouse model after intravenous administration compared to strains with wild-type lipid A. Furthermore, fliC - and fljB -Double mutants were constructed in wild-type Salmonella typhimurium strains containing asd, purI / purM, and msbB deletions. The resulting strains are examples of strains attenuated against bacterial inflammation by modification of lipid A to reduce TLR2 / 4 signaling and deletion of the flagellin subunit to reduce TLR5 recognition and inflammasome induction. The deletion of the flagellin subunit combined with LPS modification enables greater host tolerance, directs an immunostimulatory response to the production of immunostimulatory proteins, and / or induces an antitumor response, thereby enabling the delivery of RNA interference to desired targets in the TME that promote an adaptive immune response against tumors.

[0300] d. Gene deletion in the LPS biosynthesis pathway Lipopolysaccharide (LPS) in Gram-negative bacteria is the main component of the outer layer of the bacterial membrane. It consists of three main parts: lipid A, non-repeating core oligosaccharide, and O antigen (or O polysaccharide). The O antigen is the outermost part of LPS and acts as a protective layer against bacterial permeability; however, the sugar composition of the O antigen varies widely between strains. Lipid A and core oligosaccharide vary less and are more generally conserved within the same species of strain. Lipid A is the part of LPS that contains endotoxin activity. Generally, it is a disaccharide decorated with multiple fatty acids. These hydrophobic fatty acid chains fix LPS to the bacterial membrane, while the rest of LPS protrudes from the cell surface.

[0301] Lipid A domains are responsible for the toxicity of most Gram-negative bacteria. Generally, LPS in the blood is recognized as a prominent pathogen-associated molecular pattern (PAMP) and induces a severe pro-inflammatory response. LPS is a ligand for membrane-bound receptor complexes including CD14, MD2, and TLR4. TLR4 is a transmembrane protein signaled via the MyD88 and TRIF pathways to stimulate the NFκB pathway, leading to the production of pro-inflammatory cytokines such as TNF-α and IL-1β, which can result in potentially lethal endotoxin shock. In the cytosol of mammalian cells, LPS binds 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).

[0302] The composition of lipid A and the toxicogenicity of lipid A variants are well supported by the literature. For example, monophosphorylated lipid A is far less inflammatory than lipid A with multiple phosphate groups. The number and length of acyl chains in lipid A can also have a significant impact on the degree of toxicity. Standard lipid A from E. coli has six acyl chains, and this hexa-acylation is potently toxic. Lipid A from Salmonella typhimurium is similar to that of E. coli; it is a glucosamine disaccharide with four primary and two secondary hydroxyacyl chains ((Raetz and Whitfield (2002) Annu. Rev. Biochem. 71:635-700). As mentioned above, Salmonella typhimurium msbB -The mutant does not undergo terminal myristoylation of LPS and primarily produces pentaacylated lipid A, which is significantly less toxic than hexaacylated lipid A. Modification of lipid A with palmitic acid is catalyzed by palmitoyltransferase (PagP). Transcription of the pagP gene is regulated by the phoP / phoQ system, which is activated, for example, by low concentrations of magnesium in SCV. Therefore, the acyl content of Salmonella typhimurium is variable and can be hexa- or penta-acylated in wild-type bacteria. Salmonella typhimurium's ability to palmitize its lipid A increases its resistance to antimicrobial peptides secreted into phagolysosomes.

[0303] In wild-type Salmonella typhimurium, pagP expression results in heptacylated lipid A. msbB - In mutants (where the terminal acyl chain of lipid A cannot be added), pagP induction results in hexaacylated LPS (Kong et al. (2011) Infection and Immunity 79(12):5027-5038). Hexaacylated LPS has been shown to be the most pro-inflammatory. For example, this pro-inflammatory signaling has been studied by other groups, such as by the deletion of pagP to produce only hexaacylated LPS (Felgner et al. (2016) Gut Microbes 7(2):171-177; Felgner et al. (2018) Oncoimmunology 7(2): e1382791), but this can be poorly tolerated due to the TNF-α-mediated pro-inflammatory properties of LPS and paradoxically low adaptive immunity (Kocijancic et al. (2017) Oncotarget 8(30):49988-50001).

[0304] LPS is a potent TLR4 agonist that induces TNF-α and IL-6. 1E9CFU / m 2In the IV VNP20009 clinical trial, dose-restricted toxicity (Toso et al. (2002) J. Clin. Oncol. 20(1):142-152) was cytokine-mediated (fever, hypotension), with serum TNF-α levels >100,000 pg / ml and IL-6 levels >10,000 pg / ml at 2 hours. Despite msbB deletion and reduced pyrogenicity in VNP20009, LPS may still be toxic at high doses, possibly due to the presence of hexaacylated LPS. Therefore, pagP - / msbB - Because this strain cannot produce hexaacylated LPS, it is more resistant to high doses, reaching 1E9 CFU / m³ in humans. 2 This allows for administration at the above dose. Higher doses may increase tumor colony formation and enhance the therapeutic effect of immunostimulant bacteria.

[0305] Here, Salmonella bacteria, such as Salmonella typhimurium, are engineered to delete both flagellin subunits fliC and fljB to reduce pro-inflammatory signaling. For example, as shown here, a Salmonella strain lacking msbB, which results in TNF-alpha-induced reduction, is combined with fliC and fljB knockout. This results in a Salmonella strain with a combination of TNF-alpha-induced reduction and TLR5 recognition reduction. These modifications are associated with pagP - This may include other combinations of genome modifications described herein, and the resulting bacterial strains can be optionally transformed with immunostimulatory plasmids (encoding immunostimulatory proteins) containing CpG. The resulting bacteria exhibit reduced pro-inflammatory signaling but possess strong antitumor activity.

[0306] Examples of attenuated Salmonella strains shown here include exemplary strains of Salmonella typhus that can produce only pentaacylated LPS, contain a deletion in the msbB gene (which inhibits terminal myristoylation of lipid A, as described above), and are further modified by a deletion in pagP (which inhibits palmitoylation). When strains modified to produce pentaacylated LPS are further modified to express therapeutic products such as heterologous immunostimulatory proteins and / or interfering RNAs against immune checkpoints, for example, they may exhibit lower levels of pro-inflammatory cytokines, improved blood stability, improved resistance to complement fixation, increased sensitivity to antimicrobial peptides, enhanced tolerability, and increased antitumor immunity.

[0307] For example, PAGP is provided here. - Mutants can also be constructed in Salmonella typhimurium VNP20009 or other strains described herein, or in asd, msbB, purI / purM, and fliC / fljB deletion strains of wild-type Salmonella typhimurium. The resulting strains are examples of strains attenuated for bacterial inflammation by modification of lipid A to reduce TLR2 / 4 signaling, deletion of a flagellin subunit to reduce TLR5 recognition and inflammasome induction, and deletion of pagP to produce pentaacylated LPS. The deletion of the flagellin subunit in combination with LPS modification allows for greater tolerability in the host and greater stability in the blood and resistance to complement fixation, improving transport to tumor sites for delivery of RNA interference to desired targets in the TME to direct immunostimulatory responses toward the production of some gene product such as immunostimulatory proteins and / or induce antitumor responses and promote adaptive immune responses against tumors.

[0308] e. Deletion of genes necessary for biofilm formation Bacteria and fungi can form multicellular structures called biofilms. Bacterial biofilms are secreted and encapsulated within a mixture of cell wall-associated polysaccharides, glycoproteins and glycolipids, as well as extracellular DNA, collectively known as extracellular polymers. These extracellular polymers protect bacteria from multiple invaders, including desiccant, antibiotics, and antimicrobial peptides. Bacterial biofilms enable surface colonization and are a significant cause of infection in prosthetic devices such as infusion ports and catheters. Biofilms can also form in tissues during the course of infection, which prolongs bacterial persistence and shedding, limiting the effectiveness of antibiotic treatment. Chronic bacterial persistence in biofilms increases tumorigenesis, for example, in Salmonella typhi infections of the gallbladder (Di Domenico et al. (2017) Int. J. Mol. Sci. 18:1887).

[0309] Salmonella typhimurium biofilm formation is regulated by CsgD. CsgD activates the csgBAC operon, which leads to increased production of the curuli ciliary subunits CsgA and CsgB (Zakikhany et al. (2010) Molecular Microbiology 77(3):771-786). CsgA is recognized as PAMP by TLR2 and induces IL-8 production from human macrophages (Tukel et al. (2005) Molecular Microbiology 58(1):289-304). Furthermore, CsgD indirectly increases cellulose production by activating the adrA gene, which encodes diguanylate cyclase. The small molecule cyclic diguanosine monophosphate (c-di-GMP) produced by AdrA is a ubiquitous secondary messenger found in almost all bacterial species. Increased AdrA-mediated increase in c-di-GMP increases the expression of the cellulose synthase gene bcsA, which subsequently increases cellulose production via stimulation of the bcsABZC and bcsEFG operons. The reduced ability of immunostimulatory bacteria such as Salmonella typhimurium to form biofilms can be achieved by deletion of genes involved in biofilm formation, such as csgD, csgA, csgB, adrA, bcsA, bcsB, bcsZ, bcsE, bcsF, bcsG, dsbA, or dsbB (Anwar et al. (2014) PLoS One 9(8):e106095).

[0310] Salmonella typhimurium forms biofilms on solid tumors as a defense against phagocytosis by host immune cells. Salmonella variants that cannot form biofilms are more rapidly taken up by host phagocytic cells and eliminated from infected tumors (Crull et al. (2011) Cellular Microbiology 13(8):1223-1233). This increased intracellular localization within phagocytic cells reduces the persistence of extracellular bacteria and enhances the effectiveness of plasmid delivery, expression, and release of encoded therapeutic products to TMEs, as well as gene knockdown by RNA interference, as described here. Immunostimulatory bacteria engineered to reduce biofilm formation have increased elimination rates from tumors / tissues, thus increasing treatment tolerability, preventing colonization of prosthetics in patients, and thereby increasing the therapeutic benefit of these strains. Adenosine mimetic can inhibit Salmonella typhimurium biofilm formation, demonstrating that high adenosine concentrations in the tumor microenvironment may contribute to tumor-associated biofilm formation (Koopman et al. (2015) AntiMicrob Agents Chemother 59:76-84). Attenuated viable bacterial strains, such as Salmonella typhimurium provided herein, which also inhibit biofilm formation by including purI disruption (and therefore colony-forming adenosine-rich tumors) and deletion of one or more genes necessary for biofilm formation, are engineered to deliver plasmids encoding therapeutic products such as immunostimulatory proteins and interfering RNAs, including cytosolic DNA / RNA sensors and gain-of-function variants thereof and other cytokines, in order to stimulate a strong antitumor immune response.

[0311] The adrA gene encodes diguanylate cyclase, which produces c-diGMP, necessary for Salmonella typhimurium biofilm formation. c-diGMP binds to the host cytosolic protein STING and acts as an agonist. Immunostimulatory bacteria with reduced c-diGMP production due to adrA deletion, contrary to common sense, bacterial mutants (including adrA mutants) such as Salmonella typhimurium mutants that cannot form biofilms have been shown to have reduced therapeutic efficacy in mouse tumor models (Crull et al. (2011) Cellular Microbiology 13(8):1223-1233). Several human alleles of STING are resistant to binding to the bacterial-produced 3'3' CDN (Corrales et al. (2015) Cell Reports 11:1018-1030).

[0312] As described herein, bacterial strains such as Salmonella typhimurium strains, which are engineered to be adenosine-dependent and whose ability to induce pro-inflammatory cytokines is reduced by modification of LPS and / or deletion of flagellin and / or deletion of genes necessary for biofilm formation, are further modified to deliver other therapeutic anti-cancer products, such as interfering RNA and cytosolic DNA / RNA sensors and gain-of-function variants thereof (e.g., STING and others) and immunostimulatory proteins including cytokines, in order to promote a strong anti-tumor immune response.

[0313] f. Salmonella manipulated to avoid Salmonella-containing vacuoles (SCVs) Salmonella strains such as Salmonella typhimurium are intracellular pathogens that primarily replicate in membrane-bound compartments called Salmonella-containing vacuoles (SCVs). Salmonella typhimurium has been shown to evade replication in some epithelial cell lines, albeit infrequently, by entering the cytosol, a site where replication is possible. Salmonella strains engineered to evade SCVs with high efficiency can more efficiently deliver macromolecules such as plasmids to the host cell cytosol because the lipid bilayer of the SCV acts as a voltage barrier. Plasmid release into the host cytosol enables the expression of therapeutic products encoded by the plasmid, which is under the control of host recognition regulatory signals such as eukaryotic promoters. This increases the efficiency of therapeutic product production and delivery, improving the therapeutic index of the bacteria, especially when the bacteria are phagocytosed by tumor-resident immune cells.

[0314] Provided herein are Salmonella strains and methods with enhanced SCV avoidance. As described below and elsewhere in this specification, this is achieved by deletion of genes necessary for Salmonella-induced fiber (SIF) formation. These mutants exhibit increased SCV avoidance and can replicate in the cytosol of host cells. For example, plasmid delivery enhancement using the sifA mutant of Salmonella typhimurium has been demonstrated. The sifA gene encodes the SPI-2 T3SS-2 secretory effector protein, which mimics or activates the RhoA family of host GTPases (Ohlson et al. (2008) Cell Host & Microbe 4:434-446). Genes encoding other secretory effectors involved in SIF formation can be targeted. These include, for example, sseJ, sseL, sopD2, pipB2, sseF, sseG, spvB, and steA. Enhanced avoidance in Salmonella typhimurium by inhibiting SIF formation allows the viable bacteria to be released into the cytosol, where replication is possible.

[0315] Another method to enhance SCV evasion in Salmonella typhimurium and increase the delivery of macromolecules such as plasmids to the cytosol is the expression of heterologous hemolysins that cause pore formation or rupture of the SCV membrane. One such hemolysin is the listeriolisin O protein (LLO) from Listeria monocytogenes, encoded by the hlyA gene. LLO is a cholesterol-dependent pore-forming cell lysin secreted from Listeria monocytogenes, primarily responsible for phagosome evasion and entry into the cytosol of host cells. Secretion of LLO from Salmonella typhimurium can result in bacterial evasion and replication in the cytosol. To prevent intact Salmonella typhimurium from evading SCV and replicating in the cytosol, the nucleotide encoding the secretion signal sequence can be removed from the gene, producing cytoLLO. In this way, active LLO is contained within the Salmonella typhimurium cytosol, and LLO is released only when the bacteria undergo lysis (e.g., asd - (Due to intracellular DAP deletion in the strain). Bacterial lysis in the SCV allows plasmid release and cytoLLO accumulation, which creates a pore in the SCV, enabling delivery of the plasmid to the host cell cytosol, where the encoded therapeutic product can be expressed.

