Genetically engineered immunostimulatory bacterial strains and uses thereof

Abstract

To provide a delivery immunostimulatory bacterium having enhanced colonization to a tumor, a tumor microenvironment and / or a tumor indigenous immunocyte, and enhanced anti-tumor activity.SOLUTION: Provided is an immunostimulatory bacterium including a plasmid coding one or a plurality of therapeutic products to be used in treatment of cancer, where the therapeutic product is an anti-cancer therapeutic product, a genome of the immunostimulatory bacterium is modified, hence a bacterium lacks a flagellum, and a wild-type bacterium includes a flagellum.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications The present application claims the benefit of priority to International Patent Application No. PCT / US2018 / 041713, filed July 11, 2018, published January 17, 2019 as WO2019 / 014398, each entitled "ENGINEERED IMMUNOSTIMULATORY BACTERIAL STRAINS AND USES THEREOF," and co-pending U.S. Patent Application No. 16 / 033,187, filed July 11, 2018, published January 17, 2019 as U.S. Patent Application Publication No. 2019 / 0017050A1, each to Applicant Actym Therapeutics, Inc., Inventors Christopher D. Thanos, Laura Hix Glickman, and Justin Skoble.

[0002] Applicant Actym Therapeutics, Inc., inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, and Alexandre Charles Michel Iannello, claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 789,983, filed January 8, 2019, entitled "Engineered Immunostimulatory Bacterial Strains and Uses Thereof." Applicant Actym Therapeutics, Inc., inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, and Alexandre Charles Michel Iannello, claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 828,990, filed April 3, 2019, entitled "Salmonella Strains Engineered To Colonize Tumors And The Tumor Microenvironment."

[0003] The immunostimulatory bacteria provided in each of these applications can be modified as described herein, and such bacteria are incorporated herein by reference. Where possible, the subject matter of each of these applications is incorporated by reference in its entirety.

[0004] INCORPORATION-BY-REFERENCE OF SEQUENCE LISTINGS PROVIDED ELECTRONICALLY An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic application was created on July 10, 2019, is 457 kilobytes in size, and is titled 1704SEQPC.txt. [Background technology]

[0005] The field of cancer immunotherapy has made great progress, as evidenced by the clinical success of anti-CTLA4, anti-PD-1, and anti-PD-L1 immune checkpoint antibodies (see, e.g., Buchbinder et al. (2015) J. Clin. Invest. 125: 3377-3383; Hodi et al. (2010) N. Engl. J. Med. 363(8):711-723; and Chen et al. (2015) J. Clin. Invest. 125:3384-3391). Tumors have evolved a complete immunosuppressive environment, which leads to multiple mechanisms for evading immune surveillance, reprogramming antitumor immune cells to suppress immunity, and persistent mutational resistance to current cancer therapies (see, e.g., Mahoney et al. (2015) Nat. Rev. Drug Discov. 14(8):561-584). Designing immunotherapies that overcome immune tolerance and escape while simultaneously limiting the autoimmune-related toxicities of current immunotherapies presents a challenge to the field of immuno-oncology. Therefore, additional innovative immunotherapies and other treatments are needed. Summary of the Invention [Means for solving the problem]

[0006] Bacteria for anti-cancer therapy are provided that are modified to be immunostimulatory. The immunostimulatory bacteria provided herein offer a multifaceted approach to anti-tumor therapy. Bacteria provide a platform with many avenues for eliciting anti-tumor immunostimulatory activity. The bacteria provided herein, for example, Salmonella species, are fine-tuned to have potent anti-tumor activity by enhancing their ability to accumulate in or target tumors, tumor-resident immune cells, and / or the tumor microenvironment (TME). This is achieved, for example, by modifications that alter the types of cells they can infect (tropism), their virulence, their ability to evade immune systems such as complement, and / or the environment in which they can replicate. Immunostimulatory bacteria can also encode, for example, products that enhance or trigger immune responses and therapeutic products. The immunostimulatory bacteria provided herein can be administered systemically based on their improved colonization of tumors, tumor microenvironments, and / or tumor-resident cells, and their resistance to complement and other antibacterial immune responses.

[0007] The genomes of the bacteria provided herein are modified to increase accumulation in tumors and in tumor-resident immune cells, and also in the tumor microenvironment, by deleting or disabling genes responsible for infection or invasion of non-tumor cells, such as epithelial cells, and / or by reducing the cytopathogenicity of the bacteria, particularly to immune cells and tumor-resident immune cells.

[0008] Bacteria inherently stimulate the immune system, and bacterial infection induces immune and inflammatory pathways and responses, some of which are desirable for antitumor treatment, some of which are undesirable. Modification of bacteria by deleting or modifying genes and products that result in undesirable immune responses and by adding or modifying genes and products that induce desirable immunostimulatory antitumor responses improves the antitumor activity of bacteria.

[0009] Bacteria can accumulate in tumor cells and tissues and replicate there, thereby lysing the cells. Bacteria can migrate from the administration site and accumulate in other tumors and tumor cells, resulting in an abscopal effect. The bacteria provided herein are modified so that they preferentially infect and accumulate in tumor-resident immune cells, tumors, and tumor microenvironments.

[0010] All of these properties of bacteria are utilized herein to ensure the production of immunostimulatory bacteria with multiple anti-tumor activities and properties that can act individually and synergistically.

[0011] Compositions, uses thereof, and methods for modulating immune responses to treat diseases, such as cancer, are provided. The compositions include the immunostimulatory bacteria provided herein. Methods for treatment and uses of the bacteria for treatment are also provided. Subjects for treatment include humans and other primates, pets such as dogs and cats, and other animals such as horses.

[0012] Pharmaceutical compositions comprising the immunostimulatory bacteria and methods and uses thereof for treating diseases and disorders, particularly proliferative disorders such as tumors, including solid tumors and hematological malignancies, are provided.

[0013] Also provided are methods for inhibiting the growth or reducing the volume of solid tumors by administering the immunostimulatory bacteria or pharmaceutical compositions, or using the compositions for treatment. For example, methods are provided for administering or using compositions comprising an effective amount, in a single dosage, of attenuated Salmonella spp. to a subject, such as a human patient, with a solid tumor cancer. It will be understood that any modifications to the bacterial genome, such as antitumor therapies, and other modifications to the bacterial genome and plasmids described, can be combined in any desired combination.

[0014] Immunostimulatory bacteria are provided that have enhanced colonization of tumors, tumor microenvironments, and / or tumor-resident immune cells, and have enhanced anti-tumor activity. The immunostimulatory bacteria are modified by deleting or modifying the gene encoding flagella so that functional flagella are not produced, and / or by deleting or modifying pagP so that an inactive PagP product is produced. As a result, the immunostimulatory bacteria are able to bind flagellin. - (fliC - / fljB - ) and / or pagP - Alternatively, or in addition, the immunostimulatory bacteria may be pagP - / msbB - It could be.

[0015] The immunostimulatory bacteria can be flagellin-deficient, e.g., flagellin-deficient due to deletion or disruption of the gene(s) encoding flagella. For example, immunostimulatory bacteria are provided that contain a deletion of the genes encoding one or both of the flagellin subunits fliC and fljB, thereby rendering the bacteria flagella-deficient, whereas wild-type bacteria express flagella. The immunostimulatory bacteria can also have a deletion or modification of the gene encoding endonuclease I (endA), such that endA activity is inhibited or eliminated.

[0016] Immunostimulatory bacteria may have additional genomic modifications such that these bacteria are adenosine or purine auxotrophs. - , purI - and msbB - The immunostimulatory bacteria, such as Salmonella spp., may be modified to encode an immunostimulatory protein that confers anti-tumor activity in the tumor microenvironment, and / or the bacteria are modified to preferentially infect and / or induce cell death of immune cells in the tumor microenvironment or tumor-resident immune cells less than other cells. Methods of inhibiting the growth or reducing the volume of solid tumors by administering the immunostimulatory bacteria are also provided.

[0017] Flagellin-mediated translation of immune-stimulating bacterial genomes - (fliC - / fljB - ) and / or pagP - The present invention provides a method for increasing tumor colonization by immunostimulatory bacteria, such as Salmonella spp., by modifying the cells to become

[0018] The bacteria also contain plasmids encoding therapeutic products such as antitumor agents, proteins that enhance a subject's immune response, and inhibitory RNA (RNAi) that targets immune checkpoints. For example, the plasmids can encode immunostimulatory proteins such as cytokines, chemokines, and costimulatory molecules that enhance antitumor responses in a subject. The bacteria contain plasmids encoding anticancer therapeutics, such as RNAs including microRNAs, shRNAs, and siRNAs, as well as antibodies and their antigen-binding fragments, designed to suppress, inhibit, disrupt, or otherwise silence immune checkpoint genes and products and other targets involved in immunosuppressive pathways. The bacteria can also encode tumor antigens and tumor neoantigens on plasmids to stimulate an immune response against tumors. The encoded proteins are expressed under the control of a promoter that is recognized by eukaryotes, such as mammals and animals, or viruses.

[0019] Immunostimulatory bacteria are provided that contain a plasmid encoding a therapeutic product, such as an anti-cancer therapeutic, and the genome of the immunostimulatory bacteria is modified so that it preferentially infects tumor-resident immune cells and / or so that it induces less cell death of tumor-resident immune cells.

[0020] 1. An immunostimulatory bacterium comprising a plasmid encoding a product, generally a therapeutic product such as an anti-cancer therapeutic product, under the control of a eukaryotic promoter, wherein the genome of the immunostimulatory bacterium is modified so that the bacterium expresses flagellin. - (fliC - / fljB -) and / or pagP - and whereby the wild-type bacterium has a flagellum. - (fliC - / fljB - ) and pagP - These immunostimulatory bacteria exhibit increased colonization of tumors, tumor microenvironment, and / or tumor-resident immune cells, resulting in increased anti-tumor activity.

[0021] One of these immunostimulatory bacteria is flagellin - (fliC - / fljB - ), such that the therapeutic product is an anti-cancer product. In some embodiments, the bacterium is - (fliC - / fljB - ) and the product is an anti-cancer therapeutic protein or nucleic acid.

[0022] One of these immunostimulatory bacteria is one in which the therapeutic product is a TGF-beta antagonist polypeptide, and the genome of the immunostimulatory bacterium is modified so that the bacterium preferentially infects tumor-resident immune cells and / or so that it induces less cell death of tumor-resident immune cells (reduced pyroptosis), thereby delivering the TGF-beta antagonist polypeptide to the tumor microenvironment by accumulating in the tumor or tumor microenvironment or tumor-resident immune cells. The TGF-beta antagonist can be selected from anti-TGF-beta antibodies, anti-TGF-beta receptor antibodies, and soluble TGF-beta antagonist polypeptides. The nucleic acid encoding the TGF-beta antagonist polypeptide can include a nucleic acid encoding a signal sequence for secretion of the encoded polypeptide, so that it is released into tumor cells, tumor-resident immune cells, and / or the tumor microenvironment.

[0023] In other embodiments of any of the immunostimulatory bacteria provided herein, the plasmid encodes an immunostimulatory protein that confers, enhances, or contributes to an anti-tumor immune response in the tumor microenvironment. Exemplary immunostimulatory proteins that confers or contributes to anti-tumor immunity in the tumor microenvironment include IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-15, IL-36 gamma, IL-2 with attenuated binding to IL-2Ra, IL-15 / IL-15R alpha chain complex, IL-18, IL-21, IL-23, IL-36 gamma, IL-2 modified to not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β ... The antibodies may be one or more of terferon-gamma, CCL3, CCL4, CCL5, proteins involved in or causing or promoting T cell recruitment / persistence, CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), members of the B7-CD28 family, CD47 antagonists, TGF-beta polypeptide antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily.

[0024] In other embodiments of the immunostimulatory bacteria provided herein, the therapeutic product is an antibody or an antigen-binding fragment thereof. Exemplary of such are Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, disulfide-stabilized Fv (dsFv), nanobody, diabody fragment, or single-chain antibody. The antibody or antigen-binding fragment thereof may be humanized or human. Exemplary of the antibody or antigen-binding fragment thereof is an antagonist of PD-1, PD-L1, CTLA-4, VEGF, VEGFR2, or IL-6.

[0025] The immunostimulatory bacteria provided herein, including those described above, can comprise a plasmid encoding a therapeutic product under the control of a eukaryotic promoter, and the genome of the immunostimulatory bacteria is modified so that the bacteria expresses pagP. - / msbB - and flagellin - (fliC - / fljB - ) may also be used.

[0026] Exemplary immunostimulatory bacteria are those comprising a plasmid encoding an immunostimulatory protein, which when expressed in a mammalian subject confers or contributes to anti-tumor immunity in the tumor microenvironment; the immunostimulatory protein is encoded on the plasmid in the bacterium under the control of a eukaryotic promoter; and the genome of the immunostimulatory bacterium is modified so that it preferentially infects tumor-resident immune cells. In other embodiments, the immunostimulatory bacterium comprises a sequence of nucleotides encoding an immunostimulatory protein, which when expressed in a mammalian subject confers or contributes to anti-tumor immunity in the tumor microenvironment; the immunostimulatory protein is encoded on a plasmid in the bacterium under the control of a eukaryotic promoter; and the genome of the immunostimulatory bacterium is modified so that it induces less cell death of tumor-resident immune cells. Exemplary immunostimulatory proteins include cytokines and chemokines and other immunostimulatory proteins, such as IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-36 gamma, IL-2 with reduced binding to IL-2Ra, IL-15 / IL-15R alpha chain complex, IL-18, IL-21, IL-23, IL-36 gamma, IL-2 modified to not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-alpha ... Examples include one or more of interferon-β, interferon-γ, CCL3, CCL4, CCL5, proteins involved in or causing or promoting T cell recruitment / persistence, CD40, CD40 ligand, CD28, OX40, OX40 ligand, 4-1BB, 4-1BB ligand, members of the B7-CD28 family, CD47 antagonists, TGF-beta polypeptide antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily.

[0027] These immunostimulatory bacteria can include one or more modifications to the genome of the immunostimulatory bacteria such that the bacteria preferentially infect tumor-resident immune cells and / or induce less cell death of tumor-resident immune cells. The immunostimulatory bacteria can also include mutations in the genome that reduce virulence or infectivity of non-immune cells in the host.

[0028] Modification of the bacterial genome involves pagP - or pagP - and flagellin - (fliC - / fljB - In another embodiment, the immunostimulatory bacteria comprises purI. - (purM - ), msbB - , purD - , flagellin - (fliC - / fljB - ), pagP - , adrA - , CsgD - , QseC - and hilA - one or more of, for example, flagellin - (fliC - / fljB - ) / pagP - / msbB - / purI - , or flagellin - (fliC - / fljB - ) / pagP - / msbB - / purI - / hilA - In other embodiments, the immunostimulatory bacteria is hilA. - and / or flagellin - (fliC - / fljB - ) or pagP - or pagP - / msbB -or the immunostimulatory bacteria are hilA - or immunostimulatory bacteria contain flagellin - (fliC - / fljB - ) and pagP - The genomic modifications, among other properties, increase targeting or colonization of the tumor microenvironment and / or tumor-resident immune cells, and / or render the bacteria substantially or completely resistant to inactivation by complement. These properties improve the use of bacteria as therapeutic agents and allow for systemic administration.

[0029] In the immunostimulatory bacteria provided herein, the nucleic acid encoding the therapeutic product is operably linked to the nucleic acid encoding the secretion signal for expression, so that the immunostimulatory protein is secreted when expressed in the host.The therapeutic product can be a protein such as an immunostimulatory protein, or a nucleic acid such as a CRISPR cassette or RNAi.

[0030] In all embodiments, the immunostimulatory protein may be adenosine auxotrophic, or may be adenosine and adenine auxotrophic. The immunostimulatory bacteria provided herein can comprise a genome modification such that the bacteria preferentially infect tumor-resident immune cells, and / or the genome of the immunostimulatory bacteria is modified such that it induces less cell death (reduced pyroptosis) in tumor-resident immune cells, such that the immunostimulatory bacteria delivers the encoded therapeutic product to the tumor or by accumulating in the tumor microenvironment or in tumor-resident immune cells.

[0031] In immunostimulatory bacteria, the plasmid encodes a therapeutic product under the control of a eukaryotic promoter, and thus it is expressed in eukaryotic hosts, such as humans or other mammals.Generally, therapeutic products are anti-cancer therapeutic drugs, such as anti-cancer therapeutic proteins, that stimulate the host's immune system.Other therapeutic products include antibodies and their antigen-binding fragments, and nucleic acids, such as RNAi.These products can be designed to inhibit, suppress, or disrupt targets, such as immune checkpoints, and other targets that impair the ability of the subject's immune system to recognize tumor cells.

[0032] The unmodified immunostimulatory bacteria can be a wild-type strain or an attenuated strain. The genomic modifications provided and described herein attenuate the bacteria in the tumor microenvironment or outside the tumor, and the modifications alter, among other characteristics, the infectivity of the bacteria. Exemplary bacteria that can be modified as described herein are Salmonella, such as Salmonella typhimurium strains. Exemplary Salmonella typhimurium strains include attenuated and wild-type strains, such as those derived from strains designated AST-100, VNP20009, YS1646 (ATCC #202165), RE88, SL7207, χ8429, χ8431, and χ8468, or the wild-type strain having ATCC accession number 14028.

[0033] An immunostimulatory bacterium comprising a plasmid encoding a product under the control of a eukaryotic promoter, as discussed above, wherein the genome of the immunostimulatory bacterium is modified so that the bacterium expresses pagP. - / msbB -Deletion of msbB alters the acyl composition of the lipid A domain of lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, such that the bacteria produce predominantly pentaacylated LPS rather than the more toxic and pro-inflammatory hexaacylated LPS. In wild-type S. typhimurium, expression of pagP results in heptaacylated lipid A, whereas expression of msbB results in heptaacylated lipid A. - In the mutant, induction of pagP results in hexaacylated LPS. - / msbB - The mutant produces only pentaacylated LPS, resulting in reduced induction of pro-inflammatory cytokines and enhanced tolerability, which allows for higher doses to be administered to humans. Higher doses lead to increased colonization of tumors, tumor-resident immune cells, and the tumor microenvironment. Due to the resulting changes in the bacterial membrane and structure, host immune responses, such as complement activity, are altered to prevent the bacteria from being eliminated upon systemic administration. For example, pagP - / msbB - The mutant strains are shown herein to have increased resistance to complement inactivation and enhanced stability in human serum. These bacteria also express flagellin - (fliC - / fljB - ), which further enhances tolerability, resistance to complement inactivation, and tumor / TME / tumor-resident immune cell colonization. The bacteria can also contain other modifications described herein, including modifications that alter the cells they can infect, resulting in accumulation in the tumor microenvironment, tumor, and tumor-resident immune cells. Thus, the immunostimulatory bacteria provided herein can be administered systemically and exhibit high levels of tumor, tumor microenvironment, and / or tumor-resident immune cell colonization. The immunostimulatory bacteria can be purI - (purM - ) and asd - , msbB - , and flagellin - (fliC- / fljB - ) and pagP - It may be one or more of either or both of.

[0034] Immunostimulatory bacteria produce aspartate-semialdehyde dehydrogenase - (asd - ), for example, all or part of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (asd) is disrupted or deleted, so that endogenous asd is not expressed, resulting in the expression of aspartate-semialdehyde dehydrogenase - (asd - These immunostimulatory bacteria can be engineered to encode aspartate-semialdehyde dehydrogenase (asd) on a plasmid under the control of a bacterial promoter, thus allowing the bacteria to be produced in vitro.

[0035] Immunostimulatory bacteria can be made auxotrophic for specific nutrients, such as adenosine and adenine, that are abundant or accumulate in the tumor microenvironment. They can also be engineered to be auxotrophic for such nutrients, reducing or eliminating their replication ability. Inactivated / deleted bacterial genomic genes can be complemented by providing them on a plasmid under the control of a promoter recognized by the host.

[0036] For expression in eukaryotic hosts, such as humans, the encoded product on the plasmid is under the control of eukaryotic regulatory sequences, including eukaryotic promoters such as promoters recognized by RNA polymerase II or III. These include mammalian RNA polymerase II promoters. Viral promoters can also be used. Exemplary viral promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the SV40 promoter, the Epstein-Barr virus (EBV) promoter, herpesvirus promoters, and adenovirus promoters. Other RNA polymerase II promoters include, but are not limited to, the elongation factor-1 (EF1) alpha promoter, the UbC promoter (lentivirus), the PGK (3-phosphoglycerate kinase) promoter, and synthetic promoters, such as the CAGG (or CAG) promoter. The synthetic CAG promoter contains the cytomegalovirus (CMV) early enhancer element (C), a promoter (A) that is the first exon and first intron of the chicken beta-actin gene, and a splice acceptor (G) from the rabbit beta-globin gene. Other strong regulatable or constitutive promoters can be used. Regulatory sequences also include terminators, enhancers, and secretory and other transport signals.

[0037] The plasmids contained in the immunostimulatory bacteria can be present at low or medium copy numbers, e.g., by selection of an origin of replication that results in a medium to low copy number, such as a low copy number origin of replication. It is shown herein that the antitumor activity and other properties of the bacteria are improved when the plasmids are present at low to medium copy numbers, where a medium copy number is less than or less than about 150 copies, more than or about 20 copies, or between 20 or 25 copies and 150 copies, and a low copy number is less than 25 copies, or less than 20 copies, or less than about 25 copies, or less than about 20 copies.

[0038] 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 so that they induce less cell death of tumor-resident immune cells (reduced pyroptosis), so that the immunostimulatory bacteria accumulate in the tumor or in the tumor microenvironment or in tumor-resident immune cells.

[0039] As discussed above, the genomes of immunostimulatory bacteria also reveal that these bacteria express CD45 + The bacteria are modified to preferentially infect immune cells, such as tumor-resident immune cells, such as myeloid cells, such as cells that are resistant to HIV-1, and / or the genome is modified so that these bacteria induce less cell death (reduced pyroptosis) in tumor-resident immune cells than unmodified cells. As a result, the immunostimulatory bacteria deliver the therapeutic product(s) encoded on the plasmid by accumulating in the tumor, or in the tumor microenvironment, or in tumor-resident immune cells, or by accumulating to a greater extent than unmodified ones. The bacteria are modified to preferentially infect immune cells, such as myeloid cells, such as cells that are resistant to HIV-1, and / or the genome is modified so that these bacteria induce less cell death in tumor-resident immune cells than unmodified cells (reduced pyroptosis). As a result, the immunostimulatory bacteria deliver the therapeutic product(s) encoded on the plasmid by accumulating in the tumor, or in the tumor microenvironment, or in tumor-resident immune cells, or by accumulating to a greater extent than unmodified ones. - (fliC - / fljB - ), pagP - , and msbB - and may contain other modifications described herein. The bacterium may be adenosine auxotrophic, and / or purI. - (purM - ) and / or asd - It could be.

[0040] The immunostimulatory bacteria provided herein may be modified in the bacterial genome such that the bacteria induce less cell death of tumor-resident immune cells, and / or the cells more effectively target tumors, the tumor microenvironment, or tumor-resident immune cells, such as tumor-resident CD45 + These may include modifications of the bacterial genome that accumulate in cells, bone marrow cells, and bone marrow cells.

[0041] For example, the immunostimulatory bacteria can contain a deletion or modification of one or more genes or operons involved in SPI-1 entry (and / or SPI-2), such that the immunostimulatory bacteria do not invade or infect epithelial cells. Exemplary genes that can be deleted or inactivated are 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. Loss of the ability to infect epithelial cells can also be achieved by genetically engineering the immunostimulatory bacteria herein to contain a knockout or deletion of a gene encoding a protein involved in SPI-1-independent invasion, such as one or more of the genes selected from among rck, pagN, hlyE, pefI, srgD, srgA, srgB, and srgC. Similarly, the immunostimulatory bacteria can contain a deletion of a gene and / or operon in SPI-2, for example, to engineer the bacteria to escape the Salmonella-containing vacuole (SCV). These genes include, for example, sifA, sseJ, sseL, sopD2, pipB2, sseF, sseG, spvB, and steA.

[0042] The immunostimulatory bacteria provided herein can also comprise a sequence of nucleotides encoding an immunostimulatory protein that confers or contributes to anti-tumor immunity in tumor microenvironments when expressed in a mammalian subject, and this immunostimulatory protein is encoded on a plasmid in the bacteria under the control of a eukaryotic promoter.Exemplary promoters include, but are not limited to, elongation factor-1 (EF1) alpha promoter, or UbC promoter, or PGK promoter, or CAGG or CAG promoter.

[0043] In addition, the genome of the immunostimulatory bacteria is modified so that it preferentially infects tumor-resident immune cells. This is achieved by deleting or disrupting bacterial genes involved in bacterial invasiveness or infectivity and / or inducing cell death. The bacteria are modified to preferentially infect tumor-resident immune cells over unmodified bacteria and / or induce less cell death in tumor-resident immune cells than other cells that the bacteria can infect.

[0044] The immunostimulatory bacteria can also encode therapeutic products such as inhibitory RNA (RNAi), immunostimulatory proteins, such as cytokines, chemokines, and costimulatory molecules, other proteins that enhance the immune response in a subject, and other antitumor agents that confer or contribute to antitumor immunity when expressed in a mammalian subject. The therapeutic product is encoded on a plasmid in the bacteria under the control of a eukaryotic promoter. The genome of the immunostimulatory bacteria is modified so that it induces less cell death of tumor-resident immune cells. The plasmid is generally present at low or medium copy number.

[0045] Also provided are immunostimulatory bacteria that encode an immunostimulatory protein on a plasmid in the bacterium under the control of a eukaryotic promoter, which when expressed in a mammalian subject, confers or contributes to anti-tumor immunity in the tumor microenvironment. The immunostimulatory bacteria can be modified to have reduced pathogenicity, thereby reducing infection of epithelial and / or other non-immune cells compared to bacteria without the modification. These include modifications of the type 3 secretion system (T3SS) or type 4 secretion system (T4SS), for example, modifications of the SPI-1 pathway of Salmonella as described and exemplified herein. The bacteria can be modified to induce less cell death, for example, by deleting or disrupting the nucleic acid encoding lipid A palmitoyltransferase (PagP), which reduces the virulence of the bacteria.

[0046] The genome of the immunostimulatory bacteria provided herein can be modified to increase or promote infection of immune cells, particularly immune cells in the tumor microenvironment, such as phagocytes. This includes reducing infection of non-immune cells, such as epithelial cells, or increasing infection of immune cells. Bacteria can also be modified to reduce pyroptosis in immune cells. Numerous modifications of bacterial genomes can increase infection of immune cells and / or reduce pyroptosis. The immunostimulatory bacteria provided herein include such modifications, for example, deletion and / or disruption of genes involved in the SPI-1 T3SS pathway, such as disruption or deletion of hilA, and / or disruption / deletion of genes encoding flagellin, rod protein (PrgJ), needle protein (PrgI), and QseC.

[0047] Immunostimulatory bacteria are purI - (purM - ), msbB - , purD - , flagellin - (fliC - / fljB - ), pagP - , adrA - , CsgD - , QseC - and hilA - one or more of, in particular, flagellin - (fliC - / fljB - ) and / or pagP - , and / or msbB - / pagP - For example, the immunostimulatory bacteria may contain a genomic mutation, e.g., a gene deletion or perturbation, that reduces the virulence or infectivity of non-immune cells in the host. For example, the immunostimulatory bacteria may contain a pagP - In another example, the immunostimulatory bacteria may be hilA- and / or flagellin- - (fliC - / fljB- ) and pagP - Thus, for example, the immunostimulatory bacteria can encode an immunostimulatory protein, such as a cytokine, and the bacteria can be engineered so that they accumulate in the tumor microenvironment (TME) and express the cytokine therein, thereby delivering the immunotherapeutic anti-tumor product to an environment in which it has beneficial activity and avoiding harmful or toxic side effects from expression in other cells / environments. The nucleic acid encoding the immunostimulatory protein can be operably linked for expression to a nucleic acid encoding a secretion signal, such that upon expression in the host, the immunostimulatory protein is secreted into the tumor microenvironment.

[0048] The immunostimulatory bacteria provided herein include any of the strains and bacteria described in co-pending U.S. patent application Ser. No. 16 / 033,187, or published International Application No. PCT / US2018 / 041713 (published as WO2019 / 014398), which have been further modified to express immunostimulatory proteins in immune cells in the tumor microenvironment or tumor-resident immune cells, and / or to preferentially infect those cells and / or to be less virulent in those cells, as described and exemplified herein.

[0049] Immunostimulatory bacteria contain disrupted or deleted endogenous genes encoding aspartate-semialdehyde dehydrogenase (asd), resulting in the lack of expression of endogenous asd. - (asd - The immunostimulatory bacteria can be modified to encode aspartate-semialdehyde dehydrogenase (asd) on a plasmid under the control of a bacterial promoter for growth of the bacteria in vitro such that the bacteria replicate poorly in vivo.

[0050] The immunostimulatory bacteria provided herein can encode an immunostimulatory protein on a plasmid as a therapeutic product. The immunostimulatory protein can be a cytokine such as a chemokine, or a costimulatory molecule. Exemplary 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, causing, or promoting T cell recruitment / persistence, CD40, CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), members of the B7-CD28 family, and members of the tumor necrosis factor receptor (TNFR) superfamily.