[0316] Salmonella strains such as Salmonella typhimurium strain VNP20009, which are engineered to express cytoLLO to enhance plasmid delivery for the expression of therapeutic products such as STING protein and its variants and other immunostimulatory proteins, may increase the therapeutic efficacy of immunostimulatory bacteria. This is advantageous because the bacteria are engineered for accumulation in tumor-resident immune cells as described herein, thereby releasing therapeutic products directly into the tumor microenvironment.

[0317] g. Deletion of SPI-1 and SPI-2 genes and / or other genes to eliminate the ability of bacteria to infect epithelial cells, including hair loss. As described above, the pathogenesis of certain bacterial species, including Salmonella species such as Salmonella typhimurium, involves gene clusters called Salmonella pathogenic islands (SPIs). Salmonella typhimurium is an intracellular pathogen that can be rapidly taken up by bone marrow cells such as macrophages or induce its uptake by non-phagocytic cells such as epithelial cells. Once inside a cell, it can replicate within the Salmonella-containing vacuole (SCV), and in some epithelial cells, it can escape to the cytosol. The two most characteristic pathogenic islands are SPI-1, which is responsible for mediating bacterial invasion of non-phagocytic cells such as epithelial cells, and SPI-2, which is necessary for replication within the SCV (Agbor and McCormick (2011) Cell Microbiol. 13(12):1858-1869). SPI-1 and SPI-2 encode macromolecular structures known as type 3 secretion systems (T3SS), which are capable of translocating effector proteins across the host membrane (Galan and Wolf-Watz (2006) Nature 444:567-573).

[0318] i. Salmonella pathogenic island 1 (SPI-1) SPI-1-dependent host cell invasion Invasion-associated Salmonella pathogenicity island 1 (SPI-1), which includes the type 3 secretion system (T3SS), is responsible for the translocation of effector proteins into the host cell cytosol, triggering actin rearrangement that leads to Salmonella uptake. Salmonella invades non-phagocytic intestinal epithelial cells using the type 3 secretion system (T3SS) encoded by SPI-1, forming needle-like structures that directly inject effector proteins into the host cell cytosol. These effector proteins result in rearrangement of the eukaryotic cell cytoskeleton, promoting intestinal epithelial invasion and also inducing pro-inflammatory cytokines. The SPI-1 locus consists of 39 genes encoding components of this invasion system (see, e.g., Kimbrough et al. (2002) Microbes Infect. 4(1):75-82). The SPI-1 gene contains numerous operons, including sitABCD, sprB, avrA, hilC, orgABC, prgKJIH, hilD, hilA, iagB, sptP, sicC, iacP, sipADCB, sicA, spaOPQRS, invFGEABCIJ, and invH. The operons, genes, and their functions are described and illustrated, for example, by Kimbrough et al. ((2002) Microbes Infect. 4(1):75-82). The SPI-1 gene includes, but is not limited to, avrA, hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP.

[0319] T3SS is a complex that plays a major role in the infectivity of Gram-negative bacteria by directly injecting bacterial protein effectors into host cells in an ATP-dependent manner. The T3SS complex passes through the internal and external bacterial membranes and forms a pore in the eukaryotic cell membrane upon contact with the host cell. They consist of an efflux apparatus, a needle complex, and a translocon at the tip of the needle (see, e.g., Kimbrough et al. (2002) Microbes Infect. 4(1):75-82). The needle complex contains the needle-shaped protein PrgI, which is a basal body that fixes the complex to the bacterial membrane, and other proteins including PrgH, PrgK, and InvG and InvH, PrgJ (rod-shaped protein) and InvJ. The translocon that forms a pore in the host cell is a complex of proteins SipB, SipC, and SipD. The efflux apparatus that enables the translocation of effector proteins consists of proteins SpaP, SpaQ, SpaR, SpaS, InvA, InvC, and OrgB. The cytosolic sorting platform that establishes a specific order of protein secretion consists of the proteins SpaO, OrgA, and OrgB (see, for example, Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0320] Effectors translocated to host cells by T3SS-1 (T3SS of SPI-1) include SipA, SipC, SopB, SopD, SopE, SopE2, and SptP, which are essential for cellular invasion. For example, Salmonella typhimurium sipA mutants show a 60-80% reduction in invasion, sipC deletion reduces invasion by 95%, and sopB deletion reduces invasion by 50% (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 that bind to secretory proteins and maintain them in a secretory-capable conformation 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, InvI, IacP, and IagB (see, for example, Kimbrough et al. (2002) Microbes Infect. 4(1):75-82).

[0321] SPI-1 T3SS is essential for crossing the gastrointestinal epithelial layer, but is not important for infection when bacteria are injected non-enterally. Injection of certain proteins (e.g., PrgI and PrgJ) and the needle complex itself can also induce inflammasome activation and phagocytic pyroptosis. This pro-inflammatory cell death can limit the initiation of a strong adaptive immune response by directly inducing the death of antigen-presenting cells (APCs) and modifying the cytokine environment to inhibit memory T cell production. Therefore, SPI-1-dependent invasive inactivation via inactivation or knockout of one or more genes involved in SPI-1 eliminates the ability of 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 or more of the following: avrA, hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP.

[0322] SPI-1-independent host cell invasion Salmonella mutants lacking T3SS-1 have been shown to invade numerous cell lines / types via a T3SS-1-independent invasion mechanism involving several proteins, including invasin Rck, PagN, and HlyE. The rck operon contains six open reading frames: pefI, srgD, srgA, srgB, rck, and srgC. pefI is the transcriptional regulator of the pef operon involved in the biosynthesis of Pef pili. These pili are involved in biofilm formation in mouse pups, adhesion to the mouse small intestine, and fluid accumulation. SrgA oxidizes the disulfide bond of PefA, the major structural subunit of Pef pili. srgD encodes a putative transcriptional regulator; SrgD works with PefI to induce synergistic negative regulation of flagellar gene expression. srgB encodes a putative outer membrane protein, and srgC encodes a putative transcription regulator (see, for example, Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0323] Rck is a 17kDa outer membrane protein encoded by the highly pathogenic plasmids of *S. enteritidis* and *S. typhimurium*. It induces adhesion and invasion of epithelial cells and confers high levels of resistance to complement neutralization by inhibiting the formation of membrane attack complexes. Rck mutants show a 2-3-fold reduction in epithelial cell invasion compared to wild-type strains, while Rck overexpression increases invasion. Rck induces cell entry via a receptor-mediated process and promotes local actin remodeling and weak, tightly packed membrane expansion. Therefore, *Salmonella* can enter cells through two distinct mechanisms: a 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).

[0324] 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, such as an acidified macrophage phagosome environment or low Mg... 2+ The concentration is sensed by PhoQ, and then PhoP is activated to regulate specific genes. Deletion of pagN in Salmonella typhimurium has been shown to result in a threefold reduction in intestinal cell invasion without altering cell adhesion. While the PagN-mediated invasion mechanism is not fully understood, actin polymerization has been shown to be necessary for invasion. Studies have shown that PagN is necessary for Salmonella survival in BALB / c mice, and that pagN mutants are less competitive in colonizing the mouse spleen than the parent strain. Because pagN is activated by PhoP, SPI-1 island encoding T3SS-1 is largely expressed in downregulated cells. Therefore, bacteria that emerge from epithelial cells or macrophages can have optimal PagN expression levels, but have low T3SS-1 expression, which can then mediate interactions with other cells encountered after host cell destruction, demonstrating the role of PagN in Salmonella pathogenesis (see, for example, Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0325] hlyE shares over 90% sequence identity with E. coli HlyE(ClyA) hemolysin. When exported from bacterial cells via outer membrane vesicle release, the HlyE protein lyses epithelial cells and is involved in epithelial cell invasion. HlyE is also 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).

[0326] Consequently, the elimination of the bacteria's ability to infect epithelial cells can also be achieved by manipulating the immunostimulant bacteria described herein, including knockout or deletion / disruption of genes encoding proteins involved in SPI-1-independent invasion, such as one or more of the genes rck, pagN, hlyE, pefI, srgD, srgA, srgB, and srgC.

[0327] The immunostimulatory bacteria provided herein include those with deletions or disruptions in the hilA gene and / or other genes in the T3SS pathway. When these bacteria are administered intravenously or intratumorally, infection converges towards phagocytic cells such as macrophages and dendritic cells, which do not require SPI-1 T3SS for uptake. This enhances the safety profile of the immunostimulatory bacteria provided herein. It prevents off-target cellular invasion and inhibits fecal-oral transmission. In addition to reducing Salmonella uptake by non-phagocytic cells such as epithelial cells, the deletion or disruption of genes in this pathway also extends the lifespan of phagocytic cells by inhibiting inflammasome activation and macrophage pyroptosis, thus inducing less cell death in human macrophages compared to bacteria that do not contain deletions in this pathway. For example, a deletion of genes in the SPI-1 pathway (e.g., needle and rod proteins) inhibits pyroptosis by inflammasome activation, while TLR5 signaling can be maintained. This, in turn, enables the long-term secretion of encoding proteins such as STING protein or other therapeutic / anti-cancer products encoded by the immunostimulatory bacteria provided herein, allowing macrophage transport to tumors and thus improving the effectiveness of the immunostimulatory bacteria.

[0328] As described herein, immunostimulatory bacteria are provided that do not infect epithelial cells but retain the ability to infect phagocytic cells, including tumor-resident immune cells, thereby being modified to efficiently target immunostimulatory bacteria and encoded therapeutic products to the tumor microenvironment. This is achieved by deletion or knockout of any protein in SPI-1, including but not limited to deletions of one or more of avrA, hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP, as well as one or more of rck, pagN, hlyE, pefI, srgD, srgA, srgB, and srgC.

[0329] Immunostimulatory bacteria that do not infect epithelial cells can be further modified, as described herein, to encode immune-stimulating therapeutic products, such as type I interferon-inducing products (e.g., cytosolic DNA / RNA sensors and their GOF variants), and also to encode immunostimulatory proteins such as cytokines. The bacteria generally have an asd deletion to make them non-replicable in mammalian hosts. For example, strains of Salmonella typhimurium are provided that are modified by deletions of one or more SPI-1 genes, and by one or more purI deletions, msbB deletions, and asd deletions, and further modified by delivery of plasmids encoding immune-stimulating proteins such as type I interferon and / or immune-stimulating cytokine-inducing cytosolic DNA / RNA sensors and their gain-of-function variants.

[0330] For example, bacteria are provided that lack regulatory genes (e.g., hilA or invF), structural T3SS-1 genes (e.g., invG or prgH), and / or T3SS-1 effector genes (e.g., sipA or avrA) necessary for the expression of the SPI-1-associated type 3 secretion system (T3SS-1). As described above, this secretion system is responsible for injecting effector proteins into the cytosol of non-phagocytic host cells, such as epithelial cells, which trigger bacterial uptake; deletion of one or more of these genes eliminates infection / invasion of epithelial cells. Deletion of one or more genes, such as hilA, provides immunostimulatory bacteria that can be administered intravenously or intratumorally, resulting in infection of phagocytes that do not require SPI-1 T3SS for uptake, and also extends the lifespan of these phagocytes. hilA mutations also reduce the amount of pro-inflammatory cytokines and increase the tolerability of treatment and the quality of the adaptive immune response.

[0331] In addition to or separately from this, immunostimulatory bacteria may include knockouts or deletions of genes to inactivate products involved in SPI-1-independent infection / invasion, such as one or more of the genes pagN, hlyE, pefI, srgD, srgA, srgB, and srgC, thereby reducing or eliminating the bacteria's ability to infect epithelial cells.

[0332] As described herein, genes involved in the SPI-1 pathway and bacterial flagella activate inflammasomes in phagocytes (immune cells) and induce pyroptosis. Knockout or disruption of the SPI-1 gene and flagellar-encoding genes reduces or eliminates pyroptosis and also eliminates epithelial cell infection and increases phagocyte infection. Therefore, immunostimulatory bacteria may include knockouts or deletions to induce inactivation of gene products that induce cell death of tumor-resident immune cells, such as genes encoding proteins directly recognized by inflammasomes; these include fljB, fliC, prgI, and prgJ. As shown herein (see, for example, Example 6), flagellar elimination (i.e., in Salmonella, fliC - / fljB -), reducing pyroptosis in mouse macrophages and human monocytes, making infection in epithelial cells impossible, and limiting bacterial uptake to tumor resident immune / bone marrow cells.

[0333] Therefore, immunostimulatory bacteria that accumulate in phagocytic cells, particularly tumor-resident immune cells, are provided, expressing products encoded in plasmids controlled by eukaryotic regulatory signals such as RNA polymerase II. These products include those that induce immune responses, such as through pathways that increase the expression of type I interferon, thereby increasing the host immune response in the tumor microenvironment. Immunostimulatory bacteria can also encode other products, including immunostimulatory proteins such as IL-2, further enhancing the immune response in the tumor microenvironment.

[0334] ii. Salmonella pathogenic island 2 (SPI-2) Salmonella also possesses Salmonella pathogenic island 2 (SPI-2), which encodes other T3SSs that are activated after bacterial invasion of host cells, disrupting phagosome maturation and leading to the formation of specialized salmonella-containing vacuoles (SCVs) that are commensal to Salmonella during intracellular survival and replication. The SPI-2 T3SS effectors include SseB, SseC, SseD, and SpiC, which are responsible for assembling the F-actin coat around the intracellular bacterium; this actin coat promotes the fusion of SCVs with actin-containing or actin-propelled vesicles and prevents fusion with undesirable compartments. SifA is responsible for the formation of salmonella-inducing filaments (SIFs), which are tubules that connect individual SCVs in infected cells. SifA is essential for maintaining SCV integrity, and sifA variants are released into the host cell cytosol. SseF and SseG are components of SPI-2 T3SS that are involved in SCV repositioning and cellular transport processes, directing substances necessary for bacterial survival and replication to the SCVs. SseF and SseG are also involved in SIF formation. Other SPI-2 T3SS effectors include PipB2, SopD2, and SseJ, which are involved in SIF and SCV formation and vacuolar integrity maintenance; SpvC, SseL, and SspH1, which are involved in host immune signaling; and SteC, SspH2, SrfH / SseI, and SpvB, which are involved in the formation of the SCV F-actin network during the migration of infected phagocytic cells, as well as the inhibition of actin polymerization and P-body deassembly in infected cells (Coburn et al. (2007) Clinical Microbiology Reviews 20(4):535-549; Figueira and Holden (2012) Microbiology 158:1147-1161).