[0051] The immunostimulatory bacterium can optionally comprise a sequence of nucleotides encoding an inhibitory RNA (RNAi) that inhibits, suppresses, or disrupts the expression of an immune checkpoint. The RNAi can be encoded on a plasmid in the bacterium. The nucleotides encoding the immunostimulatory protein, which may encode the RNAi, can also be on a plasmid that is present at low to medium copy numbers.

[0052] The immunostimulatory bacteria can also encode therapeutic products, such as RNAi or CRISPR cassettes, that inhibit, suppress, or disrupt the expression of immune checkpoints or other targets, such that the inhibition, suppression, or disruption enhances anti-tumor immunity in a subject, and the RNAi or CRISPR cassette is encoded on a plasmid in the bacteria. Other therapeutic products include, for example, antibodies that bind to immune checkpoints and inhibit their activity.

[0053] RNAi includes all forms of double-stranded RNA, and can be used to silence the expression of target nucleic acid. RNAi includes shRNA, siRNA, and microRNA (miRNA). Any of these forms can be interchanged in the embodiments disclosed and described herein. Generally, RNAi is encoded on a plasmid in bacteria. The plasmid can contain other heterologous nucleic acids that code for the desired product that modulates or adds activity, or for the product to bacteria, or for other such products that can modulate the immune system of the subject that will be treated with bacteria. Bacterial genes can also be added, deleted, or disrupted. These genes can also code for the product that allows bacteria to grow and reproduce, or for the product that modulates the host's immune response to bacteria.

[0054] The immunostimulatory bacteria provided herein can be adenosine auxotrophic, or adenosine and adenine auxotrophic.

[0055] Bacterial species carrying the plasmids described herein and suitable for the modifications described herein include, but are not limited to, Salmonella, Shigella, Listeria, or E. coli, and Bifidobacteria, such as Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, and Salmonella enteritidis.

[0056] Examples of bacterial species include Salmonella, Shigella, E. coli, Bifidobacterium, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, Chlamydia, and Hemophilus influenzae. and strains of the genera Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, and Erysipelothrix, or attenuated or modified strains thereof of any of the preceding lists of bacterial strains.

[0057] Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix. For example, Rickettsia Rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Franciesella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus sornnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, and Agrobacterium tumerfacium.

[0058] Exemplary Salmonella strains herein include, in particular, Salmonella typhimurium strains. Salmonella can be wild-type or attenuated. Exemplary attenuated strains include strains designated YS1646 (ATCC #202165) or VNP20009. Other strains include RE88, SL7207, χ8429, χ8431, and χ8468. Exemplary wild-type or non-attenuated strains include, for example, the wild-type strain deposited under ATCC 14028, or a strain having all of the identifying characteristics of ATCC 14028. The modifications herein attenuate any strain by limiting the cells the bacteria can infect or replicate in.

[0059] These strains can be further modified to encode immunostimulatory and / or immunomodulatory proteins. For example, the immunostimulatory bacteria can encode immunostimulatory proteins, such as cytokines, that enhance immune responses in the tumor microenvironment. The immunostimulatory bacteria can also be modified, as described herein, to preferentially infect immune cells in the tumor microenvironment, or to infect resident immune cells, and / or to induce less cell death of such immune cells. These sequences and descriptions are provided in the detailed description, examples, and sequence listing. The immunostimulatory bacteria can be derived from an attenuated strain of bacteria, or can be attenuated based on modifications described herein, such as deletion of asd, so that replication is limited in vivo.

[0060] It will be understood that when bacterial genes are modified and referred to herein, they will be referred to in terms of their designation (name) in Salmonella species, which are exemplary of bacteria capable of producing immunostimulatory bacteria. Those skilled in the art will appreciate that other bacterial species have corresponding proteins, but their designations or names may differ from those in the Salmonella genus. However, the general disclosure herein can be applied to other bacterial species. For example, as shown herein, deletion or inactivation of flagellin (fliC) in Salmonella species can be used to produce immunostimulatory bacteria. - / fljB - Deletion or inactivation of pagP and / or pagP results in increased tumor colonization. Similar genes encoding flagella or similar functions for infection can be modified in other bacterial species to achieve increased tumor colonization. Similarly, inactivation / deletion of bacterial products, such as the products of pagP and / or msbB described herein, can reduce complement activation and / or other inflammatory responses, thereby increasing targeting of tumors, tumor-resident immune cells, and the tumor microenvironment. Corresponding genes in other bacterial species involved in activation of the complement pathway or other inflammatory pathways can be deleted, as exemplified herein for Salmonella.

[0061] The immunostimulatory bacteria provided herein encode inhibitors of various genes that reduce anti-tumor immune responses, and / or express genes and / or gene products that contribute to anti-tumor immune responses, and / or products that stimulate the immune system, such as immunostimulatory proteins, such as cytokines, chemokines, and costimulatory molecules, thereby being immunostimulatory. Adenosine auxotrophy is immunostimulatory. Other therapeutic products that can be encoded on plasmids include nucleic acids, such as inhibitory RNAs (RNAi), such as shRNAs, microRNAs, or siRNAs, aimed at disrupting or inhibiting the expression of TREX1, PD-L1, VISTA (a gene encoding a V-domain Ig suppressor of T cell activation), TGF-beta, and CTNNB1 (a gene encoding β-catenin), among others, and combinations thereof, as well as combinations of these with any RNAi that inhibit, suppress, or disrupt the expression of other immunosuppressive genes whose expression is activated or enhanced by the tumor or tumor microenvironment (TME). Expression of these RNAs utilizes two independent immunostimulatory pathways, resulting in enhanced tumor colonization in a single treatment modality. The effect of this combination is enhanced by the strains provided herein, which are adenosine auxotrophic, providing preferential accumulation in or recruitment into an adenosine-rich immunosuppressive tumor microenvironment. Depletion of adenosine in such a TME further enhances the immunostimulatory effect. Such combinations of features in any of the bacterial strains known or that can be genetically engineered for therapeutic administration provide similar immunostimulatory effects.

[0062] One of the targets is TGF-beta, which has three isoforms: 1, 2, and 3. One of the targets is TGF-beta, specifically isoform 1, but not isoforms 2 and 3. Toxicity is associated with the inhibition of isoforms 2 and 3. For example, cardiac valve toxicity is associated with the inhibition of isoform 2. Isoform 1 is present in most cancers (see, for example, the TCGA database). It is advantageous to inhibit only isoform 1. RNAi can be advantageously used for this purpose because it can be designed to recognize targets very specifically. With regard to TGF-beta, specific inhibition of isoform 1 can be achieved by targeting a sequence unique to isoform 1 that is not present in isoforms 2 or 3, or by selecting a sequence that targets isoforms 1 and 3, but not 2. Immunostimulatory bacteria are also provided in which the plasmid encodes an shRNA or microRNA that specifically inhibits, suppresses or disrupts the expression of TGF-beta isoform 1, but not TGF-beta isoform 2 or TGF-beta isoform 3; or in which the plasmid encodes an shRNA or microRNA that specifically inhibits, suppresses or disrupts the expression of TGF-beta isoforms 1 and 3, but not isoform 2.

[0063] RNAi, such as miRNA- or shRNA-mediated gene disruption of PD-L1 by immunostimulatory bacteria provided herein, also improves colonization of tumors, TME, and / or tumor-resident immune cells. Knocking out PD-L1 has been shown to promote S. typhimurium infection. For example, it has been observed that PD-L1 provides protection against S. typhimurium infection when PD-L1 knockout mice are challenged with bacterial loads at least 10 times higher than wild-type mice (see, for example, Lee et al. (2010) Immunol. 185:2442-2449).

[0064] The engineered immunostimulatory bacteria provided herein, such as S. typhimurium immunostimulatory bacteria, include multiple synergistic modalities to induce cold tumor immune reactivation, promote tumor antigen-specific immune responses, and simultaneously inhibit immune checkpoint pathways that tumors exploit to subvert and evade persistent anti-tumor immunity. Adenosine auxotrophy and enhanced vascular perturbation are included in embodiments. This improvement in tumor targeting and enhanced vascular perturbation via adenosine auxotrophy enhances efficacy while simultaneously localizing inflammation and limiting systemic cytokine exposure and autoimmune toxicity observed with other immunotherapy modalities.

[0065] Heterologous therapeutic proteins and other products, such as immunostimulatory proteins, antibodies, and RNA, are expressed on a plasmid under the control of a promoter recognized by the eukaryotic host cell transcription machinery, such as the 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 can be regulated. The therapeutic product is encoded on a plasmid that is stably expressed by the bacterium. Exemplary of such bacteria are globally attenuated Salmonella strains, such as adenosine auxotrophs, that have been attenuated by passage or other methods or based on the modifications described herein. Exemplary of Salmonella strains are modified S. typhimurium strains with defective asd genes. These bacteria can be modified to contain a functional asd gene on an introduced plasmid, which maintains selection for the plasmid so that an antibiotic-based plasmid maintenance / selection system is not required. The asd-deficient strain, which does not contain a functional asd gene on a plasmid, is autolytic within the host.

[0066] For the environment of tumor cells, promoters can be selected, such as promoters that are expressed in the tumor microenvironment (TME), promoters that are expressed under hypoxic conditions, or promoters that are expressed under conditions where the pH is less than 7.

[0067] Plasmids can exist as many or few copies. This can be controlled by selecting elements such as the origin of replication. Low, medium, and high copy number plasmids and origins of replication are well known to those skilled in the art and can be selected. In the immunostimulatory bacteria embodiments herein, plasmids can exist at low to medium copy numbers, such as about 150 or less copies, to low copy numbers, such as less than about 25 or about 20 or 25 copies. Exemplary origins of replication are those derived from pBR322, p15A, pSC101, pMB1, colE1, colE2, pPS10, R6K, R1, RK2, and pUC.

[0068] The contemplated plasmid may contain an RNAi gene, such that the RNA inhibits, suppresses, or disrupts the expression of an immune checkpoint or other target and its product. The plasmid may also contain a nucleic acid sequence encoding the signal-less listeriolysin O (LLO) protein (cytoLLO), a CpG motif, a DNA nuclear targeting sequence (DTS), and a retinoic acid-inducible gene-I (RIG-I) binding element. The immunostimulatory bacterium containing the nucleic acid 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 contained within a bacterial gene encoded on the plasmid, or the CpG motif may be part of a bacterial gene encoded on the plasmid. For example, the CpG-containing gene may be asd encoded on a plasmid. The immunostimulatory bacterium provided herein may contain one or more of a CpG motif, an asd gene selection marker for plasmid maintenance, and a DNA nuclear targeting sequence.

[0069] The immunostimulatory bacteria provided herein can encode two or more different RNA molecules that inhibit, suppress or disrupt the expression of an immune checkpoint and / or an RNA molecule that encodes an inhibitor of a metabolite that is immunosuppressive or in an immunosuppressive pathway.

[0070] The immunostimulatory bacteria provided herein contain aspartate-semialdehyde dehydrogenase, which allows growth in diaminopimelic acid (DAP)-supplemented media but limits replication in vivo when administered to a subject for treatment. - (asd - Such bacteria may be self-limiting and may be advantageous for treatment. The bacteria may be aspartate-semialdehyde dehydrogenase (asd) because all or part of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (asd) is disrupted or deleted, resulting in the endogenous asd not being expressed. - In other embodiments, the gene encoding aspartate-semialdehyde dehydrogenase can be contained on a plasmid for expression in vivo.

[0071] Any of the immunostimulatory bacteria provided herein can generally contain a nucleic acid containing a CpG motif or a CpG island on a plasmid, and the CpG motif is recognized by Toll-like receptor 9 (TLR9).The nucleic acid encoding a CpG motif or island is abundant in prokaryotes, and therefore the CpG motif can be contained in or be part of a bacterial gene and encoded on a plasmid.For example, the bacterial gene asd contains an immunostimulatory CpG.

[0072] The immunostimulatory bacteria provided herein can be auxotrophic for adenosine, or adenosine and adenine. Any of the bacteria herein can be adenosine trophic, which can advantageously increase anti-tumor activity because adenosine accumulates in many tumors and is immunosuppressive.

[0073] If the wild-type bacterium contains flagella, the immunostimulatory bacterium provided herein can be flagellin-deficient. These can be made flagellin-deficient by disrupting or deleting all or part of the gene(s) encoding flagella. For example, immunostimulatory bacteria are provided that have deletions of genes encoding one or both of the flagellin subunits fliC and fljB, thereby making the bacterium flagellum-deficient.

[0074] The immunostimulatory bacteria provided herein can contain a nucleic acid encoding a listeriolysin O (LLO) protein, cytoLLO, that lacks a periplasmic secretion signal sequence, resulting in accumulation in the cytoplasm. This mutation is expressed as asd - LLO is advantageously combined with bacteria. LLO is a cholesterol-dependent pore-forming hemolysin derived from Listeria monocytogenes that mediates bacterial phagocytic escape. When autolytic strains are introduced into tumor-bearing hosts, such as humans, the bacteria are engulfed by phagocytic immune cells and enter the vacuole. In this environment, the absence of DAP prevents bacterial replication and leads to bacterial autolysis within the vacuole. Subsequent lysis releases the plasmid, and accumulated LLO forms pores in the cholesterol-containing vacuolar membrane, allowing delivery of the plasmid into the host cell cytosol.

[0075] The immunostimulatory bacteria can contain a DNA nuclear targeting sequence (DTS), such as the SV40 DTS, encoded on a plasmid.

[0076] The immunostimulatory bacteria can have a deletion or modification of the gene encoding endonuclease-1 (endA) such that endA activity is inhibited or eliminated. Exemplary of these are immunostimulatory bacteria that contain one or more of a CpG motif, an asd gene selection marker for maintaining a plasmid, and a DNA nuclear targeting sequence.

[0077] The immunostimulatory bacteria can contain nucleic acids on a plasmid encoding two or more different RNA molecules that inhibit, suppress or disrupt expression of an immune checkpoint, or RNA molecules that encode inhibitors of metabolites that are immunosuppressive or in an immunosuppressive pathway.

[0078] The nucleic acid encoding the RNAi, such as shRNA or miRNA or siRNA, can comprise a transcription terminator downstream of the nucleic acid encoding the RNA.In all embodiments, the RNAi encoded on the plasmid in the immunostimulatory bacterium can be a short hairpin RNA (shRNA) or microRNA (miRNA).

[0079] The immunostimulatory bacteria can further encode therapeutic products, such as RNAi, or antibodies or other binding proteins that inhibit expression of immune checkpoints and other targets, such as 3' repair exonuclease 1 (TREX1), PD-1, PD-L1 (B7-H1), VEGF, TGF-beta isoform 1, beta-catenin, CTLA-4, PD-L2, PD-2, IDO1, IDO2, SIRPα, CD47, VISTA (B7-H5), LIGHT, HVEM, CD28, LAG3, TIM3, TIGIT, galectin-9, CEACAM1, CD155, CD112, CD226, CD244 (2B4), B7-H2, and B7-H3, which inhibit, suppress, disrupt, or silence expression of these targets, and which are immunostimulatory. , ICOS, GITR, B7-H4, B7-H6, CD27, CD40, CD40L, CD48, CD70, CD80, CD86, CD137 (4-1BB), CD200, CD272 (BTLA), CD160, CD39, CD73, A2a receptor, A2b receptor, HHLA2, ILT-2, ILT-4, gp49B, PIR-B, HLA-G, ILT-2 / 4, OX40, OX-40L, KIR, TIM1, TIM4, and STAT3, stabilin-1 (CLEVER-1), DNASE II, and RNASE H2. Cluster of differentiation 47 (CD47), also known as integrin-associated protein (IAP), is a transmembrane receptor that belongs to the immunoglobulin superfamily of proteins. CD47 is ubiquitously expressed on cells and serves as a marker for self-recognition that inhibits phagocytosis. CD47 mediates its effects through interactions with several other proteins, including thrombospondin (TSP) and signal-regulatory protein-alpha (SIRPα). The interaction of SIRPα on phagocytes with CD47 on target cells helps ensure that the target cells are not engulfed by phagocytes. Certain cancers exploit CD47-based immune evasion mechanisms by increasing CD47 expression on the cell surface of cancer cells, thereby evading clearance by the immune system.Targeting CD47-expressing cells in a subject results in toxicity. Encoding a CD47 inhibitory molecule, such as an antibody or antibody fragment, e.g., a nanobody (see, e.g., Sockolosky et al. (2016) Proc. Natl. Acad. Sci. USA 113:E2646-E2654), on a plasmid in the immunostimulatory bacteria provided herein results in expression of the anti-CD47 product in the tumor microenvironment or tumor. The anti-CD47 antibody fragment is encoded in bacteria, such as E. coli, that are administered intratumorally (see, e.g., Chowdhury et al. (2019) Nature Medicine 25:1057-1063). The bacteria provided herein have improved targeting and colonization of the tumor microenvironment, tumor, and / or tumor-resident immune cells, thereby enabling more effective delivery of anti-CD47 antibodies or antibody fragments. The immunostimulatory bacteria provided herein can be administered systemically to colonize tumors and the tumor microenvironment.

[0080] Immunostimulatory bacteria are provided in which the plasmid contains a nucleotide sequence encoding a therapeutic product that inhibits an immune checkpoint or other immunosuppressive target. Targets include, but are not limited to, TREX1, PD-L1, VISTA, TGF-beta isoform 1, beta-catenin, SIRP-alpha, VEGF, RNase H2, DNase II, CLEVER-1 / stabilin-1, and CD47. Other targets that may be inhibited, suppressed, or disrupted include CTLA-4, PD-L2, PD-1, PD-2, IDO1, IDO2, LIGHT, HVEM, CD28, LAG3, TIM3, TIGIT, galectin-9, CEACAM1, CD155, CD112, CD226, CD244 (2B4), B7-H2, B7-H3, ICOS, GITR, B7-H4, B7-H6, CD27, CD40, and CD40. The target polypeptide may be selected from among any of L, CD48, CD70, CD80, CD86, CD137 (4-1BB), CD200, CD272 (BTLA), CD160, CD39, CD73, A2a receptor, A2b receptor, HHLA2, ILT-2, ILT-4, gp49B, PIR-B, HLA-G, ILT-2 / 4, OX40, OX-40L, KIR, TIM1, TIM4, and STAT3. Exemplary of these are one of human PD-L1 (SEQ ID NO: 31), human beta-catenin (SEQ ID NO: 32), human SIRPα (SEQ ID NO: 33), human TREX1 (SEQ ID NO: 34), human VISTA (SEQ ID NO: 35), human TGF-beta isoform 1 (SEQ ID NO: 193), and human VEGF (SEQ ID NO: 194). The RNA can target or include a sequence in an immune checkpoint nucleic acid set forth in any of SEQ ID NOs: 1-30, 36-40, and 195-217. The plasmid in any of the immunostimulatory bacteria can also encode a sequence of nucleotides that is an agonist of retinoic acid-inducible gene I (RIG-I) or a RIG-I binding element.

[0081] The immunostimulatory bacteria can include one or more of the gene deletions, for example, the bacteria can include deletions of purI - (purM - ), msbB -, purD - , flagellin - (fliC - / fljB - ), pagP - , adrA - , CsgD - and hilA - The immunostimulatory bacteria may be one or more of: - For example, the immunostimulatory bacteria may comprise a purI deletion, an msbB deletion, an asd deletion, and an adrA deletion, and may also comprise a CsgD deletion. Exemplary bacterial gene deletions / modifications are any of the following: one or more mutations in genes that alter lipopolysaccharide biosynthesis selected from among one or more of rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; and / or one or more mutations introducing a suicide gene and selected from one or more of sacB, nuk, hok, gef, kil or phlA; and / or one or more mutations introducing a lysis gene, selected from one or both of hly and cly; and / or Mutations in one or more virulence factor(s) selected from among IsyA, pag, prg, iscA, virG, plc and act; and / or one or more of the following mutations in genes that modify the stress response: recA, htrA, htpR, hsp, and groEL; and / or Mutations in min that disrupt the cell cycle; and / or One or more of the mutations in genes selected from among cya, crp, phoP / phoQ and ompR that disrupt or inactivate regulatory functions.

[0082] The immunostimulatory bacteria may be a strain of Salmonella, Shigella, E. coli, Bifidobacterium, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, or Erysipelothrix, or an attenuated or modified strain thereof of any of the preceding lists of bacterial strains.

[0083] Exemplary immunostimulatory bacteria are those in which the plasmid contains one or more of a nucleic acid sequence encoding the signal-sequence-less listeriolysin O (LLO) protein (cytoLLO), a CpG motif, a DNA nuclear targeting sequence (DTS), and a retinoic acid-inducible gene-I (RIG-I) binding element.

[0084] Other exemplary immunostimulatory bacteria include those that are adenosine auxotrophic and contain a deletion in the gene(s) encoding the flagellum; a deletion in endA; a plasmid encoding CytoLLO; a nuclear localization sequence; and an asd plasmid complementation system, encoding an RNA that inhibits, suppresses, or disrupts expression of an immune checkpoint or other target, the inhibition, suppression, or disruption enhancing an anti-tumor immune response in a subject.

[0085] Such immunostimulatory bacteria include strains of the genus Salmonella, e.g., wild-type Salmonella Typhimurium strains, such as the strain deposited under ATCC Accession No. 14028, or a strain having all of the identifying characteristics of the strain deposited under ATCC Accession No. 14028. Other strains include, for example, the strains designated AST-100, VNP20009, or attenuated Salmonella Typhimurium strains selected from among strains YS1646 (ATCC #202165), RE88, SL7207, χ8429, χ8431, and χ8468.

[0086] The immunostimulatory bacteria may comprise one or more of a purI deletion, an msbB deletion, an asd deletion, and an adrA deletion, in addition to modifications that increase accumulation in tumor cells, the TME, and / or tumor-resident immune cells, and / or that decrease immune cell death, and may encode an immunostimulatory protein or other therapeutic product as described herein. The immunostimulatory bacteria may also comprise: one or more mutations in genes that alter lipopolysaccharide biosynthesis selected from among one or more of rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; and / or one or more mutations introducing a suicide gene and selected from one or more of sacB, nuk, hok, gef, kil, and phlA; and / or one or more mutations introducing a lysis gene, selected from one or both of hly and cly; and / or Mutations in one or more virulence factor(s) selected from among IsyA, pag, prg, iscA, virG, plc and act; and / or one or more mutations in a gene or genes that alter the stress response, selected from among recA, htrA, htpR, hsp, and groEL; and / or Mutations in min that disrupt the cell cycle; and / or One or more mutations that disrupt or inactivate a regulatory function selected from among cya, crp, phoP / phoQ and ompR.

[0087] The strain was msbB - , asd - , hilA - and / or flagellin - (fliC - / fljB - ), and / or pagP- In particular, the strain may be one or more of: - (fliC - / fljB - ), e.g., flagellin - (fliC - / fljB - ), msbB - , purI - / M - and asd - and / or HilA -The bacterium may be adenosine auxotrophic, or adenosine and adenine auxotrophic. The therapeutic product, e.g., an RNAi and / or immunostimulatory protein, and / or an antibody or fragment thereof, is expressed under the control of a promoter recognized by the host, such as the RNAP III promoter or RNAP II promoter described herein. Immunostimulatory bacteria include Salmonella, Shigella, E. coli, Bifidobacterium, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, Chlamydia, and Haemophilus. The bacterial strain may be a strain of the genera Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, or Erysipelothrix, or an attenuated or modified strain thereof from any of the preceding lists of bacterial strains. Generally, the bacterial strain is one that is attenuated in the host. A Salmonella strain, such as S. typhimurium, is a good example of a bacterium. Exemplary strains include Salmonella typhimurium strains derived from the strain designated AST100, VNP20009 or strains YS1646 (ATCC #202165), RE88, SL7207, χ8429, χ8431, χ8468 and the wild-type strain ATCC #14028.

[0088] Immunostimulatory bacteria. Compositions containing bacteria are provided. Such compositions include bacteria and a pharmaceutically acceptable excipient or vehicle. Immunostimulatory bacteria include any of those described herein, those described in the patents / applications incorporated herein, or those known to those skilled in the art. The bacteria encode a therapeutic product, generally an anti-cancer product, such as an inhibitor of an immune checkpoint, or an immunostimulatory protein, such as a cytokine, chemokine, or costimulatory molecule, that increases anti-tumor activity in the tumor microenvironment or tumor. The bacterial genome can be modified to have increased infectivity of immune cells and / or reduced infectivity of non-immune cells and / or a reduced ability to induce cell death in immune cells. Thus, the bacteria are modified as described herein to accumulate in tumors or tumor microenvironments or tumor-resident immune cells and / or to deliver immunostimulatory proteins and other therapeutic products that promote anti-tumor activity. The immunostimulatory bacteria can further comprise a plasmid encoding a therapeutic anti-cancer product, e.g., an RNAi, e.g., miRNA or shRNA, or a CRISPR cassette, that targets immune checkpoints or otherwise enhances the anti-tumor activity of the bacteria.

[0089] A single dose is therapeutically effective for treating a disease or disorder for which immune stimulation provides treatment. Exemplary of such stimulation are immune responses, including, but not limited to, one or both of a specific immune response and a non-specific immune response, both specific and non-specific 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.

[0090] Pharmaceutical compositions comprising any of the immunostimulatory bacteria are provided. Uses thereof for treating cancer and methods for treating cancer are also provided. The methods and uses include treating a subject with cancer, and include administering the immunostimulatory bacteria or pharmaceutical compositions to a subject, such as a human. A method for treating a subject with cancer is provided, comprising administering the immunostimulatory bacteria.

[0091] The methods and uses include combination therapy in which a second anti-cancer agent or treatment is administered. The second anti-cancer agent is a chemotherapeutic agent that induces cytoplasmic DNA, or radiation therapy, or an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, or an anti-immune checkpoint inhibitor such as CAR-T cells, or other therapeutic cells such as stem cells, TIL cells, and cells engineered for cancer therapy. The combination therapy can also include an anti-VEGF, anti-VEGFR, or anti-VEGFR2 antibody, or a fragment thereof, or an anti-IL6 antibody, or a fragment thereof, or an oncolytic virus therapy, or a cancer vaccine.

[0092] Administration can be by any suitable route, such as parenteral, and can include additional agents that can facilitate or enhance delivery. Administration can be oral, or rectal, or by aerosol to the lung, or intratumoral, intravenous, intramuscular, or subcutaneous.

[0093] Cancer includes solid tumors and hematological malignancies, such as, but not limited to, lymphoma, leukemia, gastric cancer, and cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectal, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix, and liver.

[0094] The immunostimulatory bacteria can be formulated into compositions for administration, such as suspensions. They can be dried and stored as powders. Combinations of the immunostimulatory bacteria with other anti-cancer agents are also provided.

[0095] Provided is a combination therapy for treating cancer and malignant tumors.Immunostimulatory bacteria can be administered before or simultaneously with other cancer therapies, including radiation therapy, chemotherapy, particularly genotoxic chemotherapy that causes cytosolic DNA, and immunotherapy, such as anti-checkpoint inhibitor antibodies, including anti-PD-1 antibody, anti-PD-L1 antibody and anti-CTLA4 antibody, and other such immunotherapies.Other cancer therapies also include anti-VEGF, anti-VEGFR, anti-VEGFR2 or anti-IL6 antibody or its fragment, cancer vaccine, and oncolytic virus.

[0096] Administration can be by any suitable route, including systemic or localized or topical, e.g., parenteral, including, for example, orally or rectally, or by aerosol to the lung, or intratumorally, intravenously, intramuscularly, or subcutaneously.

[0097] Also provided are methods for increasing colonization of tumors, tumor-resident immune cells, and / or the tumor microenvironment by immunostimulatory bacteria. The methods include, for example, administering the bacteria to tumors containing flagellin. - (fliC - / fljB - ) and / or pagP - Such modifications include modifying the bacterial genome to enhance tumor / tumor microenvironment / tumor-resident immune cell colonization. Such modifications are shown herein to significantly enhance tumor / tumor microenvironment / tumor-resident immune cell colonization.