[0335] The immunostimulatory bacteria described herein may contain deletions or modifications to any of the SPI-2 T3SS genes that affect the formation or integrity of related structures such as SCVs and SIFs. These mutants increase the frequency of SCV avoidance and enable replication in the cytosol. For example, immunostimulatory bacteria such as Salmonella species engineered to avoid SCVs are more efficient at delivering macromolecules such as plasmids to the host cell cytosol because the lipid bilayer of the SCV acts as a voltage barrier. Enhanced bacterial avoidance from SCVs by inhibiting SIF formation releases viable bacteria into the cytosol, a site where encoded therapeutic products or proteins can be replicated and expressed under the control of host cell mechanisms (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 deletions or mutations in genes necessary for Salmonella-induced fiber (SIF) formation, including, for example, sifA, sseJ, sseL, sopD2, pipB2, sseF, sseG, spvB, and steA.

[0336] Immunostimulating bacteria that can evade SCV can be further modified, as described herein, to encode immune system-stimulating products, such as products that induce type I interferon, and also to encode cytokines. These bacteria generally have an asd deletion and are unable to replicate in mammalian hosts.

[0337] h. Endonuclease-1 (endA) mutations to increase plasmid delivery The endA gene (e.g., SEQ ID NO: 250) is an endonuclease (e.g., SEQ ID NO: 251) that mediates double-stranded DNA (dsDNA) degradation in the periplasm of Gram-negative bacteria. The most common strains of laboratory E. coli have mutations in the endA gene, which allows for high plasmid DNA yields. -This gene is conserved across species. To promote intact plasmid DNA delivery, the endA gene in engineered immunostimulant bacteria is deleted or mutated to inhibit endonuclease activity. Examples of such mutations include the E208K amino acid substitution (Durfee, et al. (2008) J. Bacteriol. 190(7):2597-2606) or the corresponding mutation in the species of interest. EndA containing E208 is conserved across bacterial species, including Salmonella (see, e.g., SEQ ID NO: 251). Therefore, the E208K mutation can be used to eliminate endonuclease activity in other species, including Salmonella species. Those skilled in the art can introduce other mutations or deletions to eliminate endA activity. The implementation of this mutation, deletion, or disruption of the gene to eliminate endA activity in immunostimulant bacteria, such as Salmonella, increases the efficiency of intact plasmid DNA delivery, thereby increasing the expression of encoded therapeutic products and enhancing antitumor efficacy.

[0338] i. RIG-I binding sequence As described above, type I interferons (IFN-α, IFN-β) are signature cytokines induced by different TLR-dependent and TLR-independent signaling pathways. One TLR-independent type I IFN pathway involves host recognition of single-stranded (ss) and double-stranded (ds) RNAs in the cytosol. These are sensed via RNA helicases, including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), and phosphorylation of the IFN-β promoter-stimulating factor 1 (IPS-1) adapter protein of the IRF-3 transcription factor, leading to the induction of type I IFN (Ireton and Gale (2011) Viruses 3(6):906-919). RIG-I recognizes dsRNA and ssRNA carrying 5'-triphosphate. This portion can bind directly to RIG-I or be synthesized from a poly(dA-dT) template by polyDNA-dependent RNA polymerase III (Pol III) (Chiu, YH et al. (2009) Cell 138(3):576-91). The poly(dA-dT) template, containing two AA dinucleotide sequences, is generated at the U6 promoter transcription start site of a common lentiviral shRNA cloning vector. Subsequent deletion in the plasmid inhibits type I IFN activation (Pebernard et al. (2004) Differentiation. 72:103-111). The RIG-I binding sequence can be incorporated into the plasmid provided herein; this inclusion can increase immunostimulation by inducing type I IFN production, which increases the antitumor activity of the immunostimulant bacteria described herein.

[0339] j. DNase II inhibition Another nuclease responsible for the degradation of foreign and self-DNA is DNase II, an endonuclease located in the endosomal compartment that degrades post-apoptotic DNA. Deletion of DNase II (Dnase2a in mice) leads to the accumulation of endosomal DNA that evades cytosolic transmission and activates cGAS / STING signaling (Lan YY et al. (2014) Cell Rep. 9(1):180-192). In humans, DNase II deletion is associated with autoimmune type I interferonopathy. In cancer, dysent tumor cells phagocytosed by tumor-resident macrophages inhibit 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). Therefore, embodiments of the immunostimulatory bacterial strains provided herein, which encode products capable of inhibiting DNase II in the tumor microenvironment, can induce the accumulation of apoptotic tumor DNA taken up into cells in the cytosol, which is a site where they can act as potent cGAS / STING agonists.

[0340] k. RNase H2 inhibition TREX1 (3-prime repair exonuclease 1) and DNase II function in clearing abnormal DNA accumulations, while RNase H2 similarly functions in eliminating pathogenic accumulations of RNA:DNA hybrids in the cytosol. Deletion of RNase H2 also contributes to the autoimmune phenotype of Ecardi-Goutier syndrome (Rabe, B. (2013) J. Mol. Med. 91:1235-1240). Deletion of RNase H2 and subsequent accumulation of RNA:DNA hybrids or genomically embedded ribonucleotide substrates has been shown to activate cGAS / STING signaling (MacKenzie et al. (2016) EMBO J. Apr. 15;35(8):831-44). Therefore, embodiments of immunostimulatory bacterial strains encoding products that inhibit or reduce RNase H2 expression, thereby inhibiting RNase H2, result in tumor-derived RNA:DNA hybrids and their derivatives that activate cGAS / STING signaling and enhance antitumor immunity.

[0341] l. Stabilin-1 / CLEVER-1 inhibition Another molecule primarily expressed in monocytes and involved in immune regulation is stabilin-1 (also known by the gene names STAB1, CLEVER-1, and FEEL-1). Stabilin-1 is a type I transmembrane protein that is upregulated in endothelial cells and macrophages, particularly tumor-associated macrophages, after inflammation (Kzhyshkowska et al. (2006) J. Cell. Mol. Med. 10(3):635-649). Upon inflammatory activation, stabilin-1 acts as a scavenger, aiding in wound healing and the removal of apoptotic bodies, thus preventing tissue damage such as hepatic fibrosis (Rantakari et al. (2016) Proc. Natl. Acad. Sci. USA 113(33):9298-9303). Upregulation of stabilin-1 has been shown to directly inhibit antigen-specific T cell responses, and siRNA-mediated knockdown in monocytes has been shown to enhance pro-inflammatory function (Palani, S. et al. (2016) J. Immunol. 196:115-123). Therefore, embodiments of immunostimulatory bacterial strains encoding products that inhibit or reduce stabilin-1 / CLEVER-1 expression in the tumor microenvironment enhance the pro-inflammatory function of tumor-resident macrophages.

[0342] m. CpG motif and CpG Island Unmethylated cytidine-phosphate-guanosine (CpG) motifs are dominant in bacterial genomic DNA, but not in vertebrates. Pathogenic DNA and synthetic oligodeoxynucleotide (ODN)-containing CpG motifs activate host defense mechanisms and lead to innate and acquired immune responses. Unmethylated CpG motifs contain a central unmethylated CG dinucleotide and adjacent regions. In humans, four distinct classes of CpG ODNs have been identified based on differences in structure and the nature of the immune responses they induce. K-type ODNs (also called B-type) generally contain 1 to 5 CpG motifs on a phosphorothioate backbone. D-type ODNs (also called A-type) contain a mixed phosphodiester / phosphorothioate backbone, have one CpG motif, a palindromic sequence that allows for the formation of a stem-loop structure, and poly-G motifs adjacent to the 3' and 5' ends. C-type ODNs have a phosphorothioate backbone and contain multiple palindromic CpG motifs capable of forming stem-loop structures or dimers. P-class CpG ODNs have a phosphorothioate backbone and contain multiple CpG motifs with double palindromes capable of forming hairpins at the GC-rich 3' end (Scheiermann and Klinman (2014) Vaccine 32(48):6377-6389). For the purposes of this text, CpGs are encoded in plasmid DNA; they can be introduced as motifs or into genes.

[0343] Toll-like receptors (TLRs) are key receptors for pathogen-associated molecular pattern (PAMP) sensitization and activation of innate immunity against pathogens (Akira et al. (2001) Nat. Immunol. 2(8):675-680). TLR9 is a hypomethylated CpG motif in prokaryotic DNA that is not naturally present in mammalian DNA (McKelvey et al. (2011) J. Autoimmunity 36:76-86). Recognition of the CpG motif by phagocytosis of pathogens into endosomes by a subset of immune cells induces IRF7-dependent type I interferon signaling, activating innate and adaptive immunity.

[0344] Immunostimulatory bacterial strains, such as Salmonella species and Salmonella typhimurium, carrying plasmids containing CpG island / motifs are provided herein. These bacteria can activate TLR9 and induce type I IFN-mediated innate and adaptive immunity. As illustrated herein, bacterial plasmids containing hypomethylated CpG islands can induce innate and adaptive antitumor immune responses and, when combined with encoding products such as gain-of-function variant STING proteins, may have synergistic or enhanced antitumor activity. For example, the asd gene (see, e.g., SEQ ID NO: 48) encodes high-frequency hypomethylated CpG islands. CpG motifs may be incorporated into immunostimulatory bacteria in combination with therapeutic products such as STING proteins and their variants described herein or evident therefrom, thereby enhancing or improving antitumor immune responses by regulating TLRs such as TLR9.

[0345] Immunostimulatory CpGs can generally be incorporated into plasmids by encapsulating nucleic acids encoding gene products from bacterial genes (e.g., asd) and adding nucleic acids containing CpG motifs. The plasmids here may contain CpG motifs. Examples of CpG motifs are known (see, for example, U.S. Patents 8,232,259, 8,426,375 and 8,241,844). These are, for example, 10-100, 10-20, 10-30, 10-40, 10-50, or 10-75 base pair lengths, with a general formula: (CpG) n (Here, n is the number of iterations.) It contains synthetic immunostimulatory oligonucleotides having the following properties.

[0346] Generally, at least one or two repeats are used; non-CG bases may be incorporated. Those skilled in the art are very familiar with the common use of CpG motifs for inducing immune responses by modifying TLRs, particularly TLR9.

[0347] 5. Modifications that increase the uptake of Gram-negative bacteria such as Salmonella by immune cells and reduce immune cell death. Immunostimulatory bacteria provided herein, such as strains of Salmonella typhimurium, can be modified to increase uptake by immune cells, such as tumor resident immune cells, and decrease uptake by non-immune cells, such as epithelial cells. Bacteria can also be modified to reduce immune cell death, such as by reducing macrophage pyroptosis. Numerous bacterial genome modifications can result in either increased immune cell infection or decreased pyroptosis, or both. Immunostimulatory bacteria provided herein include modifications such as the disruption or deletion of hilA, rod-shaped proteins, and / or needle-shaped proteins, for example, deletion and / or disruption of genes involved in the SPI-1 T3SS pathway and / or disruption / deletion of other bacterial genes encoding flagellin. These modifications allow bacteria to accumulate in tumor resident immune cells, which are the site where they can express encoded therapeutic products and release them directly into the tumor microenvironment, thereby increasing therapeutic efficacy. Furthermore, for example, reducing pyroptosis extends the lifespan of tumor-resident macrophages, enabling efficient production of encoded therapeutic products and activation of the immune response in the tumor microenvironment, thereby enhancing the effectiveness of bacterial antitumor therapy.

[0348] a. Bacterial uptake by immune cells The genomes of immunostimulatory bacteria provided herein may be modified to increase or promote infection of immune cells, such as phagocytes, particularly in the tumor microenvironment. 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, may derive from the activity of encoding genes in pathways that promote host cell invasion. Salmonella invasion-associated Salmonella pathogenicity island 1 (SPI-1) is one example. SPI-1 is a type 3 secretory apparatus (T3SS) responsible for the translocation of effector proteins into the cytosol of host cells. These proteins can trigger actin rearrangement leading to Salmonella uptake. T3SS effectors mediate the uptake of Salmonella typhimurium into non-phagocytic host cells, such as epithelial cells. SPI-1 T3SS is essential for translocation of the intestinal epithelium, but is not important for infection, for example, when bacteria are injected non-enterally. SPI-1 mutants have a defect in epithelial cell invasion and dramatically reduced oral pathogenicity, but are taken up normally by phagocytic cells such as macrophages (Kong et al. (2012) Proc. Natl. Acad. Sci. USA 109(47):19414-19419).

[0349] The immunostimulatory bacterial strains provided herein, such as Salmonella typhimurium, can be engineered to have mutations in the SPI-1 T3SS gene, thereby inhibiting uptake by epithelial cells and converging on immune cells such as tumor-associated macrophages, thereby enhancing the anti-tumor immune response. Furthermore, as shown herein (see, for example, Example 6), flagellar elimination makes infection in epithelial cells impossible and restricts bacterial uptake to tumor resident immune / bone marrow cells. Therefore, in these embodiments, immunostimulatory bacteria can be modified by deletion or disruption of the gene in SPI-1 T3SS and / or deletion or disruption of the gene encoding flagella, thereby inhibiting or reducing infection by non-phagocytic cells (e.g., epithelial cells) and increasing or limiting infection to tumor resident bone marrow cells. Such bacteria can also be modified with plasmids encoding therapeutic products such as type I IFNs and immunostimulatory cytokines, further enhancing the anti-tumor immune response and therapeutic efficacy through the expression of these therapeutic products in tumor resident immune cells.