[0098] The terms and expressions employed are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are contemplated. [Brief explanation of the drawings]

[0099] [Figure 1]Figure 1 shows a schematic diagram of the process used to delete the asd gene from strain YS1646. The asd gene from S. typhimurium strain YS1646 was deleted using the lambda-derived Red recombination system described by Datsenko and Wanner [Proc Natl Acad Sci USA 97:6640-6645 (2000)]. [Figure 2] Figure 2 shows the level of tumor establishment in injected and distant tumors after IT administration of AST-104. BALB / c mice (6–8 weeks old) were implanted with double CT26 (2 × 10 cells) subcutaneous flank tumors in the right and left flanks (n = 10 per group). Mice with established tumors were injected IT into the right flank with 5 × 10 CFU of the YS1646 strain (AST-104) containing the TREX1 shRNA plasmid. Thirty-five days after tumor implantation (12 days after the final AST-104 administration), three mice were sacrificed. The injected and distant tumors were homogenized (GentleMACs™, Miltenyi Biotec) and plated on LB plates to count the number of colony-forming units (CFU) per gram of tumor tissue. The figure shows the mean CFU per gram of tissue, ±SD. [Figure 3]Figure 3 demonstrates that CpG-scrambled plasmids have immunostimulatory antitumor properties. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of YS1646 strain (AST-100), YS1646 strain (AST-103) containing a scrambled shRNA control plasmid, or PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) × 100. The figure shows the mean tumor growth for each group ± SEM. **p<0.01, Student's t-test. [Figure 4] Figure 4 shows a schematic diagram of the process used to delete the fliC gene. The flic gene was deleted from the chromosome of S. typhimurium strain AST-101 (an asd deletion strain of YS1646) using the lambda-derived Red recombination system described by Datsenko and Wanner [Proc Natl Acad Sci USA 97:6640-6645 (2000)]. [Figure 5] Figure 5 shows that flagellin deletion strains grow normally in LB. The figure shows the growth of strains AST-108 ASD(pATI-shTREX1) and AST-112 ASD / FLG(pATI-shTREX1) in LB broth at 37°C, as measured by OD600 using a Spectramax 96-well plate reader (Molecular devices). [Figure 6]Figure 6 shows that flagellin knockout improves antitumor efficacy. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the asd / fljB / fliC knockout strain (AST-113) containing the pATI shTREX1 plasmid, or the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid, or PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 – (mean test tumor volume / mean control tumor volume) × 100. The figure shows the mean tumor growth for each group ± SEM. *p<0.05, Student's t-test. [Figure 7] Figure 7 shows that flagellin knockout mice exhibit an increased IFN-gamma signature. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice bearing established tumors were intravenously injected with 5 × 10 CFU of the asd / fljB / fliC knockout strain (AST-113) containing the pATI shTREX1 plasmid, the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid, or a PBS control. Six hours after the first injection, mice were bled, and systemic serum cytokines were analyzed using a Luminex 200 instrument (Luminex Corporation) and a mouse cytometric bead array (BD Bead Array, FACS Fortessa, FCAP software, all BD Biosciences). *P < 0.05, **p < 0.01, ***p < 0.001, Student's t-test. [Figure 8]Figure 8 shows that flagellin is not required for tumor establishment. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the asd / fljB / fliC knockout strain (AST-113) containing the pATI shTREX1 plasmid, the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid, or a PBS control. On day 35 (D35) after tumor implantation (12 days after the last dose of the genetically engineered Salmonella therapy), three mice per group were sacrificed, and tumors were homogenized (GentleMACs™, Miltenyi Biotec) and plated on LB plates to count the number of colony-forming units (CFU) per gram of tumor tissue. The figure shows the mean colony-forming units (CFU) per gram of tissue, ± SD. [Figure 9] Figure 9 shows that the cytoLLO-expressing strains grow normally in vitro. The figure shows the growth of strains AST-110 [YS1646 with the asd deletion containing (pATI-shTREX1)] and AST-115 [YS1646 with the asd deletion and cytoLLO expression cassette knock-in containing (pATI-shTREX1)] in LB broth at 37°C, as measured by OD using a Spectramax 96-well plate reader (Molecular devices). [Figure 10]Figure 10 shows that AST-115 (ASD knockout + CytoLLO knock-in strain harboring shTREX1 plasmid) exhibits robust single-dose efficacy in a murine CT26 tumor model. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of AST-115 [YS1646 (containing pATI-shTREX1) with an asd deletion and a cytoLLO expression cassette knock-in at the asd locus] or PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) x 100. The figure shows the mean tumor growth for each group ± SEM. **p<0.01, Student's t-test. [Figure 11] Figure 11 shows that strain YS1646 requires tumor microenvironment levels of adenosine for growth. Growth of strain YS1646 (purI- / msbB-) and the wild-type parent strain ATCC14028 in LB broth at 37°C, as measured by OD600 using a Spectramax 96-well plate reader (Molecular devices), is shown. [Figure 12] Figure 12 shows that ASD, FLG, and CytoLLO engineered strains require high adenosine for growth. Growth of strains AST-117 (YS1646 Δasd containing a low-copy shTREX-1 plasmid), AST-118 (YS1646 Δasd / ΔfilC / ΔfljB containing a low-copy shTREX-1 plasmid), and AST-119 (YS1646 Δasd:LLO containing a low-copy shTREX-1 plasmid) in LB broth at 37°C is shown, as measured by OD using a Spectramax 96-well plate reader (Molecular devices). [Figure 13]Figure 13 shows that strains with a low-copy origin of a replicating asd-encoding plasmid have superior growth kinetics to strains with a high-copy origin of a replicating asd-encoding plasmid. Growth of strains YS1646, AST-117 (YS1646 Δasd containing a low-copy shTREX-1 plasmid with a functional asd gene), AST-104 (YS1646 containing a low-copy pEQ shTREX-1 plasmid lacking the asd gene), and AST-110 (YS1646 Δasd containing a high-copy pATI-shTREX-1 plasmid with a functional asd gene) in LB broth at 37°C, as measured by OD using a Spectramax 96-well plate reader (Molecular devices), is shown. [Figure 14] Figure 14 shows that strains carrying a low-copy asd plasmid are more compatible with mouse tumor cells than strains carrying a high-copy asd plasmid. The intracellular growth of strains AST-117 (YS1646 Δasd containing a low-copy shTREX-1 plasmid carrying a functional asd gene) and AST-110 (YS1646 Δasd containing a high-copy pATI-shTREX-1 plasmid carrying a functional asd gene) is shown in B16F.10 mouse melanoma cells and CT26 mouse colon carcinoma cells. 5 × 10 cells in 24-well dishes were infected with S. typhimurium strains at an MOI of 5. Thirty minutes after infection, the medium was replaced with medium containing gentamicin to kill extracellular bacteria. At the indicated time points, cell monolayers were lysed by osmotic shock, and cell lysates were diluted and plated on LB agar to enumerate CFU. [Figure 15]Figure 15 shows that the asd gene complementation system results in plasmid retention in S. typhimurium-infected tumors in vivo. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of either the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid or YS1646 (AST-104) containing the pEQ shTREX-1 plasmid lacking the asd gene. Three mice per group were sacrificed 35 days after tumor implantation (12 days after the final dose of genetically engineered Salmonella therapy). Tumors were homogenized using a GentleMACs™ homogenizer (Miltenyi Biotec) and plated on LB agar plates or LB agar plates containing 50 μg / mL kanamycin. The figure shows the percentage of kanamycin-resistant CFU in tumor tissue homogenates ±SD. [Figure 16] Figure 16 shows the improved therapeutic efficacy of the asd gene complementation system and the strain (AST-110) containing a plasmid carrying shTREX1. BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid or the asd knockout strain (AST-109) containing the pATI scrambled plasmid, or the YS1646 strain (AST-104) containing the pEQ-shTREX-1 plasmid lacking the asd gene, or PBS control, on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 - (mean test tumor volume / mean control tumor volume) × 100. The figure shows the mean tumor growth for each group, ± SEM. [Figure 17] Figure 17 shows that the strain containing the low-copy shTREX1 plasmid (AST-117) has superior antitumor properties compared to the strain containing the high-copy plasmid (AST-110). BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid with a high copy number replication origin or the asd knockout strain (AST-117) containing the pATI shTREX1 plasmid with a low copy number replication origin, or PBS control, on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1-(mean test tumor volume / mean control tumor volume) x 100. The figure shows the mean tumor growth for each group, ±SEM. *p<0.05, Student's t-test. [Figure 18]Figures 18A and 18B show that the AST-117 low-copy plasmid strain colonizes tumors better than the AST-110 high-copy plasmid strain, with a higher tumor-to-spleen colonization ratio. BALB / c mice (6-8 weeks old) were implanted with a single CT26 (2 x 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 x 10 CFU of either the asd knockout strain (AST-110) containing the pATI shTREX1 plasmid with a high copy number replication origin or the asd knockout strain (AST-117) containing the pATI shTREX1 plasmid with a low copy number replication origin. On day 35 after tumor implantation (12 days after the final dose of genetically engineered Salmonella therapy), three mice per group were sacrificed, and tumors were homogenized using a GentleMACs™ homogenizer (Miltenyi Biotec) and plated on LB plates to count the number of CFU per gram of tumor tissue. Figure 18A shows the mean CFU per gram of tumor tissue ±SD. Figure 18B shows the tumor-to-spleen colonization ratio. [Figure 19] Figure 19 shows that the autolytic strain (AST-120) cannot grow in the absence of DAP. The figure shows the growth over time of the Δasd:cytoLLO strain (AST-120) containing the pEQU6-shTREX1 plasmid, which does not contain the asd gene, in LB broth alone or in LB broth supplemented with 50 μg / mL DAP, as measured by OD using a Spectramax 96-well plate reader (Molecular devices). [Figure 20]Figure 20 shows the antitumor activity of the autolytic strain (AST-120). BALB / c mice (6–8 weeks old) were implanted with a single CT26 (2 × 10 cells) subcutaneous flank tumor (n = 9 per group). Mice with established tumors were intravenously injected with 5 × 10 CFU of the Δasd:cytoLLO strain (AST-120) containing the pEQU6-shTREX1 plasmid, which does not contain the asd gene, or with PBS control on the days indicated by the arrows. Tumor measurements were performed using electronic calipers (Fowler, Newton, MA). Tumor volume was calculated using the modified ellipsoid formula 1 / 2 (length × width 2). Mice were euthanized according to IACUC regulations when tumor size reached >20% of body weight or when necrotic. TGI was calculated as 1 – (mean test tumor volume / mean control tumor volume) × 100. The figure shows the mean tumor growth for each group, ± SEM. *p<0.05, Student's t-test. [Figure 21] FIG. 21 shows proteins that act downstream of HilA in the SPI-1 pathway. [Figure 22-1] FIG. 22 shows the SPI-1 T3SS and the functional classification of the proteins encoded by SPI-1 (adapted from Kimbrough and Miller (2002) Microbes Infect. 4(1):75-82). [Figure 22-2] Same as above. [Figure 23] Figure 23 shows the effect of SPI-1 T3SS on macrophages. Flagellin is detected by NAIP5 / 6, and rod and needle proteins are detected by NAIP1 / 2, which leads to activation of the NLRC4 inflammasome and caspase-1, resulting in the release of IL-1β and IL-18, and pyroptosis. [Figure 24] FIG. 24 shows T3SS-1-mediated entry of bacteria into epithelial cells and SCVs. [Figure 25]FIG. 25 shows the recognition of bacterial flagellin by TLR5 and LPS by TLR4, and the roles that flagellin, LPS, HilA, PrgI, and PrgJ play in host cell infection, cytokine release, inflammasome activation, and pyroptosis. DETAILED DESCRIPTION OF THE INVENTION

[0100] overview A.Definition B. Overview of Immunostimulatory Bacteria C. Cancer immunotherapy 1. Immunotherapy 2. Adoptive Immunotherapy 3. Cancer vaccines and oncolytic viruses D. Bacterial cancer immunotherapy 1. Bacterial treatment 2. Comparison of immune responses to bacteria and viruses 3. Treatment with Salmonella a. Tumor-tropic bacteria. b. Salmonella enterica serovar vertifimurium C. Attenuation of the bacteria i.msbB-mutant ii.purI-mutant iii. Attenuating mutation combinations iv. VNP20009 and other attenuated and wild-type S. typhimurium strains v. S. typhimurium genetically engineered to deliver macromolecules 4. Enhancing immunostimulatory bacteria to increase the therapeutic index ASD gene deletion B adenosine auxotrophy C. flagellin-deficient strain d. Salmonella genetically engineered to escape Salmonella-containing vacuoles (SCVs) e. Deletions in Salmonella genes required for biofilm formation f. Deletion in a gene in the LPS biosynthetic pathway g. Deletion of the SPI-1 and SPI-2 genes h. Endonuclease (endA) mutation to increase plasmid delivery i. RIG-I inhibition j.DNASE II inhibition k.RNase H2 inhibition l. Stabilin-1 / CLEVER-1 inhibition 5. Immunostimulatory proteins 6. Modifications that increase immune cell uptake of Gram-negative bacteria such as Salmonella and reduce immune cell death 7. Bacterial culture conditions E. Bacterial attenuation and colonization 1. Deletion of flagellin (fliC) - / fljB - ) 2. Detection of genes in the LPS biosynthetic pathway 3. Settlement F. Construction of Exemplary Plasmids Encoding Therapeutic Proteins 1. Immunostimulatory proteins 2. Antibodies and antibody fragments 3. Interfering RNA (RNAi) a.shRNA B microRNA 4. Origin of Replication and Plasmid Copy Number 5. CpG motifs and CpG islands 6. Plasmid Maintenance / Construct Selection 7. RNA polymerase promoter 8. DNA nuclear targeting sequence 9. CRISPR G. Tumor-Targeted Immunostimulatory Bacteria Contain RNAi Against Exemplary Immune Target Genes to Stimulate Antitumor Immunity 1.TREX1 2.PD-L1 3. VISTA 4.SIRPα 5. β-catenin 6. TGF-β 7. VEGF 8. Additional Exemplary Checkpoint Targets H. Pharmaceutical Production, Compositions, and Formulations 1.Manufacturing a. Cell bank production b. Manufacturing of APIs c. Manufacturing of drug products 2. Composition 3. Preparation a. Liquids, injections, emulsions b. Heat-stable dry formulation 4. Compositions for other routes of administration 5. Dosage and Administration 6. Packaging and Articles of Manufacture I. Methods of Treatment and Use 1. Tumor 2. Administration 3. Monitoring J. Working Example

[0101] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention(s) pertain. All patents, patent applications, published applications and literature, GenBank sequences, databases, websites and other publications mentioned throughout this disclosure are incorporated by reference in their entirety unless otherwise noted. In the event that there are multiple definitions for terms in this specification, those in this section shall prevail. When referring to a URL or other such identifier or address, it should be understood that such identifiers may change and specific information on the Internet may be overwhelmed, but equivalent information may be found by searching the Internet. Reference thereto evidences the availability and general dissemination of such information.

[0102] As used herein, a therapeutic bacterium is a bacterium that, when administered to a subject, such as a human, confers an effect on a therapy, such as a cancer or anti-tumor therapy.

[0103] As used herein, immunostimulatory bacteria are therapeutic bacteria that, when introduced into a subject, accumulate in immune-privileged tissues and cells, such as tumors, and replicate and / or express products that are immunostimulatory or result in immune stimulation. For example, immunostimulatory bacteria are attenuated in a host by being unable to replicate and / or express products (or having reduced replication / product expression) except primarily in immune-privileged environments, and / or by having reduced toxicity or pathogenicity and / or by having encoded products that reduce toxicity or pathogenicity. The immunostimulatory bacteria provided herein are modified to encode a product(s) or exhibit characteristics or properties that make them immune-stimulatory. Such products, properties, and characteristics include, but are not limited to, at least one of: immune-stimulatory proteins, such as cytokines, chemokines, or costimulatory molecules; RNAi, such as siRNA (shRNA and microRNA) or CRISPR, that target, disrupt, or inhibit immune checkpoint genes, such as TREX1 and / or PD-L1; or immune checkpoint inhibitors, such as anti-immune checkpoint antibodies. The immunostimulatory bacteria can also include modifications that render the bacteria auxotrophic for metabolites that are immunosuppressive or in an immunosuppressive pathway, such as adenosine.