[0350] b. Macrophage pyroptosis The macrophage NLRC4 inflammasome, which plays a role in innate immunity and antimicrobial responses, is a large multiprotein complex that recognizes cytosolic pathogens and provides autocatalytic activation of caspase-1. Activation of caspase-1 induces the maturation and release of pro-inflammatory cytokines IL-1β and IL-18, leading to pyroptosis, a rapid inflammatory form of macrophage cell death. This pro-inflammatory cell death can modify the cytokine environment to limit the initiation of a strong adaptive immune response directly induced by antigen-presenting cell (APC) death and inhibit the production of memory T cells.

[0351] Infection with Gram-negative bacterial type 3 or 4 secretion systems, such as Salmonella typhimurium and Pseudomonas aeruginosa, induces activation of the NLRC4 inflammasome via recognition of bacterial ligands such as needle-shaped proteins, rod-shaped proteins, and flagellin, following translocation to the host cell cytosol by the Salmonella pathogenic island-1 type III secretion system (SPI-1 T3SS). Pyroptosis is not limited to macrophages; caspase-1-dependent death has been observed in post-infection dendritic cells in 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 here, knockout of genes in the Salmonella genome involved in inducing pyroptosis enhances the antitumor immune response. This prevents the loss of immune cells, including macrophages, after bacterial infection. For example, genes encoding HilA, rod-shaped protein (PrgJ), needle-shaped protein (PrgI), flagellin, and / or QseC can be knocked out / destroyed in the immunostimulatory bacteria provided herein.

[0352] i. Flagellin As described above, in some bacterial species, including Salmonella, flagellin, in addition to SPI-1 T3SS, is required for the induction of pyroptosis in macrophages and is detected and activated by the macrophage NLRC4 inflammasome. Flagellin, a major component of flagella, is recognized by TLR5. Salmonella encodes two flagellin genes, fliC and fljB; elimination of the flagellin subunit reduces pyroptosis in macrophages. For example, Salmonella typhimurium lacking fliC and fljB shows a significant decrease in IL-1β secretion compared to the wild type, but bacterial cellular uptake and intracellular replication remain unaffected. This indicates that flagellin plays a significant role in inflammasome activation. Furthermore, Salmonella typhimurium strains engineered to constitutively express FliC were found to induce macrophage pyroptosis (see, for example, 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).

[0353] The genome of immunostimulatory bacteria can be modified to delete, disrupt, or mutate the flagellin genes fliC and fljB in Salmonella typhimurium, thereby reducing cell death of tumor-resident immune cells such as macrophages and enhancing the antitumor immune response of immunostimulatory bacteria.

[0354] ii. SPI-1 protein The SPI-1 protein also activates the NLRC4 inflammasome in macrophages, activating caspase-1 and leading to cell death via pyroptosis. These effectors include, but are not limited to, rod-shaped proteins (PrgJ) and needle-shaped proteins (PrgI), for example.

[0355] Rod-shaped protein (PrgJ) The NLRC4 inflammasome is also detected in demargallerated Salmonella typhimurium. A flagellin-independent response is indicated by the detection of PrgJ, an SPI-1 T3SS rod-shaped protein, in Salmonella typhimurium. Delivery of purified PrgJ protein to the macrophage cytosol leads to rapid NLRC4-dependent caspase-1 activation and IL-1β secretion, similar to the effect induced by flagellin (Miao et al. (2010) Proc. Natl. Acad. Sci. USA 107(7):3076-3080). Therefore, mutations or knockout of the PrgJ coding gene in Salmonella typhimurium can reduce macrophage pyroptosis, which enhances the antitumor immune effect of immunostimulant bacteria by preserving immune cells that are susceptible to death by the bacterium.

[0356] Needle-shaped protein (PrgI) PrgI, the SPI-1 T3SS needle-shaped protein in Salmonella typhimurium, is also recognized and activated by the NLRC4 inflammasome. Delivery of Salmonella typhimurium PrgI to the cytosol of human primary monocyte-derived macrophages leads to IL-1β secretion and subsequent cell death. Salmonella mutants expressing PrgI but not flagellin showed inflammasome activation in primary monocyte-derived macrophages at a later time point than flagellin-expressing strains (Kortmann et al. (2015) J. Immunol. 195:815-819). Thus, the immunostimulatory bacteria provided herein can be modified to mutate or delete the gene encoding the needle-shaped protein in Salmonella typhimurium, inhibiting immune cell pyroptosis and enhancing antitumor immune effects.

[0357] iii. QseC The sensor protein QseC is a highly conserved membrane histidine sensor kinase found in many Gram-negative bacteria that responds to the environment and regulates the expression of several pathogenic factors. These pathogenic factors include, for example, the flhDC gene, which encodes the master regulator of flagellar biosynthesis in Salmonella typhimurium; the sopB gene, which encodes a protein that plays a role in the invasion of non-phagocytic cells, the regulation of early maturation and transport of Salmonella-containing vacuoles (SCVs), and the inhibition of SCV-lysosome fusion; and the sifA gene, which is necessary for SCV maintenance and membrane integrity.

[0358] Selective inhibition of QseC by LED209 has been shown to partially protect mice from death after infection with Salmonella typhimurium or Mycobacterium tularensis by inhibiting QseC-mediated activation of pathogenicity-related gene expression (e.g., flhDC, sifA, and sopB) without suppressing Salmonella typhimurium proliferation. QseC blockade was found to inhibit macrophage caspase-1 activation, IL-1β release, and Salmonella typhimurium-induced pyroptosis by inhibiting excessive inflammasome activation in infected macrophages. QseC inhibition also suppresses flagellar gene expression and motility, and inhibits Salmonella typhimurium invasion and replication in epithelial cells (Li et al. (2016) Scientific Reports 6:37447). Therefore, modifications of immunostimulatory bacteria here, which involve mutations or knockouts in the gene encoding QseC, may enhance the antitumor immune response by converging Salmonella typhimurium infection to non-epithelial cells and reducing cell death in immune cells, such as by preventing pyroptosis in macrophages.

[0359] 6. Bacterial culture conditions Bacterial culture conditions can affect gene expression. Salmonella typhimurium can induce rapid pro-inflammatory caspase-dependent cell death in macrophages within 30-60 minutes of infection via a mechanism involving SPI-1 and its related T3SS-1, but this has not been shown in epithelial cells (Lundberg et al (1999) Journal of Bacteriology 181(11):3433-3437). This cell death is now known to be mediated by inflammasome activation, which subsequently activates caspase-1, promoting the maturation and release of IL-1β and IL-18, initiating a novel form of cell death called pyroptosis (Broz and Monack (2011) Immunol. Rev. 243(1):174-190). This pyroptosis activity can be induced using log-phase bacteria, whereas quiescent bacteria do not induce this rapid cell death in macrophages. The SPI-1 gene is induced during the bacterial logarithmic growth phase. Therefore, rapid pyroptosis of macrophages can be prevented by collecting Salmonella typhimurium for therapeutic use during the quiescent phase. Macrophages are important mediators of the innate immune system and act to secrete cytokines that are crucial for establishing a proper antitumor response. Furthermore, limiting the secretion of pro-inflammatory cytokines such as IL-1β and IL-18 improves the tolerability of administered Salmonella typhimurium therapeutic agents. The immunostimulatory Salmonella typhimurium provided here, collected during the quiescent phase, is used to induce an antitumor response.

[0360] 7. Increased tumor colony formation VNP20009 is a microbial cancer treatment agent based on attenuated Salmonella typhimurium, developed for the treatment of cancer. VNP20009 is attenuated via deletions of the genes msbB and purI (purM). The purI deletion makes the microorganism nutrient-dependent on purines or adenosine. The deletion of the msbB gene reduces toxicity associated with bacterial lipopolysaccharide (LPS) by inhibiting the addition of the terminal myristyl group to the lipid A domain, resulting in less toxicity from lipid A (Khan et al. (1998) Mol. Microbiol. 29:571-579).

[0361] The ability of VNP20009 to colonize tumors differs between mice and humans. Systemic administration of VNP20009 resulted in tumor colonization in mice; on the other hand, systemic administration of VNP20009 in human patients resulted in little to no tumor colonization. In mice, VNP20009 was found to exhibit high levels 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. 18:219-33). However, in a phase 1 clinical trial in patients with advanced melanoma, VNP20009 was hardly detectable in human tumors after 30-minute intravenous infusion (see Toso et al. (2002) J. Clin. Oncol. 20:142-52). Patients who entered a follow-up clinical trial evaluating the long 4-hour infusion of VNP20009 were also shown to be undetectable after tumor biopsy (Heimann et al. (2003) J. Immunother. 26:179-180). Colony formation of VNP20009 derivatives was detected after intratumoral administration (Nemunaitis et al. (2003) Cancer Gene Ther. 10:737-44). Direct intratumoral administration of VNP20009 to human tumors resulted in tumor colonization, demonstrating that human tumors can colonize at high levels, and that differences in tumor colonization between mice and humans occur only after systemic administration.

[0362] Strains such as VNP20009 are inactivated by human complement, leading to low tumor colony formation. Strains with improved resistance to complement are provided herein. These strains may also carry nucleic acids encoding therapeutic products, including, but not limited to, low or medium copy number plasmids and cytokines, type I interferon-producing and other protein-stimulating therapeutic gene / product variants, which include modifications to the bacterial genome and optionally provide for replication (asd under eukaryotic promoter control) as described elsewhere herein.

[0363] The table below summarizes bacterial genotypes / modifications, their functional effects, and benefits / advantages. [Table 4]

[0364] The strains provided herein are ΔFLG and / or ΔpagP so as to be flagellate-less. Furthermore, the strains may be one or more of ΔpurI (ΔpurM), ΔmsbB, and Δasd (in bacterial genomes). Plasmids are modified to encode a product under the control of a host recognition promoter (e.g., a eukaryotic promoter such as the RNA polymerase II promoter, including those derived from eukaryotes and animal viruses). Plasmids may optionally encode asd to enable in vivo replication, as well as nucleic acids (e.g., CpG) and gene products having other beneficial functions as described elsewhere herein.

[0365] Immunostimulating bacteria are derived from suitable bacterial strains, including attenuated and wild-type or other non-attenuated strains. Bacterial strains may be attenuated by well-known or standard methods, or, by modifications provided herein, strains that are attenuated in such a way that their colony-forming ability is limited primarily to immune-privileged tissues and organs, particularly to immune and tumor cells, including solid tumors. Bacteria include, but are not limited to, strains of Salmonella, Shigella, Listeria, Escherichia coli, and Bifidobacteria. For example, species include Shigella sonnei, Shigella flexneri, Shigella shigai, Listeria monocytogenes, Salmonella typhi, Salmonella mucinosa, Salmonella trityphi, and Bacillus gertner's. Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Cytrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix. For example, this includes Rickettsia japonica, Rickettsia typhus, Rickettsia scrub typhus, Rickettsia sibirica, Bacillus subtilis bronchoseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas euclenophila, Aeromonas salmonicida, Bacillus tularensis, Mycobacterium pseudotuberculosis, Cytrobacter freundii, Chlamydia pneumoniae, Haemophilus somnus, Mycobacterium bovine malformation, Mycobacterium intracellulare, Bacillus regalis, Rhodococcus equii, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelas swine, Yersinia enterocolitica, Rosaliamea quintana, and Agrobacterium tumelfasium.

[0366] Examples of immunostimulatory bacteria provided herein are Salmonella species. Examples of bacteria for modification described herein are wild-type Salmonella strains, such as a strain possessing all identified features of a strain deposited with ATCC as deposit number 14028. Modified Salmonella typhimurium strains are provided, such as strain YS1646 (ATCC Catalog # 202165; also referred to as VNP20009, see also International PCT application publication WO99 / 13053), which is plasmid-modified to complement asd gene knockout and for antibiotic-free plasmid maintenance. The strain is then modified for flagellin gene deletion and / or pagP deletion. The strain is also considered purine-, particularly adenosine-dependent, and asd - and msbB - The asd gene can be provided to a plasmid for replication in a eukaryotic host. These deletions and plasmids are described elsewhere in this specification. Any of the nucleic acids encoding therapeutic products and immunostimulant proteins and other products described elsewhere in this specification and / or known to those skilled in the art can be incorporated into the plasmid. Plasmids generally exist at low to medium copy numbers, as described elsewhere in this specification. Therapeutic products include gain-of-function variants of cytosolic DNA / RNA sensors that promote antitumor immune responses in the tumor microenvironment, and other such products described herein, which can constitutively induce / induce immunostimulant proteins such as type I IFN expression and other cytokines.

[0367] E. Gain-of-function mutations in non-human STING proteins and proteins for stimulating immune responses in the tumor microenvironment. Immunostimulating bacteria are provided, which contain a sequence of nucleotides encoding a gene product that is a therapeutic product, particularly an anti-cancer product, that promotes or stimulates an antitumor or antiviral immune response. The therapeutic product is a product referred to as a cytosolic DNA / RNA sensor, which induces an immune response when exposed to nucleic acids such as RNA, DNA, nucleotides, dinucleotides, cyclic nucleotides, cyclic dinucleotides, and other such molecules in the cytosol of a cell. The immunostimulating bacteria here are modified products that induce an increased or constitutive immune response and do not require the presence of a DNA / RNA product in the cytosol. An example is an encoding protein containing a gain-of-function mutation that enhances the immune response in the tumor microenvironment. Not only are immunostimulating bacteria provided, but other delivery media can be used to deliver such immunostimulating proteins or encoding proteins. These delivery media include exosomes, vectors, and viruses. For example, oncolytic viruses, particularly cytosolic viruses such as vaccinia virus, can also be modified to express gain-of-function products. Encoding gain-of-function products can be delivered to exosomes, liposomes, and other suitable media that are generally targeted to tumors.

[0368] Immunostimulatory bacteria encoding gain-of-function products (and / or other therapeutic products) include immunostimulatory bacteria that preferentially infect tumors, including tumor resident immune cells, and / or immunostimulatory bacteria whose genomes have been modified to deliver constitutively active proteins and / or therapeutic products to the cellular and tumor microenvironment so that the bacteria induce hypocellular death in tumor resident immune cells, thereby causing the immunostimulatory bacteria to accumulate in tumor cells and tumor resident immune cells, thereby stimulating an immune response against the tumor. Immunostimulatory bacteria can further encode tumor antigens in a target to enhance a response against a particular tumor. Any of the immunostimulatory bacteria provided herein and described above and below may be modified to encode such gain-of-function products. The products are encoded in plasmids under the control of a promoter and some other desired regulatory sequence recognized in eukaryotic targets such as humans or other animals or mammals. Generally, nucleic acids encoding gain-of-function products are under the control of the RNA polymerase II promoter.

[0369] Therapeutic products, including STING proteins and other proteins in the type I interferon signaling pathway described herein, as well as gain-of-function variants of other anticancer products, are expressed under the control of eukaryotic promoters. 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 the respiratory multinucleated virus (RSV) promoter; cellular promoters such as EIF-1a; inducible chimeric promoters such as tet-CMV; and tissue-specific promoters (Chang et al. (2013) Cold Spring Harb Protoc; doi:10.1101 / pdb.prot075853).

[0370] Furthermore, any of the bacteria described herein regarding modification, such as strains of Salmonella, Shigera, Escherichia coli, Bifidobacteria, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Cytrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, and Erysipelothrix or their attenuated or modified strains, as well as exosomes, liposomes, and oncolytic viruses, may be modified by encoding them on plasmids containing nucleic acids that encode gain-of-function products under the control of a host-recognized RNA polymerase promoter, or on plasmids in bacteria. The gain-of-function products are expressed in the target cells. Immunostimulatory bacteria include those modified to accumulate in or preferentially infect tumors and tumor-resident immune cells, as described herein. For example, immunostimulatory bacteria encoding gain-of-function products that result in the expression or constitutive expression of type I IFNs such as interferon (IFN)-beta, are modified to reduce or eliminate their ability to infect epithelial cells, but to enable them to infect phagocytic cells, including tumor-resident immune cells, and / or modify the bacteria so as not to kill the infected phagocytic cells.

[0371] The immunostimulatory bacteria referred to here may encode products called cytosolic DNA / RNA sensors that induce an immune response when exposed to nucleic acids such as RNA, DNA, nucleotides, dinucleotides, cyclic nucleotides, cyclic dinucleotides, and other such molecules in the cytosol of a cell. The immunostimulatory bacteria referred to here encode modified products that constitutively induce an immune response and do not require the presence of DNA / RNA and other nucleotides in the cytosol. An example of such a product is a component of the pathway that induces type I interferon expression. The products referred to here are modified DNA / RNA sensors with constitutive or increased activity (gain-of-function products) so that type I interferon is expressed or produced in the absence of nucleotides, dinucleotides, cyclic nucleotides, cyclic dinucleotides, and other such ligands in the cytosol of a cell. Expression of these modified products in cells, particularly tumor cells and tumor-resident immune cells, results in the constitutive expression of type I interferon, including interferon-β, in the tumor microenvironment. Because immunostimulant bacteria and oncolytic viruses (and other delivery media described herein) expressing 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 response and enhanced therapeutic efficacy in the tumor microenvironment.

[0372] Examples of gene products that can be encoded in immunostimulatory bacteria and other media include, but are not limited to, proteins that sense or involve 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, 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 can be known or identified and delivered to the tumor microenvironment by immunostimulatory bacteria or other vectors and delivery media such as exosomes or liposomes, including by cell infection or targeting and binding to tumor cells. Gain-of-function mutations include those identified from individuals with impairments resulting from constitutive type I interferon expression. Examples of gain-of-function products occur in subjects with interferonopathy. As described above, mutations can also be identified by screening for those that produce gain-of-function products.

[0373] The immunostimulatory bacteria described here encode proteins such as STING, including non-human STING proteins and variants of the STING protein that have lower NF-κB signaling activity than human STING, as well as other DNA / RNA sensors that constitutively induce an immune response and do not require the presence of DNA / RNA or other nucleotide ligands in the cytosol. An example of this is a component of the pathway that induces type I interferon expression.

[0374] The nucleic acids encoding identified gain-of-function mutant products can be further modified to improve their expression properties. Modifications include codon optimization that increases transcription efficiency, such as reducing GC or CpG dinucleotide content, removing potential splicing sites, negative CpG islands, Shine-Dalgano (SD) sequence substitutions, and TATA box and / or terminal signal substitutions, which increase transcription efficiency in mammals, particularly humans. Codons can also be optimized to increase translation efficiency by modifying codon use bias, reducing GC content, reducing mRNA secondary structure, removing intermediate poly-A sites, removing RNA instability motifs (AREs), removing stable free energy from mRNA, modifying internal chi sites and ribosome binding sites, and reducing RNA secondary structure.

[0375] 1. Type I interferon and pathway Type I interferon induction pathways mediated by host recognition of nucleic acids, such as single-stranded and double-stranded RNA, circular dinucleotides, and other such forms of nucleic acids, are known to induce type I IFNs. There are also Toll-like receptor (TLR)-independent type I IFN pathways mediated by host recognition of single-stranded (ss) and double-stranded (ds) RNA in the cytosol. These nucleic acids are sensed via IFN-β promoter-stimulating factor 1 (IPS-1) adapter protein-mediated phosphorylation of RNA helicases, including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), and the IRF-3 transcription factor, leading to IFN-beta induction (Ireton and Gale (2011) Viruses 3(6):906-919). As described here, proteins in these pathways can be modified or exist as variants, leading to constitutive expression of type I interferons (also called interferon type 1), including IFN-α and IFN-β. Provided herein are immunostimulatory bacteria and other delivery media, including exosomes, liposomes, and oncolytic viruses, that encode variant proteins. These delivery media may be used to treat cancer by direct administration to a subject and / or by administration to cells, allogenes, or autologous cells for use in cell therapy protocols.

[0376] Type I interferons (IFNs; also known as interferon type 1) include IFN-α and IFN-β, and are multifaceted cytokines with antiviral, antitumor, and immunomodulatory activity. IFN-β is produced by most cell types; IFN-α is produced mainly by hematopoietic cells, particularly plasmacytoid dendritic cells. Type I IFNs are produced after the sensing of pathogen-associated molecular patterns (PAMPs), including microbial and viral nucleic acids and LPS (lipopolysaccharide), by pattern recognition receptors (PRRs) and cytokines. They are potent immunomodulators that activate the adaptive immune system by being part of the innate immune response to pathogenic infections, including viruses, promoting antigen presentation, mediating DC maturation, activating cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and macrophages, and promoting high-affinity antigen-specific T and B cell responses and the development of immunological memory.

[0377] Type I IFNs exhibit antiproliferative and pro-apoptotic effects on tumors and have anti-angiogenic effects on tumor angiogenesis. They induce the expression of MHC class I molecules on the surface of tumor cells, increasing the immunogenicity of tumor cells and activating cytotoxicity against them. Type I IFNs are used as therapeutic agents for the treatment of cancer and viral infections. For example, IFN-α (marketed under the trade names Intron® / Roferon®-A) is approved for the treatment of hairy cell leukemia, malignant melanoma, AIDS-associated Kaposi's sarcoma, and follicular non-Hodgkin lymphoma; it is also used for the treatment of chronic myeloid leukemia (CML), renal cell carcinoma, neuroendocrine neoplasms, multiple myeloma, non-follicular non-Hodgkin lymphoma, tendonoid, and cutaneous T-cell lymphoma (Ivashkiv and Donlin (2014) Nat. Rev. Immunol. 14(1):36-49; Kalliolias and Ivashkiv (2010) Arthritis Research & Therapy 12(Suppl 1):S1; Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893).

[0378] The expression of type I interferons in tumors and the tumor microenvironment is an immune response designed to be induced, in particular, by immunostimulatory bacteria and other delivery vehicles. Induction or activation of type I interferons provides anti-tumor immunity for cancer treatment.

[0379] 2. Type I interferonosis and gain-of-function variants The induction of type I interferons (IFNs), pro-inflammatory cytokines, and chemokines is necessary for the development of an immune response that prevents or inhibits pathogen infection. This response may also be effective as an antitumor agent. The immunostimulatory bacteria and other delivery media provided herein encode proteins that constitutively induce type I IFNs, particularly those occurring in individuals with various diseases or disorders involving the overproduction of immune response modulators. For example, overproduction, excessive production, or incomplete negative regulation of type I IFNs and pro-inflammatory cytokines can lead to undesirable effects such as inflammatory and autoimmune diseases. Disorders involving the chronic overproduction of type I IFNs and pro-inflammatory cytokines are collectively referred to as interferonopathy (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 interferonosis include Ecardi-Goutier syndrome (AGS), infant-onset STING-associated vascular disorder (SAVI), Singleton-Merten syndrome (SMS), atypical SMS, familial chilblain-like lupus (FCL), systemic lupus erythematosus (SLE), bilateral striatal necrosis (BSN), cerebrovascular disease (CVD), hereditary contralateral pigmentary disorder (DSH), spastic paraplegia (SP), X-linked reticular disorder (XLPDR), proteasome-associated autoinflammatory syndrome (PRAAS), intracranial calcification (ICC), Mendelian mycobacterial susceptibility to infection (MSMD), and intraspinal chondrodysplasia (SPENCD) (see, e.g., Rodero et al. (2016) J. Exp. Med. 213(12):2527-2538). These phenotypes are associated with specific genotypes involving gene mutations that lead to constitutive activity of products involved in type I IFN induction.

[0380] Sustained activation of interferon signaling can be caused by: 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, RNASEH2B, RNASEH2C, and POLA1); 2) loss-of-function mutations resulting in RNA editing deficiencies and abnormal sensing of self-nucleotide RNA species in the cytosol (e.g., mutations in ADAR1); 3) gain-of-function mutations leading to constitutive activation of the cytosolic IFN signaling pathway / increased sensitivity to cytosolic nucleotide ligands (e.g., mutations in RIG-I, MDA5, and STING); 4) folding This could be due to loss-of-function mutations in MAVS leading to abnormal RNA signaling mediated by interference with the response of a missing protein (e.g., mutations in SKIV2L); 5) loss-of-function mutations in molecules responsible for limiting IFN receptor (IFNAR1 / 2) signaling leading to unregulated IFN-stimulated gene (ISG) production (e.g., mutations in USP18 and ISG15); 6) proteasome dysfunction leading to increased IFN signaling via an unknown mechanism (e.g., mutations in PSMA3, PSMB4, and PSMB8); and 7) loss-of-function mutations in TRAP / ACP5 and C1q, the mechanism by which type I IFN signaling is elusive (Rodero et al. (2016) J. Exp. Med. 213(12):2527-2538).

[0381] What is interesting here are the mutations that lead to gain-of-function (GOF) mutations. Mutations associated with gain-of-function (GOF) mutations that result in constitutive activation and / or increased sensitivity of the encoding protein or increased affinity or binding to endogenous ligands are known in STING, MDA5, and RIG-I. For example, GOF mutations in STING are associated with SAVI and FCL; GOF mutations in MDA5 are associated with AGS and SMS; and GOF mutations in RIG-I are associated with atypical SMS.

[0382] The immunostimulant bacteria and oncolytic viruses provided herein, encoding these proteins with gain-of-function mutations, utilize the constitutive activation of these proteins to increase the production of type I IFN and pro-inflammatory cytokines. Tumor-targeting immunostimulant bacteria and oncolytic viruses and other delivery vehicles encoding STING, MDA5, and / or RIG-I with gain-of-function mutations are provided herein. Such immunostimulant bacteria and other delivery vehicles increase the production of type I IFN and pro-inflammatory cytokines in the tumor microenvironment, enhancing the anti-tumor immune response of the immunostimulant bacteria and improving therapeutic efficacy. The gene encoding STING is designated TMEM173, the gene encoding MDA5 is designated IFIH1, and the gene encoding RIG-I is designated DDX58. Each gene has numerous alleles, and mutations that can occur in any of the alleles that result in gain-of-function mutations are known. The mutations listed below can occur individually or in some combination. Other mutations that result in gain-of-function mutations can be identified by routine screening / mutation protocols. The table below lists examples of gain-of-function mutations for STING / TMEM173 (SEQ ID NOs. 305-309), MDA5 / IFIH1 (SEQ ID NOs. 310), and RIG-I / DDX58 (SEQ ID NOs. 311). Other mutations may also be introduced, such as deletions or substitutions of one or more phosphorylation sites in STING, including 324-326 SLS→ALA, and other substitutions that eliminate phosphorylation sites to reduce such signaling in STING or in other proteins that use nuclear factor-κB (NF-κB) signaling.

[0383] The resulting protein may encode an immunostimulatory bacterium provided herein. The protein may be encoded in a plasmid in the immunostimulatory bacterium, or encoded in the genome of an oncolytic virus, or delivered via a delivery medium such as an exosome or liposome.

[0384] [Table 5] [Table 6]

[0385] The amino acid residues R197, D205, R310, R293, T294, E296, S272, Q273, E316, D231, R232, K236, S358, E360, S366, and R238, which are reference amino acid residues to the human STING sequence shown in SEQ ID NOs. 305-309, correspond to the amino acid residues R196, D204, R309, R292, T293, E295, S271, Q272, E315, D230, R231, K235, S357, E359, S365, and R237, respectively, which are reference amino acid residues to the mouse STING sequence shown in SEQ ID NO. 351.

[0386] 3. STING-mediated immune activation STING (interferon-stimulating agent), transmembrane protein 173 (also known as TMEM173), IRF3 activation mediator (MITA), methionine-proline-tyrosine-serine (MPYS), and endoplasmic reticulum (ER) IFN stimulator (ERIS) are 379-amino acid proteins that occur in the endoplasmic reticulum and function as signaling adapter proteins, regulating the transcription of immune response genes such as type I IFN and pro-inflammatory cytokines. Stimulation of the STING pathway activates endothelial cells and induces endothelial cell death in culture, upregulating interferon-response genes, apoptotic pathway genes, and tissue-factor expression, which are potent initiators of the coagulation cascade (Liu et al. (2014) N. Engl. J. Med. 371:507-518).