[0104] As used herein, the strain designations VNP20009 (see, e.g., WO 99 / 13053; see also U.S. Pat. No. 6,863,894) and YS1646 and 41.2.9 are used interchangeably and each refer to a strain deposited with the American Type Culture Collection (ATCC) and assigned accession number 202165. VNP20009 is an engineered, attenuated strain of Salmonella typhimurium that contains deletions of msbB and purI and was generated from the wild-type strain ATCC #14028.

[0105] As used herein, the strain designations YS1456 and 8.7 are used interchangeably and each refers to the strain deposited with the American Type Culture Collection and assigned accession number 202164 (see U.S. Patent No. 6,863,894).

[0106] As used herein, an origin of replication is a sequence of DNA on a chromosome, plasmid, or virus from which replication begins. For small DNA molecules, including bacterial plasmids and small viruses, a single origin is sufficient.

[0107] The replication origin determines the copy number of the vector, which depends on the replication origin selected. For example, if the expression vector is derived from the low-copy-number plasmid pBR322, it can be approximately 25-50 copies / cell, and if it is derived from the high-copy-number plasmid pUC, it can be 150-200 copies / cell.

[0108] As used herein, a medium copy number of a plasmid in a cell is about 150, or 150 or less, and a low copy number is 15-30, such as 20 or less. A low to medium copy number is less than 150. A high copy number is greater than 150 copies / cell.

[0109] As used herein, a CpG motif is a pattern of bases that includes an unmethylated central CpG (the "p" refers to a phosphodiester bond between consecutive C and G nucleotides) surrounded by at least one base adjacent to the central CpG (at the 3' and 5' positions). A CpG oligodeoxynucleotide is an oligonucleotide that is at least about 10 nucleotides in length and includes an unmethylated CpG. At least the C in the 5'CG3' is unmethylated.

[0110] As used herein, the RIG-I binding sequence refers to a 5' triphosphate (5' ppp) structure derived directly from a poly(dA-dT) sequence or synthesized from this sequence by RNA pol III, which can activate type I IFN through the RIG-I pathway by interacting with RIG-I. The RNA has at least four A ribonucleotides (AAAA); it can contain 4, 5, 6, 7, 8, 9, 10 or more. The RIG-I binding sequence is introduced into a plasmid in bacteria and transcribed into polyA.

[0111] As used herein, immunostimulatory protein is one that provides, promotes, strengthens or enhances immune response, particularly in tumor microenvironment, for example, in tumor and / or tumor-resident immune cells.Immunostimulatory protein includes but is not limited to cytokines, chemokines, costimulatory molecules and other immunoregulatory proteins and products.Therefore, as used herein, "immunostimulatory protein" refers to the protein that provides, shows or promotes anti-tumor response in tumor microenvironment. Exemplary of such proteins are cytokines, chemokines, and costimulatory molecules, including, but not limited to, GM-CSF, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-12p70 (IL-12p40+IL-12p35), IL-15 / IL-15R alpha chain complex, CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, molecules involved in T cell potential recruitment / persistence, CD40, CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), members of the B7-CD28 family, CD47 antagonists, TGF-beta polypeptide antagonists, and members of the TNFR superfamily.

[0112] As used herein, "cytokine" is a broad, general category of small proteins (approximately 5-20 kDa) that are important in cell signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are cell signaling molecules that aid in intercellular communication in the immune response and stimulate cell migration to sites of inflammation, infection, and trauma.

[0113] As used herein, "chemokine" refers to a chemoattractant (chemotactic) cytokine that binds to a chemokine receptor, and includes proteins isolated from natural sources as well as those produced synthetically, such as by recombinant means or chemical synthesis. Exemplary chemokines include, but are not limited to, IL-8, IL-10, GCP-2, GRO-α, GRO-β, GRP-γ, ENA-78, PBP, CTAP, and the like. 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, lungkine, Tim-1, eotaxin-1, eotaxin-2, I-309, SCYA17, TRAC, RANTES (CCL5), DC-CK-1, lymphotactin, ALP, lungkine, and fractalkine, as well as others known to those skilled in the art. Chemokines are involved in the migration of immune cells to sites of inflammation as well as in the maturation of immune cells and in the development of adaptive immune responses.

[0114] As used herein, bacteria modified to "induce less cell death of tumor-resident immune cells" or "induce less cell death of immune cells" are those that are less toxic than bacteria without the modification or have reduced virulence compared to bacteria without the modification. Exemplary of such modifications are those that reduce / eliminate pyroptosis and those that alter the lipopolysaccharide (LPS) profile of the bacteria. These modifications include one or more of the following: disruption or deletion of the flagellin gene, pagP, or one or more components of the SPI-1 pathway, such as hilA, rod protein, needle protein, and QseC.

[0115] As used herein, a bacterium "modified to preferentially infect tumor-resident immune cells" or "modified to preferentially infect immune cells" has a modification in its genome that reduces its ability to infect cells other than immune cells. Exemplary of such modifications are modifications that disrupt the type 3 or type 4 secretion apparatus, or other genes or apparatus that affect the ability of bacteria to invade non-immune cells. For example, disruption / deletion of SPI-1 components, which are not required for Salmonella infection of cells such as epithelial cells and do not affect infection of immune cells such as phagocytes.

[0116] As used herein, "modification" refers to alterations to the sequence of amino acids in a polypeptide or the sequence of nucleotides in a nucleic acid molecule, including deletions, insertions, and substitutions of amino acids or nucleotides, respectively. Methods for modifying polypeptides are routine for those skilled in the art, such as by using recombinant DNA methodologies.

[0117] As used herein, modifications to the bacterial genome or to a plasmid or gene include nucleic acid deletions, substitutions and insertions.

[0118] As used herein, RNA interference (RNAi) is a biological method in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules and inhibiting translation, thereby inhibiting expression of the targeted gene.

[0119] As used herein, the RNA molecules that act through RNAi are called inhibitory based on their silencing of the expression of targeted gene.Silencing expression means that the expression of targeted gene is reduced or suppressed or inhibited.

[0120] As used herein, RNAi-mediated gene silencing is said to inhibit, suppress, disrupt or silence the expression of targeted gene.Targeted gene comprises the nucleotide sequence corresponding to the sequence in inhibitory RNA, and thus inhibitory RNA silences the expression of mRNA.

[0121] As used herein, inhibiting, suppressing, disrupting, or silencing a targeted gene refers to a method of altering the expression, such as translation, of a targeted gene, thereby reducing the activity or expression of the product encoded by the targeted gene. Reduction includes complete or partial knockout, and is therefore performed in relation to the immunostimulatory bacteria and administration provided herein.

[0122] As used herein, small interfering RNA (siRNA) is a small piece of double-stranded (ds) RNA, usually about 21 nucleotides long, with a 3' overhang (2 nucleotides) at each end, which can be used to bind to messenger RNA (mRNA) at specific sequences and promote its degradation, thereby "interfering" with protein translation.By doing so, based on the nucleotide sequence of their corresponding mRNA, siRNA can prevent the production of specific proteins.This process is called RNA interference (RNAi), also called siRNA silencing or siRNA knockdown.

[0123] As used herein, short hairpin RNA or short hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn, which can be used to silence the expression of target genes through RNA interference (RNAi).The expression of shRNA in cells is typically achieved by delivering plasmid or through viral or bacterial vectors.

[0124] As used herein, the tumor microenvironment (TME) refers to the cellular environment in which a tumor resides, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). Conditions present include, but are not limited to, increased angiogenesis, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure, and metabolites or metabolic changes such as elevated levels of adenosine, which are indicative of tumor growth.

[0125] As used herein, human type I interferon (IFN) is a subgroup of interferon proteins that regulate the activity of the immune system. All type I IFNs bind to specific cell surface receptor complexes, such as the IFN-α receptor. Type I interferons include, among others, IFN-α and IFN-β. IFN-β proteins are produced by fibroblasts and have antiviral activity that is primarily involved in the innate immune response. Two types of IFN-β are IFN-β1 (IFNB1) and IFN-β3 (IFNB3).

[0126] As used herein, the description that nucleic acid or the RNA that it encodes targets a gene means that it inhibits, suppresses or silences the expression of the gene by any mechanism.Generally, such nucleic acid comprises at least the complementary part of the targeted gene, and this part is sufficient to form a hybrid with the complementary part.

[0127] As used herein, a "deletion," when referring to a nucleic acid or polypeptide sequence, refers to the deletion of one or more nucleotides or amino acids compared to a sequence such as a target polynucleotide or polypeptide or native or wild-type sequence.

[0128] As used herein, "insertion," when referring to a nucleic acid or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids within a target, native, wild-type, or other related sequence. Thus, a nucleic acid molecule that contains one or more insertions contains one or more additional nucleotides within the linear length of the sequence compared to the wild-type sequence.

[0129] As used herein, "addition" to nucleic acid and amino acid sequences describes the addition of nucleotides or amino acids onto either end compared to another sequence.

[0130] As used herein, " substitution " or " replacement " refers to the replacement of one or more nucleotides or amino acids with alternative nucleotides or amino acids in a native, target, wild-type or other nucleic acid or polypeptide sequence, without changing the length of the molecule (as described in the number of residues).Therefore, one or more substitutions in a molecule do not change the number of amino acid residues or nucleotides of the molecule.Amino acid replacement can be expressed in terms of the number of amino acid residues along the length of a polypeptide sequence, compared to a specific polypeptide.

[0131] As used herein, the phrase "at a corresponding position," or a nucleotide or amino acid position that "corresponds to" a nucleotide or amino acid position in a disclosed sequence as shown in a sequence listing, refers to a nucleotide or amino acid position identified in an alignment with the disclosed sequence using a standard alignment algorithm, such as the GAP algorithm, to maximize identity. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide. Generally, to identify corresponding positions, amino acid sequences are aligned to obtain the highest order match (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAM J Applied Math 48:1073).

[0132] As used herein, sequence alignment refers to the use of homology to align two or more sequences of nucleotides or amino acids. Typically, two or more sequences related by 50% or more identity are aligned. An aligned set of sequences refers to two or more sequences aligned at corresponding positions and can include aligned sequences from RNA, such as ESTs, and other cDNAs aligned with genomic DNA sequences. Related or variant polypeptide or nucleic acid molecules may be aligned by any method known to those skilled in the art. Such methods typically maximize matches, including manual alignment, using numerous available alignment programs (e.g., BLASTP), and others known to those skilled in the art. By aligning polypeptide or nucleic acid sequences, those skilled in the art can identify similar portions or positions using conserved and identical amino acid residues as a guide. Furthermore, those skilled in the art can also use conserved amino acid or nucleotide residues as a guide to find corresponding amino acid or nucleotide residues between human and non-human sequences. Corresponding positions can also be based on structural alignment, for example, by using computer-simulated protein structure alignments. In other instances, corresponding regions can be identified. Those skilled in the art can also use conserved amino acid residues as a guide to find corresponding amino acid residues between human and non-human sequences.

[0133] As used herein, the "property" of a polypeptide, such as an antibody, refers to any characteristic exhibited by the polypeptide, including, but not limited to, binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, thermotolerance, and tolerance to pH conditions. A change in the property can alter the "activity" of the polypeptide. For example, a change in the binding specificity of an antibody polypeptide can alter the ability to bind an antigen and / or various binding activities, such as affinity or avidity, or the in vivo activity of the polypeptide.

[0134] As used herein, "activity" or "functional activity" of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. Such activities can be empirically determined. Exemplary activities include, but are not limited to, the ability to interact with a biomolecule, for example, through antigen binding, DNA binding, ligand binding, or dimerization, or enzymatic activity, such as kinase activity or proteolytic activity. With respect to antibodies (including antibody fragments), activities include, but are not limited to, the ability to specifically bind a particular antigen, the affinity of antigen binding (e.g., high or low affinity), the avidity of antigen binding (e.g., high or low avidity), the on-rate, the off-rate, effector functions such as antigen neutralization or clearance, the ability to promote virus neutralization, and in vivo activities such as the ability to prevent pathogen infection or invasion or promote clearance, or to penetrate specific tissues, fluids, or cells within the body. Activity can be assessed in vitro or in vivo using recognized assays such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays measuring binding or dissociation rates, immunohistochemistry and immunofluorescence histology and microscopy cell-based assays, flow cytometry, and binding assays (e.g., panning assays).

[0135] As used herein, "binding," "bonding," or grammatical variations thereof, refers to the participation of a molecule with another molecule in any attractive interaction, resulting in a stable association in which the two molecules are in close proximity to one another. Bonding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and intermolecular interactions such as chemical compounds, including, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, e.g., drugs.

[0136] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic, such as those produced recombinantly, including any fragment thereof that contains at least a portion of the variable heavy and light chain regions of an immunoglobulin molecule sufficient to form an antigen-binding site and, when assembled, specifically bind to an antigen. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). For example, an antibody refers to an antibody comprising two heavy chains (which can be designated H and H') and two light chains (which can be designated L and L'), where each heavy chain can be a full-length immunoglobulin heavy chain or a sufficient portion thereof to form an antigen-binding site (e.g., heavy chains include, but are not limited to, VH, VH-CH1, and VH-CH1-CH2-CH3), and each light chain can be a full-length light chain or a sufficient portion thereof to form an antigen-binding site (e.g., light chains include, but are not limited to, VL and VL-CL). Each heavy chain (H and H') is paired with one light chain (L and L', respectively). Typically, an antibody minimally comprises all or at least a portion of a variable heavy (VH) chain and / or a variable light (VL) chain. An antibody can also comprise all or a portion of a constant region.

[0137] For purposes herein, the term antibody includes full-length antibodies and portions thereof, including antibody fragments such as anti-EGFR antibody fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, nanobodies (camelid antibodies), fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fd' fragments, single-chain Fvs (scFvs), single-chain Fabs (scFabs), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies also include synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intracellular antibodies. The antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subsubclass (e.g., IgG2a and IgG2b). Antibodies for human therapeutic use are generally human or humanized.

[0138] As used herein, "antibody fragment" refers to (i) monovalent and monospecific antibody derivatives that contain the variable heavy and / or light chains of an antibody or functional fragments thereof, but lack the Fc portion; and (ii) BiTEs (tandem scFvs), DARTs, diabodies, and single-chain antibodies (scDBs). Thus, antibody fragments include Fab, Fab', scFab, scFv, Fv fragments, nanobodies (e.g., antibodies derived from Camelus bactriamus, Calelus dromaderius, or Lama paccos) (see, e.g., U.S. Patent No. 5,759,808; and Stijlemans et al. (2004) J. Biol. Chem. 279:1256-1261), VHH dAbs, minimal recognition units, single-chain diabodies (scDbs), BiTEs, and DARTs. The listed antibody fragments have a molecular weight of less than 60 kDa.