[0387] Due to its role in IFN production and pro-inflammatory mechanisms, human STING is an immunotherapeutic target for cancer and infectious diseases. For example, studies have shown that direct activation of STING by its ligand, cyclic dinucleotide (CDN), can induce tumor death. Consequently, tumors with increased STING expression are directly killed by activation of the STING-mediated cell death pathway. Activation of the STING pathway in dendritic cells (DCs) promotes DC maturation, which is linked to CD8 + STING produces a memory response that prevents cancer recurrence through a T cell-mediated cytotoxic response. STING can also enhance the therapeutic efficacy of radiotherapy and chemotherapy through DNA released from the treatment. In mice, the efficacy of the Pneumovax23® vaccine is STING-dependent, as the vaccine is ineffective in a mouse model of the human HAQ loss-of-function allele. CDN loses its adjuvant activity in HAQ mice, indicating the role of STING in infection.

[0388] STING signaling boosts host immune recognition of tumor antigens, leading to a potent antitumor response. Studies have shown that STING expression and signaling are suppressed in many cancers, including colorectal cancer, and that this suppression of STING signaling inhibits the DNA damage response and antitumor T cell priming. STING expression is also found in many primary and metastatic melanomas and Burkitt lymphomas, breast cancer, leukemia, lymphomas, and HPV. +STING is also lost / deregulated in cancer, HCV or HBV-associated hepatocellular carcinoma, and herpesvirus-associated cancers. Reconstitution of STING into cancer cell lines such as 293T and MCF7, which lack intracellular DNA signaling, can reclaim intracellular pathways and induce type I IFN and other signaling pathways (see, e.g., U.S. Patent Application Publication 2018 / 0085432). As a result, STING agonists are being developed for cancer immunotherapy. U.S. Patent Application Publication 2018 / 0085432 describes the use of nucleic acid sequences encoding wild-type STING for the treatment of diseases caused by exogenous factors such as bacterial, fungal, parasitic, and viral infections, and for the modulation of the immune system for inducing antitumor responses. STING can be administered to patients for cancer treatment as polynucleotides, polypeptides, peptides, or antisense oligonucleotides in vectors expressing STING, antibodies, etc. The vectors include viral vectors (adenoviruses, adeno-associated viruses, VSVs, and retroviruses), liposomes, other lipid-containing complexes, and other macromolecular complexes that can intervene in the delivery of polynucleotides to host cells (US Publication 2018 / 0085432). US Publication 2018 / 0311343 describes the administration of mRNA encoding a constitutively active human STING polypeptide and mRNA encoding an antigen of interest, such as a t...

Claims

1. A modified interferon gene-stimulating (STING) protein derived from a non-human species, wherein the non-human STING has lower NF-κB signaling activity compared to human STING and, optionally, higher type I interferon (IFN) pathway signaling activity compared to human STING. Non-human STING proteins are modified to include one or more mutations that increase their activity or allow them to act constitutively in the absence of cytosolic nucleic acids; A mutation is the insertion, deletion, and / or substitution of an amino acid; and The STING protein may, if desired, have a deletion of the TRAF6 binding site. Modified STING protein.

2. A modified interferon gene-stimulating (STING) protein from a non-human species or a chimeric human STING protein or a variant of a chimeric STING protein, wherein the variant of the modified STING protein and the chimeric STING protein contains one or more mutations associated with gain-of-function (GOF) that result in constitutive activation and / or increased sensitivity, affinity, or binding to the encoding STING protein and / or endogenous ligand: STING proteins are modified by one or more insertions, deletions, and substitutions of one or more amino acids; Chimeric human STING proteins consist of a portion of human STING protein and a portion of non-human STING protein; Non-human or chimeric STING proteins possess IFN-beta signaling activity and attenuated activated B cell nuclear factor kappa light chain enhancer (NF-κB) signaling activity compared to human STING; and One or more mutations result in increased STING activity or constitutive activity that induces IFN-beta production. Modified STING protein.

3. The modified STING protein according to claim 1 or 2, wherein the unmodified human STING protein contains the amino acid sequence shown in any of SEQ ID NOs. 305 to 309 or is a human allele variant thereof having at least 98% sequence identity with the amino acid sequence shown in any of SEQ ID NOs. 305 to 309.

4. The STING protein is a chimera in which the C-terminal tail (CTT) region of the STING protein from type 1 is replaced with the CTT of the STING protein from type 2; The second type of STING protein has lower NF-κB signaling activity than human STING; and The TRAF6 binding site in CTT is deleted as desired. A modified STING protein according to any one of claims 1 to 3.

5. A modified STING protein according to any one of claims 1 to 4, wherein one or more mutations are associated with infant-onset STING-associated vascular injury (SAVI) of human autoinflammatory diseases.

6. A chimeric modified interferon gene-stimulating (STING) protein comprising the substitution of the CTT (C-terminal tail) region of STING from type 1 to CTT from type 2, The second type of STING protein has lower NF-κB signaling activity than human STING; and The TRAF6 binding site in CTT is missing. Modified STING protein.

7. The chimera includes a portion of the human STING protein and a portion of the non-human STING protein; and A modified STING protein according to any one of claims 2 to 6, wherein the human STING protein is a human allele variant thereof that contains the amino acid sequence shown in any of SEQ ID NOs. 305 to 309 or has at least 98% sequence identity with the amino acid sequence shown in any of SEQ ID NOs. 305 to 309.

8. A modified STING protein according to any of claims 2 and 4-7, which is a chimera containing portions of STING proteins from two species, the resulting STING protein having IFN-beta signaling activity, where the first species is human and the second species is selected from Tasmanian devil, marmoset, cattle, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, mouse, and elephant shark.

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

10. A modified STING protein according to any one of claims 1 to 9, wherein the NF-κB signaling activity of the non-human STING is less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of the NF-κB signaling activity of wild-type human STING.

11. A modified STING protein according to any one of claims 1 to 10, wherein the non-human species or secondary species is selected from Tasmanian devil, marmoset, cattle, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, mouse, and elephant shark.

12. A modified STING protein according to any one of claims 1 to 11, wherein the modification of STING is one or more mutations that, when aligned with human STING, correspond to mutations that cause interferonosis, and the sequence of human STING on which the alignment is performed is shown in any of sequence numbers 305 to 309.

13. A modified STING protein according to any one of claims 1 to 12, comprising replacing the C-terminal tail (CTT) of a secondary STING protein with a CTT that exhibits reduced NF-κB signaling activity compared to that of human STING.

14. A modified STING protein according to any one of claims 1 to 13, wherein the TRAF6 binding site in the CTT is deleted.

15. A modified sting protein according to claim 13 or 14, wherein the second CTT is derived from Tasmanian devil, marmoset, cattle, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, mouse, or elephant shark sting protein.

16. A modified STING protein according to claim 13 or 14, wherein the second CTT is derived from Tasmanian devil, marmoset, cattle, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, mouse, or elephant shark STING protein, and replaces human STING CTT.

17. A modified STING protein according to any one of claims 13 to 16, wherein the replacement CTT is selected from the following species and is an allele variant of each of these sequences having the following sequences or having at least 98% sequence identity: Table 1

18. A modified STING protein according to any one of claims 4 to 17, wherein the human STING CTT contains the sequence EKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (SEQ ID NO: 352) or is an allele variant having at least 98% sequence identity thereto.

19. A modified STING protein according to any one of claims 4 to 18, wherein the modified STING protein is a chimera in which human STING CTT is replaced with CTT from 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 ID NO: 353) or an allele variant having at least 98% sequence identity thereto.

21. A modified STING protein according to any one of claims 1 to 20, comprising a deletion of the TRAF6 binding site.

22. The modified STING protein according to claim 21, wherein STING is human STING and the TRAF6 binding site contains the amino acid residue DFS at the C-terminus.

23. A modified STING protein according to any one of claims 1 to 22, comprising modifications that increase type I interferon signaling activity or constitutively enhance activity in the absence of cytosolic nucleic acids.

24. The modified STING protein of claim 23, wherein the modification corresponds to a mutation that causes interferonosis when aligned with human STING as a reference, and where human STING has the sequence shown in any of SEQ ID NOs. 305 to 309.

25. Modifications that increase activity or make it constitutively active are based on the sequence of human STING shown in any of SEQ ID NOs: 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 / Q A modified STING protein according to any one of claims 1 to 24, wherein the substitution is one or more amino acids corresponding to one or more of 273A, 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.

26. A modified STING protein according to any one of claims 1 to 25, wherein the non-human STING protein is an allele variant thereof having at least 98% sequence identity with the Tasmanian devil STING protein of SEQ ID NO: 331 or the STING protein of SEQ ID NO:

331.

27. A modified STING protein according to any one of claims 1 to 26, comprising a substitution corresponding to C206Y or R284G based on the sequence of human STING shown in any of sequence numbers 305 to 309.

28. A modified STING protein according to claim 27, comprising the amino acid sequence shown in SEQ ID NO: 332 or 333.

29. A modified STING protein according to any one of claims 1 to 25, wherein the sequence of the non-human STING protein is that shown in SEQ ID NOs. 331, 338, and 341-351, or the non-human STING protein is an allele variant of any of SEQ ID NOs. 331, 338, and 341-351 having at least 98% sequence identity with it.

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

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

32. An immunostimulant bacterium containing a plasmid in its unmodified form that encodes a gain-of-function variant of an immunostimulant protein that is part of the cytosolic DNA / RNA sensor pathway, resulting in the expression of type I interferon (IFN).

33. Gain-of-function (GOF) variants are constitutive active variants of immunostimulatory proteins that promote or cause interferonosis in humans; and The immunostimulatory bacterium according to claim 32, wherein 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 (reduced pyroptosis) in tumor-resident immune cells, thereby causing the immunostimulatory bacterium to accumulate in the tumor or tumor microenvironment or tumor-resident immune cells, thereby delivering constitutively active immunostimulatory proteins to host cells, stimulating or inducing the expression of type I IFNs.

34. An immunostimulatory bacterium according to claim 32 or 33, wherein the GOF variant protein contains a mutation that eliminates a phosphorylation site in an immunostimulatory protein, thereby reducing nuclear factor-kappa light chain enhancer (NF-κB) signaling in activated B cells.

35. An immunostimulatory bacterium according to any one of claims 32 to 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 the expression of type I IFN or a variant with increased activity or constitutive activity, wherein the immunostimulatory protein is STING, RIG-I, IRF-3, IRF-7, or MDA5.

37. An immunostimulatory bacterium according to any one of claims 32 to 36, wherein the immunostimulatory protein is a variant of STING, RIG-I, IRF-3, IRF-7, or MDA5 containing a gain-of-function mutation that results in increased expression of type I IFN.

38. An immunostimulatory bacterium according to any one of claims 32 to 37, wherein the immunostimulatory protein is a variant of STING, RIG-I, IRF-3, IRF-7, or MDA5, and one or more serine (S) or threonine (T) residues that are phosphorylated as a result of viral infection are substituted with aspartic acid (D) residues, and the variant protein obtained thereby is a phosphorylation mimetic that constitutively induces type I IFN.

39. The variant protein is IRF-3 and contains one or more substitutions in the residues corresponding to positions 385, 386, 396, 398, 402, 404 and 405 relative to SEQ ID NO: 312; and The residue is replaced with an aspartic acid residue. The immunostimulatory bacterium according to claim 38.

40. The immunostimulatory bacterium of claim 39, wherein the variant IRF-3 protein has substitution 396D based on SEQ ID NO:

312.

41. The immunostimulatory bacterium of claim 39, wherein the variant IRF-3 protein comprises substitution S396D / S398D / S402D / T404D / S405D or a conserved substitution based on SEQ ID NO:

312.

42. The immunostimulatory bacterium of claim 39, wherein the variant IRF-3 protein comprises substitutions S396D / S398D / S402D / T404D / S405D based on SEQ ID NO:

312.

43. An immunostimulatory bacterium according to any one of claims 32 to 42, wherein the immunostimulatory protein is a variant that, when expressed in a target, leads to constitutive expression of type I IFN.

44. An immunostimulatory bacterium according to any one of claims 30 to 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 insertions, deletions, and substitutions.

45. An immunostimulatory bacterium according to any one of claims 32 to 44, wherein the immunostimulatory protein is a variant that, when expressed in a target, leads to constitutive expression of type I IFN.

46. An immunostimulatory bacterium according to any one of claims 32 to 44, wherein an immunostimulatory protein, in its unmodified form, senses or directly or indirectly interacts with cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides to induce the expression of type I IFNs, and a variant protein induces the expression of type I IFNs in the absence of sensing or interaction with cytosolic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides.

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

48. An immunostimulatory bacterium according to any one of claims 32 to 47, wherein the immunostimulatory protein, in an unmodified form, activates or induces the expression of type I IFNs.

49. An immunostimulant bacterium according to any one of claims 32 to 48, wherein the unmodified form of the immunostimulant protein is involved in the recognition of natural cytosolic DNA / RNA that activates type I IFNs.

50. The immunostimulatory bacterium according to claim 49, wherein the type I IFN is interferon-α or interferon-β.

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

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

53. An immunostimulatory bacterium according to any one of claims 32 to 52, wherein the immunostimulatory protein, which is part of the cytosolic DNA / RNA sensor pathway, is a variant comprising a gain-of-function mutation or a mutation constitutively affecting the expression of type I interferon or other immune mediators / modulators.

54. The immunostimulatory proteins that sense cytosolic DNA / RNA are variant STING, MDA5, RIG-I, IRF-7, or IRF-3 proteins; Unmodified STING has the sequence shown in any of SEQ ID NOs. 305 to 309, unmodified MDA5 has the sequence shown in SEQ ID NO. 310, unmodified RIG-I has the sequence shown in SEQ ID NO. 311, unmodified IRF-7 has the sequence shown in SEQ ID NO. 313, unmodified IRF-3 has the sequence shown in SEQ ID NO. 312; and Modified immunostimulatory proteins have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications selected from substitutions, deletions, and insertions. The immunostimulatory bacterium according to claim 53.

55. An immunostimulatory bacterium according to any one of claims 32 to 54, comprising a gain-of-function mutation in which the immunostimulatory protein is selected from STING, MDA5, IRF-3, IRF-7, and RIG-I, and constitutively activates STING, MDA5, IRF-3, IRF-7, or RIG-I, thereby constitutively activating type I IFN expression.