[0139] As used herein, "nucleic acid" refers to at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically joined together by a phosphodiester bond. Nucleic acid analogs, such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof, are also included in the term "nucleic acid." Nucleic acids also include DNA and RNA derivatives containing, for example, nucleotide analogs or "backbone" bonds other than phosphodiester bonds, such as phosphotriester, phosphoramidate, phosphorothioate, thioester, or peptide bonds (peptide nucleic acids). The term also includes, as equivalents, derivatives, variants, and analogs of either RNA or DNA made from nucleotide analogs, single-stranded (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. With respect to RNA, the uracil base is uridine.

[0140] As used herein, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular materials, or culture medium if produced by recombinant technology, or substantially free of chemical precursors, or other chemicals if chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

[0141] As used herein, "operably linked" in reference to a nucleic acid sequence, region, element, or domain means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, such that the nucleic acids can be transcribed, translated, and express a functional fusion protein, with the leader peptide resulting in the secretion of the fusion polypeptide. In some instances, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) can be operably linked to a nucleic acid encoding a second polypeptide, such that the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript may result in the expression of one of two polypeptides. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce a fusion protein containing either the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter controls or mediates transcription of the nucleic acid.

[0142] As used herein, "synthetic," for example in connection with a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0143] As used herein, naturally occurring α-amino acid residues are those 20 α-amino acid residues found in nature, which are incorporated into proteins in humans with their cognate mRNA codons through specific recognition of a charged tRNA molecule.

[0144] As used herein, a "polypeptide" refers to two or more covalently joined amino acids. The terms "polypeptide" and "protein" are used interchangeably herein.

[0145] As used herein, "peptide" refers to a polypeptide that is from 2 to about 40 or 40 amino acids in length.

[0146] As used herein, "amino acid" refers to an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, the amino acids contained in the provided antibodies include the 20 naturally occurring amino acids (see the table below), unnatural amino acids, and amino acid analogs (e.g., amino acids in which the α-carbon has a side chain). As used herein, the amino acids present in the various amino acid sequences of the polypeptides appearing herein are identified according to their well-known three-letter or one-letter abbreviations (see the table below). Nucleotides present in various nucleic acid molecules and fragments are designated using standard single-letter designations commonly used in the art.

[0147] As used herein, "amino acid residue" refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide bonds. Amino acid residues described herein are generally in the "L" isomeric form. Residues in the "D" isomeric form can be substituted for any L-amino acid residue as long as the desired functional properties are retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide. In adherence to standard polypeptide nomenclature as set forth in J. Biol. Chem., 243:3557-59 (1968) and adopted in Federal Regulations 1.821-1.822 on Patents, Trademarks, and Copyrights, abbreviations for amino acid residues are set forth in the following table:

[0148] [Table 1]

[0149] All sequences of amino acid residues represented by formulas herein have a left-to-right orientation in the conventional direction from amino terminus to carboxyl terminus. The phrase "amino acid residue" is defined to include the amino acids listed in the Table of Correspondence above, including modified, non-natural, and unusual amino acids. A dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, or to an amino-terminal group such as NH2, or to a carboxyl-terminal group such as COOH.

[0150] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0151] Such substitutions can be made in accordance with the exemplary substitutions shown in the table below.

[0152] [Table 2]

[0153] Other substitutions are also permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.

[0154] As used herein, "naturally occurring amino acids" refers to the 20 L-amino acids that occur in polypeptides.

[0155] As used herein, the term "unnatural amino acid" refers to an organic compound that has a structure similar to a natural amino acid, but is structurally modified to mimic the structure and reactivity of a natural amino acid. Thus, unnatural amino acids include, for example, amino acids or amino acid analogs other than the 20 naturally occurring amino acids, including, but not limited to, D-stereoisomers of amino acids. Exemplary unnatural amino acids are known to those of skill in the art and include, but are not limited to, 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β-alanine / β-aminopropionic acid (Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid / piperidine acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2′-diaminopimelic acid (Dpm). , 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), allohydroxylysine (Ahyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), alloisoleucine (Aile), N-methylglycine, sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle), and ornithine (Orn).

[0156] As used herein, a DNA construct is a single- or double-stranded, linear or circular DNA molecule that contains segments of DNA linked and arranged in a manner not found in nature. DNA constructs exist as a result of human manipulation and include clones and other copies of engineered molecules.

[0157] As used herein, a DNA segment is a portion of a larger DNA molecule having specific attributes. For example, a DNA segment encoding a specific polypeptide is a portion of a long DNA molecule, such as a plasmid or plasmid fragment, that encodes the amino acid sequence of the specific polypeptide when read in the 5' to 3' direction.

[0158] As used herein, the term polynucleotide refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in units of nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The term nucleotide is used in reference to single- and double-stranded molecules where the context allows. When the term is applied to a double-stranded molecule, it is used to indicate the overall length and will be understood to be equivalent to the term base pair. Those skilled in the art will recognize that the two strands of a double-stranded polynucleotide may differ slightly in length and their ends may be staggered, and therefore, not all nucleotides within a double-stranded polynucleotide molecule may be paired. Such unpaired ends will generally not exceed 20 nucleotides in length.

[0159] As used herein, recombinant production refers to the use of well-known methods of molecular biology to express proteins encoded by cloned DNA.

[0160] As used herein, a "heterologous nucleic acid" is a nucleic acid that encodes a product (i.e., RNA and / or protein) not normally produced in vivo by the cell in which it is expressed, or a nucleic acid at a locus where it is not normally present, or a nucleic acid that encodes an agent that mediates or alters the expression of an endogenous nucleic acid, such as DNA, by affecting transcription, translation, or other regulatable biochemical processes. Heterologous nucleic acids, such as DNA, are also referred to as foreign nucleic acids. Any nucleic acid, such as DNA, that one of skill in the art would recognize or consider heterologous or foreign to the cell in which it is expressed is encompassed herein by heterologous nucleic acid, and heterologous nucleic acids include exogenously added nucleic acids that are also expressed endogenously. A heterologous nucleic acid is generally not endogenous to the cell in which it is introduced but obtained from another cell or prepared synthetically, or it is introduced into a non-naturally occurring locus, or its expression is under the control of regulatory sequence(s) different from the native regulatory sequence(s).

[0161] Examples of heterologous nucleic acids herein include, but are not limited to, nucleic acids encoding RNAi, or nucleic acids encoding immunostimulatory proteins such as cytokines that confer or contribute to anti-tumor immunity in the tumor microenvironment, or nucleic acids encoding antibodies, antibody fragments, or other therapeutic products or proteins. In immunostimulatory bacteria, heterologous nucleic acids are generally encoded on an introduced plasmid, but may also be introduced into the bacterial genome, such as a promoter that modifies the expression of bacterial products. Heterologous nucleic acids, such as DNA, include nucleic acids that can mediate in some way the expression of DNA encoding a therapeutic product, or can encode products, such as peptides or RNA, that directly or indirectly mediate in some way the expression of a therapeutic product.

[0162] As used herein, cell therapy includes the delivery of cells to a subject to treat a disease or condition. The cells, which may be allogeneic or autologous, are modified ex vivo to deliver or express a product when they are introduced into a subject, for example, by infecting the cells with an immunostimulatory bacterium provided herein.

[0163] As used herein, gene therapy involves the transfer of heterologous nucleic acid, such as DNA, into certain cells, such as target cells of a mammal, particularly a human, having a disorder or condition for which such therapy is sought. The nucleic acid, such as DNA, is introduced into the selected target in such a way that the heterologous nucleic acid, such as DNA, is expressed and produces the encoded therapeutic product(s). Gene therapy can also be used to deliver nucleic acids encoding gene products that replace defective genes or complement gene products produced by the mammal or cell into which it is introduced. The introduced nucleic acid can encode a therapeutic compound, e.g., a growth factor or its inhibitor, or a tumor necrosis factor or its inhibitor, such as its receptor, that is not normally produced in the mammalian host, or not produced in therapeutically effective amounts or at a time useful for treatment. Heterologous nucleic acid, such as DNA, encoding a therapeutic product can be modified to enhance or otherwise alter the product or its expression prior to introduction into the cells of the affected host. Gene therapy can also include the delivery of inhibitors or repressors or other modulators of gene expression.

[0164] As used herein, " expression " refers to the method by which polypeptide is produced by transcription and translation of polynucleotide.The expression level of polypeptide can be evaluated by any method known in the art, including, for example, the method of determining the amount of polypeptide produced from host cell.Such method can include, but is not limited to, ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay and Bradford protein assay to quantify polypeptide in cell lysate.

[0165] As used herein, a "host cell" is a cell used to house, maintain, reproduce, and / or propagate a vector. Host cells can also be used to express a polypeptide encoded by a vector. The nucleic acid contained in the vector is replicated when the host cell divides, resulting in amplification of the nucleic acid.

[0166] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to a vector typically includes vectors into which a nucleic acid encoding a polypeptide, or a fragment thereof, can be introduced by restriction digestion and ligation. Reference to a vector also includes vectors containing a nucleic acid encoding a polypeptide, such as a modified anti-EGFR antibody. A vector is used to introduce a nucleic acid encoding a polypeptide into a host cell to amplify the nucleic acid or to express / display the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but can be designed to integrate a gene or portion thereof into a chromosome of the genome. Vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles are well known to those skilled in the art. Vectors also include "viral vectors" or "viral vectors." Viral vectors are genetically engineered viruses that are operably linked to exogenous genes and transfer the exogenous genes into cells (as vehicles or shuttles).

[0167] As used herein, "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences and may also include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or 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 an appropriate host cell, results in expression of cloned DNA. Suitable expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic and / or prokaryotic cells and those that remain episomal or integrate into the host cell genome.

[0168] As used herein, "primary sequence" refers to the sequence of amino acid residues in a polypeptide or the sequence of nucleotides in a nucleic acid molecule.

[0169] As used herein, "sequence identity" refers to the number of identical or similar amino acids or nucleotide bases compared between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by aligning nucleic acid or protein sequences and identifying similar or identical regions. For purposes herein, sequence identity is generally determined by alignment, and identical residues are identified. Alignment can be local or global. Matches, mismatches, and gaps can be identified between the compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of the aligned sequences, allowing identical or similar features to be aligned. Generally, these can be internal and terminal gaps. When gap penalties are used, sequence identity can be determined using no penalty for terminal gaps (e.g., no penalty is applied to terminal gaps). Alternatively, sequence identity can be determined without considering gaps, as the number of identical positions / length of all aligned sequences x 100.

[0170] As used herein, "global alignment" refers to an alignment of two sequences from beginning to end, with each character in each sequence aligned only once. The alignment is produced regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on "global alignment" means that 50% of the residues are the same in the entire alignment of two compared sequences, each 100 nucleotides long. It is understood that global alignment can also be used to determine sequence identity even when the lengths of the aligned sequences are not the same. Differences at the ends of the sequences will be taken into account in determining sequence identity unless "no penalty for end gaps" is selected. Generally, global alignment is used on sequences that share significant similarity over most of their length. An exemplary algorithm for performing global alignment is the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48: 443). Exemplary programs for performing global alignments are publicly available and include the global sequence alignment tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ) and the program available at deepc2.psi.iastate.edu / aat / align / align.html.

[0171] As used herein, "local alignment" refers to aligning two sequences, but only aligning the portions of the sequences that share similarity or identity. Thus, local alignment determines whether a subsegment of one sequence exists in another sequence. If there is no similarity, no alignment will be achieved. Local alignment algorithms include BLAST or the Smith-Waterman algorithm (Adv. Appl. Math. 2: 482 (1981)). For example, 50% sequence identity based on "local alignment" means that in the full sequence alignment of two compared sequences of any length, a 100-nucleotide-long region of similarity or identity has 50% of the residues that are the same in the region of similarity or identity.

[0172] For purposes herein, sequence identity can be determined by standard alignment algorithm programs using the default gap penalty established by each supplier. Default parameters for gap programs include: (1) the unary comparison matrix (including a value of 1 for identity and 0 for non-identity) and weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745, as described in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Whether any two nucleic acid molecules have nucleotide sequences, or any two polypeptides have amino acid sequences, that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical," or other similar variations describing percentage identity, can be determined using known computer algorithms based on local or global alignments (see, e.g., wikipedia.org / wiki / Sequence_alignment_software, which provides links to many known, publicly available alignment databases and programs).Generally, for the purposes herein, sequence identity is determined using a computer algorithm based on global alignment, such as the Needleman-Wunsch global sequence alignment tool available from NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (implementing the Huang and Miller algorithm by William Pearson (Adv. Appl. Math. (1991) 12:337-357)); and the program from Xiaoqui Huang available at deepc2.psi.iastate.edu / aat / align / align.html. Typically, the full-length sequences of each polypeptide or nucleotide being compared are aligned over the full length of each sequence in global alignment. Local alignment can also be used when the sequences being compared are substantially the same length.

[0173] Thus, as used herein, the term "identity" refers to a comparison or alignment between a test and a reference polypeptide or polynucleotide. In one non-limiting example, "at least 90% identical to" refers to a percentage identity of 90 to 100% with respect to the reference polypeptide or polynucleotide. For illustrative purposes, assuming that the test and reference polypeptides or polynucleotides are compared to a length of 100 amino acids or nucleotides, identity at a level of 90% or higher indicates that 10% or less (i.e., 10 out of 100) of the amino acids or nucleotides in the test polypeptide or polynucleotide differ from those in the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed throughout the entire length of the amino acid sequence, or they can be clustered at one or more positions of varying lengths up to the maximum allowable length, for example, 10 / 100 amino acid difference (approximately 90% identity). Differences can also be due to deletion or truncation of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. At levels of homology or identity greater than about 85-90%, depending on the length of the sequences being compared, results may be independent of the program and gap parameters set; such high levels of identity can be easily assessed and often do not rely on software.

[0174] As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from a cause or condition, including but not limited to, an infection, an acquired condition, or a genetic condition, and characterized by an identifiable symptom.

[0175] As used herein, "treating" a subject exhibiting a disease or condition means that the subject's symptoms are partially or totally alleviated, or remain unchanged following treatment.

[0176] As used herein, treatment refers to any effect that alleviates the symptoms of a disease or disorder. Treatment includes prevention, therapy and / or cure. Treatment also includes any pharmaceutical use of any immunostimulatory bacteria or compositions provided herein.

[0177] As used herein, prevention refers to the prevention of potential disease and / or the prevention of worsening of symptoms or progression of disease.