56. The following are modifications involving amino acid substitutions: a) In STING, based on sequence numbers 305 to 309, 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, S27 One or more selected from 2A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R375A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A and S324A / S326A; b) In MDA5, with reference to sequence number 310, one or more of T331I, T331R, A489T, R822Q, G821S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F, and A452T; c) In RIG-I, with reference to sequence number 311, either or both of E373A and C268F; d) In IRF-3, with reference to sequence number 312, S396D; e) In IRF-7, with reference to Sequence ID No. 313, one or more of S477D / S479D, S475D / S477D / S479D and S475D / S476D / S477D / S479D / S483D / S487D; and f) Conservative amino acid substitutions that increase or constitutively promote the expression of type I interferon. An immunostimulatory bacterium of claim 54 or claim 55, selected from the above.

57. The immunostimulatory proteins, based on sequence numbers 305-309, are 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, E An immunostimulant bacterium according to any one of claims 54 to 56, which is a variant STING comprising one or more amino acid substitutions selected from 316A, 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.

58. An immunostimulant bacterium containing, in its unmodified form, nucleic acid encoding a modified STING protein, an immunostimulant protein that senses cytosolic RNA / DNA, wherein the STING protein is encoded in a plasmid and the encoding nucleic acid is manipulably linked to a regulatory sequence recognized by a eukaryotic host.

59. The immunostimulatory bacterium according to claim 58, wherein the modified STING protein does not require a cyclic dinucleotide (CDN) for activity.

60. The modification in STING is constitutive in that it does not require cGAMP for activity; The modified STING protein is encoded by the modified TMEM173 gene; Modifications include the insertion, deletion, or substitution of amino acids; and Modified STING proteins exhibit enhanced immunostimulatory activity compared to unmodified STING proteins. An immunostimulatory bacterium according to claim 58 or claim 59, comprising a sequence of nucleotides encoding a modified STING protein.

61. The amino substitutions in STING, based on SEQ ID NOs. 305-309, are 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 / E3 An immunostimulant bacterium according to claim 59 or claim 60, which is one or more selected from 16A, 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 and their conserved amino acid substitutions.

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

63. The genome of immunostimulatory bacteria is modified, which causes the bacteria to produce flagellin － (fliC － / fljB － ) and / or pagP － The immunostimulatory bacterium according to any one of claims 30 to 62.

64. The genome of immunostimulatory bacteria is modified, which causes the bacteria to produce flagellin － (fliC － / fljB － An immunostimulatory bacterium according to any one of claims 30 to 63.

65. The genome of the immunostimulatory bacterium is modified such that the bacterium is pagP － / msbB － The immunostimulatory bacterium according to any one of claims 30 to 64, which is such.

66. Bacteria flagellin － (fliC － / fljB － ) and pagP － The immunostimulatory bacterium according to any one of claims 30 to 63.

67. Immunostimulating bacteria produce aspartate semialdehyde dehydrogenase － (asd － An immunostimulatory bacterium according to any one of claims 30 to 66, wherein the bacterium is an immunostimulatory bacterium according to any one of claims 30 to 66.

68. In bacteria, all or part of the endogenous gene encoding aspartate semialdehyde dehydrogenase (ASD) is destroyed or deleted, resulting in the non-expression of endogenous ASD. － Aspartic acid semialdehyde dehydrogenase － (asd － An immunostimulatory bacterium according to any one of claims 30 to 66, wherein the bacterium is an immunostimulatory bacterium according to any one of claims 30 to 66.

69. An immunostimulatory bacterium according to any one of claims 30 to 68, encoding aspartate semialdehyde dehydrogenase (ASD) in a plasmid under the control of a bacterial promoter.

70. msbB － The immunostimulatory bacterium according to any of claims 30 to 69.

71. purI － (purM － An immunostimulatory bacterium according to any one of claims 30 to 70, wherein the bacterium is an immunostimulatory bacterium according to any one of claims 30 to 70.

72. asd － ,purI － , msbB － Flagellin － (fliC － / fljB － ) and pagP － The immunostimulatory bacterium according to any one of claims 30 to 71.

73. An immunostimulatory bacterium according to any one of claims 30 to 72, wherein one or more genes or operons involved in SPI-1-dependent invasion are deleted or inactivated, thereby preventing the immunostimulatory bacterium from invading or infecting epithelial cells.

74. The immunostimulatory bacterium of claim 73, wherein one or more of avrA, hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP are deleted or inactivated.

75. One or more genes involved in SPI-1-independent invasion are deleted or inactivated. Immunostimulant bacteria do not invade or infect epithelial cells; and One or more genes are selected from pagN, hlyE, pefI, srgD, srgA, srgB, and srgC. An immunostimulatory bacterium according to any of claims 30 to 72.

76. Modifications of the bacterial genome that reduce the induction of cell death by tumor-resident immune cells; and / or Modifications to the bacterial genome cause bacteria to preferentially infect tumor-resident immune cells compared to other cell types. An immunostimulatory bacterium according to any one of claims 30 to 75, including the above.

77. An immunostimulatory bacterium according to any one of claims 30 to 76, wherein the bacterium encodes an asd in a plasmid, and its expression is under the control of a promoter that expresses it in the tumor microenvironment (TME) of a eukaryote.

78. An immunostimulatory bacterium according to any one of claims 30 to 77, which has nutritional requirements for adenosine and adenine.

79. An immunostimulatory bacterium according to any one of claims 30 to 77, which has a nutritional requirement for adenosine.

80. An immunostimulatory bacterium according to any one of claims 30 to 79, which is nutritionally dependent on adenosine, lacks flagella, and where wild-type bacteria have flagella.

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

82. Bacteria and pagP － The immunostimulatory bacterium according to any one of claims 30 to 81.

83. Immunostimulating bacteria flagellin － (fliC － / fljB － ) and / or pagP － and purI － (purM － ), msbB － purD － adrA － , csgD － qseC － and hilA － , lppa － or lppB － An immunostimulatory bacterium according to any one of claims 30 to 82, wherein one or more of the above conditions are met.

84. Immunostimulating bacteria hilA － pagP － and flagellin － (fliC － / fljB － An immunostimulatory bacterium according to any one of claims 30 to 83.

85. An immunostimulatory bacterium according to any one of claims 30 to 84, comprising a nucleic acid encoding a CpG motif, wherein the nucleic acid encoding the CpG motif is included in or part of a bacterial gene encoded on a plasmid.

86. The immunostimulatory bacterium of claim 85, wherein the gene containing CpG is asd, and here asd is encoded on a plasmid.

87. An immunostimulatory bacterium according to any one of claims 30 to 86, which is flagellin-deficient and in which the wild-type bacterium includes a flagella.

88. An immunostimulatory bacterium according to any one of claims 30 to 87, wherein the plasmid comprises a nucleic acid encoding cytoLLO, which is a listeriolisin O(LLO) protein lacking a signal sequence.

89. An immunostimulatory bacterium according to claims 30 to 88, comprising a DNA nuclear targeting sequence (DTS) encoded on a plasmid.

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

91. An immunostimulatory bacterium according to any one of claims 30 to 90, having a deletion or modification in the gene encoding endonuclease I (endA), thereby inhibiting or eliminating endA activity.

92. An immunostimulatory bacterium according to any one of claims 30 to 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. An immunostimulatory bacterium according to any one of claims 30 to 92, wherein the immunostimulatory bacterium comprises a purI deletion, an msbB deletion, an asd deletion, and an adrA deletion.

94. An immunostimulatory bacterium according to any of claims 30 to 93, One or more mutations in a gene that alters the biosynthesis of lipopolysaccharides selected from one or more of the following: rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; and / or A suicide gene is introduced, and one or more mutations selected from one or more of sacB, nuk, hok, gef, kill and phlA are introduced; and / or Introducing a bacterial lysis gene and one or more mutations selected from either hly or cly; and / or Mutations in one or more pathogenic factors selected from IsyA, pag, prg, iscA, virG, plc, and act; and / or One or more mutations in stress response-modifying genes selected from recA, htrA, htpR, hsp, and grEL; and / or Mutations in the min that disrupt the cell cycle; and / or One or more mutations in a gene that disrupts or inactivates a regulatory function selected from cya, crp, phoP / phoQ, and ompR. Immunostimulating bacteria, including those mentioned above.

95. msbB － / asd － / purI － The immunostimulatory bacterium according to any of claims 30 to 94.

96. An immunostimulatory bacterium according to any one of claims 30 to 95, wherein prgH and / or prgK are inactivated or deleted.

97. An immunostimulatory bacterium according to any one of claims 30 to 96, wherein a nucleic acid encoding an immunostimulatory protein or STING protein on a plasmid is operably linked to a nucleic acid encoding a secretion signal, thereby causing a product to be secreted upon expression in a host.

98. An immunostimulatory bacterium according to any one of claims 30 to 97, wherein a plasmid encoding an immunostimulatory protein is present in a low or medium copy number.

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

100. An immunostimulatory bacterium according to any one of claims 30 to 98, wherein the plasmid contains a low copy number origin of replication.

101. An immunostimulatory bacterium according to any one of claims 30 to 100, wherein the plasmid is present in a low copy number.

102. An immunostimulatory bacterium according to any one of claims 98 to 101, wherein the intermediate copy number is less than 150 or about 150 and greater than 20 or about 20 or between 20 and 150.

103. An immunostimulant bacterium according to claim 101, wherein the low copy number is less than 25, less than 20, or about 25, or less than 20 copies.

104. An immunostimulatory bacterium according to any one of claims 30 to 103, wherein the plasmid replication origin is selected from origins derived from pBR322, p15A, pSC101, pMB1, colE1, colE2, pPS10, R6K, R1, RK2, and pUC.

105. An immunostimulant bacterium according to any one of claims 30 to 104, wherein an unmodified nucleic acid encoding an immunostimulant protein or STING protein that senses cytosolic RNA / DNA is located on a plasmid and is operably linked to a regulatory sequence recognized by a eukaryotic host.

106. An immunostimulatory bacterium according to any one of claims 30 to 105, wherein the plasmid encodes an immunostimulatory protein or STING under the control of a eukaryotic promoter.

107. An immunostimulatory bacterium according to claim 105 or claim 106, wherein the regulatory sequence comprises an RNA polymerase II promoter or an RNA polymerase III promoter.

108. An immunostimulatory bacterium according to claim 105 or claim 106, wherein the regulatory sequence comprises a terminator and / or promoter selected from the SV40, hGH, BGH, chicken beta-globulin, and rbGlob (rabbit globulin) genes.

109. The immunostimulatory bacterium according to 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 which 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 the cytomegalovirus (CMV) promoter, SV40 promoter, Epstein-Barr virus (EBV) promoter, herpesvirus promoter, polynuclear respiratory virus (RSV) promoter, and adenovirus promoter.

112. An immunostimulatory bacterium according to any one of claims 105 to 110, wherein the promoter that controls the expression of one or more plasmid-encoded heterologous proteins is the elongation factor-1 (EF-1) alpha promoter, the UBC promoter, the PGK promoter, the MND promoter, or the CAGG promoter.

113. An immunostimulatory bacterium according to any one of claims 105 to 110, wherein the promoter is the EF-1 alpha promoter, CMV promoter, SV40 promoter, PGK promoter, MND promoter, EIF4A1 promoter, CAG promoter, or CD68 promoter.

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

115. An immunostimulatory bacterium according to any one of claims 105 to 113, wherein the promoter is a viral promoter that is a late promoter.

116. An immunostimulatory bacterium according to any one of claims 30 to 115, wherein the bacterium is a Gram-negative bacterium.

117. The bacteria include Rickettsia rickettsiae (spot fever), Rickettsia prowazekii (typhus), Rickettsia tsutsugamuchi (scrub typhus), Rickettsia mooseri (mercident fever), Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, and Citrobacter freundii. Chlamydia freundii, Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana An immunostimulant bacterium according to any of claims 30 to 116, which is either Agrobacterium quintana or Agrobacterium tumerfacium or an attenuated or modified strain of any of the aforementioned bacterial strains.

118. The bacteria include strains of Salmonella, Shigella, Escherichia coli, Bifidobacteriae, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Cytorobacter, Chlamydia, and He An immunostimulant bacterium according to any of claims 30 to 116, which is Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, or Erysipelothrix, or an attenuated or modified strain of any of the aforementioned bacterial strains.

119. An immunostimulant bacterium according to any of claims 30 to 118, which is a weakened bacterium.

120. An immunostimulatory bacterium according to any of claims 30 to 119, which is a strain of Salmonella.

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

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

123. An immunostimulatory bacterium according to claim 120 or claim 121, wherein the unmodified Salmonella strain is attenuated.

124. The immunostimulatory bacterium of any one of claims 30 to 123, wherein the immunostimulatory bacterium is selected from strains named AST-100, VNP20009, YS1646 (ATCC #202165), RE88, SL7207, χ8429, χ8431 and χ8468, or is derived from a wild-type bacterium having all the identified characteristics of the strain deposited as ATCC deposit number 14028, or is an attenuated Salmonella typhimurium strain that is strain ATCC 14028.

125. An immunostimulatory bacterium according to any of claims 30 to 124, wherein the plasmid is encoded a) Nucleic acids encoding one or more of the gain-of-function variants of STING, RIG-I, MDA5, IRF3, and IRF7; and b) Nucleic acids encoding one or more members of the B7-CD28 family, TGF-beta polypeptide antagonists, and tumor necrosis factor receptor (TNFR) superfamily. Immunostimulating bacteria, including those mentioned above.

126. The immunostimulatory bacterium of claim 125, comprising nucleic acids encoding two proteins in a) and two proteins in b).

127. An immunostimulatory bacterium according to claim 125 or claim 126, wherein different promoters and terminators express control over each protein.

128. An immunostimulatory bacterium according to claim 125 or claim 126, wherein the gene is expressed from a single promoter, comprises a single terminator, and each protein-coding sequence comprises an intrasequence ribosome entry site (IRES), thereby making the encoding nucleic acid polycistronic.

129. An immunostimulatory bacterium according to any one of claims 125 to 128, wherein the promoter is selected from EF-1 alpha, CMV, and GAPDH promoter, and the terminator is selected from SV40, hGH, BGH, and rbGlob.

130. An immunostimulatory bacterium according to any one of claims 30 to 129, wherein the protein-coding plasmid comprises a construct comprising an enhancer, a promoter, a protein-coding open reading frame, and a poly-A tail.

131. An immunostimulatory bacterium according to any one of claims 30 to 130, wherein the protein-coding plasmid comprises a construct comprising an enhancer, a promoter, an IRES, a protein-coding open reading frame, and a poly-A tail.

132. An immunostimulatory bacterium according to any one of claims 30 to 131, wherein the protein-coding plasmid comprises a construct comprising an enhancer, a promoter, an IRES, a localization sequence, a protein-coding open reading frame, and a poly-A tail.

133. An immunostimulatory bacterium according to any one of claims 30 to 132, wherein the construct of a protein-coding plasmid comprises woodchuck hepatitis virus (WHP), a post-transcriptional regulatory element (WPRE), or a post-transcriptional regulatory element of hepatitis B virus (HPRE).

134. An immunostimulatory bacterium according to any one of claims 30 to 133, having a deletion in the SPI-2 complex.

135. An immunostimulatory bacterium according to any one of claims 30 to 134, wherein the bacterium is treated with polymyxin to reduce its pyrogenicity.

136. A delivery medium comprising a nucleic acid encoding a modified STING protein according to any of claims 1 to 29.

137. A delivery medium 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-κB signaling activity compared to human STING.

138. A delivery medium according to claim 136 or claim 137, wherein the human STING protein comprises a human allele variant having at least 98% sequence identity with the sequence shown in any of SEQ ID NOs. 305 to 309 or the amino acid sequence shown in any of SEQ ID NOs. 305 to 309.

139. A delivery medium according to any one of claims 136 to 138, wherein the type I IFN signaling activity is at least 30% or at least about 30% of the type I IFN signaling activity of wild-type human STING.

140. A delivery medium according to any one of claims 136 to 139, wherein the NF-κB signaling activity is less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of that of human STING.

141. A delivery medium according to any one of claims 136 to 140, wherein the non-human species is a Tasmanian devil, marmoset, cow, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, mouse, zebrafish, or elephant shark.

142. The delivery medium comprises a nucleic acid encoding an immunostimulatory protein that, when expressed in a target, leads to the constitutive expression of type I IFN, wherein the delivery medium is selected from nanoparticles, liposomes, exosomes, bacteria, viruses, cells, and microvesicles.

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

144. The delivery medium of claim 143, wherein an immunostimulatory protein in a signaling pathway that senses or directly or indirectly interacts with cytosolic nucleic acids, nucleotides, dinucleotides, cyclic nucleotides, or cyclic dinucleotides activates a type I IFN.

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

146. A delivery medium according to any one of claims 142 to 145, wherein the immunostimulatory protein is selected from STING, MDA5, IRF-3, IRF-7, and RIG-1.

147. The unmodified STING protein has the sequence shown in any of SEQ ID NOs: 305-309; The unmodified MDA5 protein has the sequence shown in SEQ ID NO: 310; The unmodified RIG-I protein has the sequence shown in SEQ ID NO: 311; The unmodified IRF-3 protein has the sequence shown in SEQ ID NO: 312; and The unmodified IRF-7 protein has the sequence shown in SEQ ID NO:

313. The delivery medium according to claim 146.

148. A delivery medium according to any one of claims 142 to 147, wherein the immunostimulatory protein comprises an amino acid substitution that constitutes its activity.

149. A delivery medium according to any one of claims 142 to 148, wherein an immunostimulatory protein causes interferonosis when it is produced in a subject.

150. A delivery medium according to any one of claims 142 to 149, wherein the immunostimulatory protein is selected from variant STING, variant MDA5, variant IRF-3, variant IRF-7, and variant RIG-I, each containing one or more gain-of-function mutations.

151. A delivery medium according to claim 150, wherein gain-of-function mutation results in the expression of constitutive type I interferon.

152. The mutations are as follows: a) In STING, based on sequence numbers 305 to 309, 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, S27 One or more selected from 2A / 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; b) In MDA5, with reference to sequence number 310, one or more of T331I, T331R, A489T, R822Q, G821S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F, and A452T; c) In RIG-I, with reference to sequence number 311, either or both of E373A and C268F; d) In IRF-3, with reference to Sequence ID No. 312, S396D; and e) In IRF-7, one or more of the following, based on sequence number 313: S477D / S479D, S475D / S477D / S479D, and S475D / S476D / S477D / S479D / S483D / S487D A delivery medium according to claim 150 or claim 151.

153. The delivery medium according to claim 152, wherein unmodified STING has the sequence shown in any of SEQ ID NOs. 305 to 309, unmodified MDA5 has the sequence shown in SEQ ID NOs. 310, unmodified RIG-I has the sequence shown in SEQ ID NOs. 311, unmodified IRF-3 has the sequence shown in SEQ ID NOs. 312, unmodified IRF-7 has the sequence shown in SEQ ID NOs. 313, and each modified protein has at least 95% sequence identity with the unmodified protein.

154. A delivery medium according to any of claims 136 to 153, which is a cell, an exosome, an oncolytic virus or viral vector, a liposome or other lipid-based medium or an immunostimulatory bacterium.

155. The delivery medium according to claim 154, wherein the delivery medium is a cell that is a stem cell or an immune cell.

156. A delivery medium according to any one of claims 136 to 154, wherein the medium is an exosome.

157. A delivery medium according to any one of claims 136 to 154, wherein the delivery medium is an oncolytic virus.

158. A delivery medium according to any one of claims 136 to 15, which is oncolytic, selected from oncolytic adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, rhabdovirus, papillomavirus, varicella stomatitis virus, measles virus, Newcastle disease virus, picornavirus, Sindbis virus, poxvirus, parvovirus, reovirus, coxsackievirus, influenza virus, mumps virus, poliovirus, Seneca Valley virus, Sendai virus, dengue virus, poliovirus, Semryqui forest fever virus, alphavirus, and flavivirus.

159. The delivery medium of claim 158, wherein the oncolytic virus is vaccinia virus, herpesvirus, measles virus, or reovirus.

160. A delivery medium according to any one of claims 136 to 159, wherein a nucleic acid encoding an immunostimulatory protein is expressed under the control of a eukaryotic regulatory sequence.

161. A delivery medium according to any one of claims 136 to 160, wherein a nucleic acid encoding an immunostimulatory protein is expressed under the control of a eukaryotic promoter.

162. A delivery medium according to any one of claims 136 to 161, wherein the immunostimulatory protein is a constitutively active STING protein.

163. A delivery medium according to any one of claims 136 to 162, further comprising a nucleic acid encoding an immunostimulatory protein that, when expressed in a mammalian subject, confers or contributes to antitumor immunity in the tumor microenvironment.

164. The delivery medium according to claim 163, wherein the immunostimulatory protein is a cytokine, chemokine, costimulatory protein or receptor or costimulatory receptor having a cytosolic domain deletion.

165. A delivery medium according to any one of claims 136 to 164, which is a cell.

166. The delivery medium according to claim 165, wherein the cells are stem cells.

167. The delivery medium according to claim 166, wherein the stem cells are mesenchymal stem cells (MSCs).

168. The delivery medium of claim 167, wherein the MSCs are genetically modified to express a combination of immunomodulatory cytokines.

169. The delivery medium of claim 168, wherein the cytokines are interleukin 12 (IL-12) and interleukin 21 (IL-21).

170. A delivery medium, STING protein, immunostimulatory protein, or immunostimulatory bacterium according to any one of claims 1 to 169, wherein the type I interferon is interferon-α or interferon-β.

171. Isolated cells comprising a delivery medium according to any one of claims 136 to 164.

172. The cell according to claim 171, which is an immune cell, stem cell, tumor cell, or primary cell line.

173. The cell according to claim 172, which is a hematopoietic cell.

174. A cell according to claim 172, which is a T cell.

175. A cell according to any one of claims 171 to 174, which is produced ex vivo by infecting the cell with a delivery medium.

176. Isolated cells containing immunostimulatory bacteria, Modifying immunostimulatory bacteria to preferentially infect tumor-resident immune cells and / or modifying the genome of immunostimulatory bacteria to reduce the induction of cell death in tumor-resident immune cells; and These are immune cells, stem cells, cells from primary cell lines, or tumor cells. Isolated cells.

177. Immunostimulating bacteria flagellin － (fliC － / fljB － The cell of claim 176, which is a Salmonella species.

178. Isolated cells or cultured cells comprising any immunostimulatory bacteria according to claims 30 to 135.

179. A cell according to any one of claims 176 to 178, which is a T cell or a hematopoietic cell.

180. A cell according to any one of claims 177 to 179, which is produced ex vivo by infection of the cell with an immunostimulatory bacterium.

181. A method for treating cancer, comprising administering any of the cells of claims 176 to 180 to a subject having cancer that includes a solid tumor or is a hematopoietic malignancy.

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

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

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

185. Use of any cells according to claims 176 to 180 for the treatment of cancer.

186. Use of claim 185, wherein the cancer includes solid tumors or hematopoietic malignancies.

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

188. Use of any of claims 185 to 187, wherein the cancer is selected from cancers of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectal, ovaries, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix and liver, gastric cancer, lymphoma and leukemia.

189. A pharmaceutical composition comprising a modified STING protein according to any of claims 1 to 29, an immunostimulatory bacterium according to any of claims 30 to 135, a delivery medium according to any of claims 136 to 170, or a cell according to any of claims 171 to 180, in a pharmaceutically acceptable medium.

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

191. The cell according to claim 190, which is an immune cell, stem cell, tumor cell, or primary cell line.

192. The cell according to claim 191, which is a hematopoietic cell and is a chimeric antigen myeloid cell in which the hematopoietic cell is a macrophage.

193. A cell according to claim 191, which is a T cell.

194. Cells according to any one of claims 190 to 193, produced ex vivo by infecting cells with a modified STING protein, immunostimulatory bacteria, or a delivery medium.

195. A method for treating cancer, comprising administering to a subject having cancer which includes a solid tumor or is a hematopoietic malignancy, a modified STING protein according to any of claims 1 to 29, an immunostimulatory bacterium according to any of claims 30 to 135, a delivery medium according to any of claims 136 to 170, or cells according to any of claims 171 to 180 and 190 to 194, or a pharmaceutical composition according to claim 189, or cells according to any of claims 190 to 194.

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

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

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

199. Use of any modified STING protein according to claims 1 to 29, or any immunostimulatory bacteria according to claims 30 to 135, or any delivery medium according to claims 136 to 170, or any cells according to claims 171 to 180 and 190 to 194, or the pharmaceutical composition according to claim 189, or any cells according to claims 190 to 194, for the treatment of cancer.

200. Use of claim 199, wherein cancer includes solid tumors or hematopoietic malignancies.

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

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

203. A modified STING protein, delivery medium, immunostimulatory bacteria, pharmaceutical composition, cell, method, or use according to any of claims 1 to 202, wherein the non-human STING protein is a variant of various allele STING proteins having the amino acid sequence shown in SEQ ID NOs. 331, 338, or 341-351, or having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NOs. 331, 338, or 341-351.

204. A modified STING protein, delivery medium, immunostimulatory bacteria, pharmaceutical composition, cell, method, or use according to any one of claims 1 to 202, wherein the non-human STING protein or chimera has the amino acid sequence shown in any one of SEQ ID NOs: 332-337, 339, or 340.

205. A pharmaceutical composition comprising an immunostimulatory bacterium according to any of claims 30 to 135 in a pharmaceutically acceptable medium.

206. A pharmaceutical composition according to claim 189 or claim 205, formulated for administration without dilution.

207. A pharmaceutical composition according to any one of claims 189, 205, and 206, formulated as a single dose.

208. A pharmaceutical composition according to any one of claims 189 and 205-207, formulated for non-enteral administration.

209. A method for treating cancer in a subject that includes a solid tumor or is a hematopoietic malignancy, comprising administering any of the pharmaceutical compositions of claims 189 and 205-208.

210. Use of any pharmaceutical composition of claims 189 and 205-208 for the treatment of cancers that include solid tumors or are hematopoietic malignancies in a subject.

211. An immunostimulatory bacterium according to any one of claims 30 to 135, for use in the treatment of cancers that include solid tumors or hematopoietic malignancies in a subject.

212. A method or use of any of claims 195-202 and 209-211 or an immunostimulatory bacterium, wherein the subject is a human.

213. The treatment comprises a combination therapy in which a second anticancer agent or treatment is applied, by any method or use of claims 195-202 and 209-212 or by an immunostimulatory bacterium.

214. The method or use of immunostimulatory bacteria according to claim 213, wherein a second anticancer agent or treatment is administered before, concurrently with, after, or intermittently to the immunostimulatory bacteria.

215. The method or use of claim 214 or immunostimulatory bacteria, wherein the second anticancer agent or treatment is immunotherapy.

216. Any method or use of claims 195-202 and 209-215 or immunostimulatory bacteria, wherein the administration of proteins, cells, delivery media, pharmaceutical compositions or immunostimulatory bacteria is non-enteral.

217. Any method or use of claims 195-202 and 209-216 or immunostimulatory bacteria, wherein administration is orally, rectally, or as an aerosol to the lungs or intratumor.

218. Any method or use of claims 195-202 and 209-216 or immunostimulatory bacteria, wherein the administration is intravenous, intramuscular, or subcutaneous.

219. Any method or use of claims 195-202 and 209-218 or immunostimulatory bacteria, wherein the cancer is selected from leukemia, lymphoma, gastric cancer and cancers of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectal, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix and liver.

220. The method or use of claim 215 or immunostimulatory bacteria, wherein the immunotherapy comprises the administration of an anti-PD-1 or anti-PD-L1 or anti-CTLA-4 antibody.

221. This involves modifying the genome, thereby allowing bacteria to flagellin － (fliC － / fljB － ) and / or pagP － A method to increase the ability of therapeutic bacteria to colonize tumors.

222. The method according to claim 221, wherein the therapeutic bacteria are immunostimulatory bacteria.

223. The method of claim 221 or claim 222, wherein the bacteria is a species of Salmonella.

224. Identify mutant, gain-of-function, constitutively active immune-stimulating proteins that promote interferonosis in human patients; and This involves introducing nucleic acids encoding identified proteins into tumor-targeting bacteria or viruses, thereby promoting immune stimulation of the tumor microenvironment when introduced into a target. A method for producing immunostimulatory bacteria or oncolytic viruses for cancer treatment.

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