[0178] As used herein, "prevention" or prevention, and their grammatical equivalents, refer to a method of reducing the risk or likelihood of developing a disease or condition.

[0179] As used herein, a "pharmaceutically active agent" includes any therapeutic or bioactive agent, including, but not limited to, anesthetics, vasoconstrictors, dispersing agents, and conventional therapeutic agents, including small molecule drugs and therapeutic proteins.

[0180] As used herein, a "therapeutic effect" means an effect resulting from treatment of a subject that alters, typically improves or reverses, the symptoms of a disease or condition, or cures the disease or condition.

[0181] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a drug, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. It is thus the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or disorder.

[0182] As used herein, "therapeutic efficacy" refers to the ability of a drug, compound, material, or composition comprising a compound to produce a therapeutic effect in a subject to which the drug, compound, material, or composition comprising the compound is administered.

[0183] As used herein, "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a drug, compound, material, or composition containing a compound that, when administered to a subject, will have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom, or reducing the occurrence of a viral infection. The maximum preventive effect does not necessarily occur by administering a single dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more doses.

[0184] As used herein, amelioration of symptoms of a particular disease or disorder by treatment, such as by administration of a pharmaceutical composition or other therapeutic agent, refers to any decrease in symptoms, whether permanent or temporary, long-term or transient, that can result from or be associated with administration of the composition or therapeutic agent.

[0185] As used herein, " anticancer agent " refers to any agent that is destructive or toxic to malignant cells and tissues.For example, anticancer agent includes agent that kills cancer cells, or inhibits or impairs the growth of tumor or cancer cells in particular.An exemplary anticancer agent is a chemotherapeutic agent.

[0186] As used herein, "therapeutic activity" refers to the in vivo activity of a therapeutic polypeptide. Generally, a therapeutic activity is an activity associated with the treatment of a disease or condition.

[0187] As used herein, the term "subject" refers to animals, including mammals such as humans.

[0188] As used herein, a patient refers to a human subject.

[0189] As used herein, animals include, but are not limited to, primates, including humans, gorillas, and monkeys; rodents, such as mice and rats; poultry, such as chickens; ruminants, such as goats, cows, deer, and sheep; pigs and other animals. Non-human animals exclude humans as animals considered. Polypeptides provided herein are from any source, including animals, plants, prokaryotes, and fungi. Most polypeptides are of animal origin, including mammalian origin.

[0190] As used herein, a "composition" refers to any mixture, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0191] As used herein, a "combination" refers to any association between or among two or more items. The combination can be two or more individual items, such as two compositions or two collections, a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are generally functionally related or associated.

[0192] As used herein, combination therapy refers to the administration of two or more different therapeutic substances. The different therapeutic substances or agents can be provided and administered separately, sequentially, intermittently, or can be provided in a single composition.

[0193] As used herein, a kit is a packaged combination for purposes including, but not limited to, activation, administration, diagnosis, and evaluation of a biological activity or property, and may include other elements such as additional reagents and instructions for using the combination or these elements.

[0194] As used herein, "unit dosage form" refers to physically discrete units suitable for human and animal subjects and individually packaged as known in the art.

[0195] As used herein, "single dosage formulation" refers to a formulation for direct administration.

[0196] As used herein, the term "multi-dose formulation" refers to a formulation that contains multiple doses of a therapeutic agent, and can be administered directly to provide several single doses of the therapeutic agent.Doses can be administered over minutes, hours, weeks, days, or months.Multi-dose formulations can allow for dose adjustment, dose pooling, and / or dose division.Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.

[0197] As used herein, an "article of manufacture" is a product that is manufactured and sold. As used throughout this application, this term is intended to encompass any composition provided herein that is contained in a package.

[0198] As used herein, "fluid" refers to any composition that can flow. Fluids therefore encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0199] As used herein, an isolated or purified polypeptide or protein (e.g., an isolated antibody or antigen-binding fragment thereof), or biologically active portion thereof (e.g., an isolated antigen-binding fragment), is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. A preparation can be determined to be substantially free if it appears to be free of readily detectable impurities as determined by standard analytical methods used by those skilled in the art to assess its purity, such as thin-layer chromatography (TLC), gel electrophoresis, and high-performance liquid chromatography (HPLC), or is sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as the enzymatic and biological activity, of the substance. Methods for purifying compounds and producing substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound can be a mixture of stereoisomers. In such cases, further purification may increase the specific activity of the compound. As used herein, "cell extract" or "lysate" refers to a preparation or fraction made from lysed or perturbed cells.

[0200] As used herein, "control" refers to a sample that is substantially identical to the test sample, except that it may be from a healthy volunteer not treated with the test parameter or, if it is a plasma sample, not suffering from the condition of interest. A control may also be an internal control.

[0201] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide comprising "an immunoglobulin domain" includes one or more polypeptides that are immunoglobulin domains.

[0202] As used herein, the term "or" is used to mean "and / or" unless expressly indicated to refer only to alternatives or where the alternatives are mutually exclusive.

[0203] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Thus, "about 5 amino acids" means "about 5 amino acids" and also "5 amino acids."

[0204] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when said event or circumstance occurs and instances when said event or circumstance does not occur. For example, a moiety that may be variant means that the part is variant or non-variant.

[0205] Abbreviations for any protecting groups, amino acids and other compounds used herein follow their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 11(9):1726-1732), unless otherwise indicated.

[0206] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0207] B. Overview of Immunostimulatory Bacteria Provided herein are modified bacteria, referred to as immunostimulatory bacteria, which encode therapeutic products, such as antitumor agents, immunostimulatory proteins, and inhibitory RNAs (RNAi), such as shRNAs and microRNAs (miRNAs), that accumulate and / or replicate in tumors, tumor-resident immune cells, and / or tumor microenvironment (TME) (i.e., colonize therein); and / or induce less cell death in tumor-resident immune cells; and / or target genes whose inhibition, suppression, or silencing achieves tumor therapy. The bacterial strains used for modification are all suitable for therapeutic use. Provided herein are modified immunostimulatory bacteria for use and methods for treating cancer. The bacteria are modified for such uses and methods.

[0208] The immunostimulatory bacteria provided herein are modified by deleting or modifying bacterial genes to attenuate their inflammatory response, increase their tolerance, increase their resistance to complement, increase their infectivity, accumulation, and colonization in tumors, tumor-resident immune cells, and / or TME, reduce the induction of immune cell death (e.g., reduce pyroptosis), and enhance anti-tumor immune responses in hosts treated with the bacteria. The modification can be modification of a gene encoded on a plasmid in the bacteria. For example, the bacteria can be adenosine auxotrophic or adenosine and adenine auxotrophic, and the bacteria contain a plasmid encoding a therapeutic product, such as an immunostimulatory protein, an antibody, or an RNAi that inhibits immune checkpoint genes in the host. Attenuation of the inflammatory response to the bacteria can be achieved by deleting the msbB gene, which reduces TNF-alpha in the host, and / or knocking out the flagellin gene, and / or deleting or mutating pagP. The bacteria are modified to stimulate host anti-tumor activity, for example, by adding plasmids encoding immunostimulatory proteins such as cytokines, chemokines, and costimulatory molecules, or plasmids encoding RNAi that target host immune checkpoints, and by adding nucleic acids with CpG / CpG motifs.

[0209] Bacterial strains can be attenuated strains, or strains attenuated by standard methods or by modifications provided herein such that their ability to colonize is primarily restricted to immune-privileged tissues and organs, particularly immune and tumor cells, including solid tumors. Examples of bacteria include, but are not limited to, strains of the genera Salmonella, Shigella, Listeria, E. coli, and Bifidobacterium. Examples of bacterial species include Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, and Salmonella enteritidis. Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix.For example, Rickettsia Rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Franciesella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus sornnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, and Agrobacterium tumerfacium.

[0210] Bacteria accumulate based on one or more properties, including diffusion, migration, and chemotaxis, into immune-privileged tissues or organs or environments, environments that provide nutrients or other molecules for which they are auxotrophic, and / or environments that contain replicating cells that provide a favorable environment for bacterial invasion and replication. Immunostimulatory bacteria and species that result in such treatments provided herein include species of Salmonella, Listeria, and E. coli.

[0211] The bacteria contain a plasmid encoding a therapeutic product, such as an immunostimulatory protein, or an immune checkpoint inhibitor, or other immunostimulatory or immunosuppressive blocking product. The product includes antibodies, e.g., antibody fragments and nanobodies, RNAi, e.g., one or more short hairpin (sh)RNA construct(s), microRNA, or other RNAi modalities, the expression of which inhibits, suppresses, or disrupts the expression of a target gene or otherwise enhances or reduces an immune response. The therapeutic product is expressed under the control of a eukaryotic promoter, such as an RNA polymerase (RNAP) II or III promoter. Typically, the RNAPIII (also called POLIII) promoter is constitutive and can regulate RNAPII (also called POLII). In some examples, the shRNA targets the gene TREX1 and inhibits its expression.

[0212] In some embodiments, the plasmid can encode multiple therapeutic products, including immunostimulatory proteins such as cytokines, and RNAi molecules such as shRNAs, and antibodies, including nanobodies, that inhibit two or more immune checkpoint genes, e.g., TREX1, PD-L1, VISTA, SIRPα, CTNNB1, TGF-beta, CD47, and / or VEGF, and any others known to those of skill in the art. When multiple therapeutic products are encoded, expression for each is generally under the control of a different promoter.

[0213] Among the bacteria provided herein are bacteria modified to be adenosine auxotrophic. This can be achieved by modifying or deleting genes involved in purine synthesis, metabolism, or transport. For example, disrupting the tsx gene in Salmonella species, such as Salmonella typhi, results in adenosine auxotrophy. Adenosine is immunosuppressive and accumulates at high concentrations in tumors; adenosine auxotrophy improves the antitumor activity of the bacteria, as bacteria selectively replicate in tissues rich in adenosine.

[0214] Bacteria modified to have a defective asd gene are also provided. These bacteria for in vivo use are modified to carry a functional asd gene on an introduced plasmid; this maintains selection for the plasmid so that an antibiotic-based plasmid maintenance / selection system is not required. Also provided is the use of asd-deficient strains that are genetically engineered to not contain a functional asd gene on a plasmid and thus autolyze within the host.

[0215] Also provided are bacteria modified so that they are unable to produce flagella. This can be achieved by modifying the bacteria with a deletion of a gene encoding a flagellin subunit. Modified bacteria lacking flagellin are less inflammatory and therefore better tolerated, and induce stronger anti-tumor responses.

[0216] Also provided are bacteria engineered to produce listeriolysin O (LLO), which improves plasmid delivery in phagocytic cells.

[0217] Also provided are bacteria modified to harbor low-copy CpG-containing plasmids, which may further comprise other modifications and may encode therapeutic products such as immunostimulatory proteins, antibodies and fragments thereof, and RNAi.

[0218] If the bacterium is a Salmonella species, the bacterium can also be engineered to grow in a manner that reduces expression of the virulence SPI-1 (Salmonella pathogenicity island-1) gene. In Salmonella, the genes responsible for virulence, invasion, survival, and extraintestinal spread are located in the Salmonella pathogenicity island (SPI).

[0219] The bacteria contain a plasmid encoding an RNAi, such as an shRNA or microRNA, that inhibits a checkpoint, such as PD-L1 or TREX1 alone, or TREX1 and one or more of a second immune checkpoint. The bacteria can be further modified for other desirable characteristics, including selection for maintaining the plasmid, particularly for preparing strains without antibiotics. The immunostimulatory bacteria may also encode therapeutic polypeptides, including anti-tumor therapeutic polypeptides and drugs.

[0220] Exemplary of the immunostimulatory bacteria provided herein are species of the genus Salmonella. Exemplary of bacteria for modification as described herein are genetically engineered strains of Salmonella typhimurium, such as strain YS1646 (ATCC Catalog #202165; also referred to as VNP20009, see WO 99 / 13053), which are plasmid-engineered to complement the asd gene knockout and antibiotic-free plasmid maintenance.

[0221] Modified immunostimulatory bacterial strains that have been conferred adenosine auxotrophy are provided herein, as are pharmaceutical compositions comprising such strains formulated for administration to subjects, such as humans, for use in methods of treating tumors and cancer.

[0222] Also provided are methods or uses for immunostimulatory bacteria or pharmaceutical compositions containing bacteria, in which the treatment includes a combination therapy in which a second anti-cancer agent or treatment is administered. The second anti-cancer agent or treatment can be administered before, simultaneously with, after, or intermittently with the immunostimulatory bacteria or pharmaceutical composition, and includes immunotherapy, such as antibodies or antibody fragments; oncolytic viruses; radiation / radiotherapy; and chemotherapy. The immunotherapy can include, for example, the administration of anti-PD-1 or anti-PD-L1 or anti-CTLA4 or anti-IL6 or anti-VEGF or anti-VEGFR or anti-VEGFR2 antibodies, or fragments thereof. The combination therapy can also include surgery.

[0223] The genetically engineered immunostimulatory bacteria provided herein incorporate multiple synergistic modalities to induce immune reactivation of cold tumors and promote tumor antigen-specific immune responses, while simultaneously inhibiting immune checkpoint pathways that tumors exploit to subvert and evade persistent antitumor immunity. Improved tumor targeting via adenosine auxotrophy and enhanced vascular perturbation improve efficacy while localizing inflammation and limiting systemic cytokine exposure and autoimmune toxicity observed with other immunotherapy modalities. Exemplary of such modified bacteria are S. typhimurium strains, including strain YS1646, specifically asd - Such modifications of the strain are included.

[0224] For example, immunostimulatory bacteria are provided herein that provide shRNA-mediated gene disruption of PD-L1. Gene disruption of PD-L1 has been shown to improve tumor colonization in mice. For example, it has been shown that S. typhimurium infection in PD-L1 knockout mice leads to a bacterial burden 10 times higher than that in wild-type mice (see Lee et al. (2010) Immunol. 185:2442-2449). Thus, PD-L1 is protective against S. typhimurium infection. Provided herein are immunostimulatory bacteria, such as S. typhimurium, carrying plasmids capable of RNAi-mediated gene knockdown of TREX1, PD-L1, or PD-L1 and TREX1. Such bacteria provide anti-tumor effects due to the combination of two independent pathways, resulting in an enhanced and sustained anti-tumor immune response in a single treatment.

[0225] C. Cancer immunotherapy The immunosuppressive environment found within the tumor microenvironment (TME) is a driver of tumor initiation and progression. Cancer emerges after the immune system fails to control and contain the tumor. Multiple tumor-specific mechanisms create a tumor environment that forces the immune system to tolerate tumors and their cells instead of eliminating them. The goal of cancer immunotherapy is to aid the immune system's natural ability to eliminate tumors. Acute inflammation associated with microbial infections has been linked to spontaneous tumor regression based on centuries of observation.

[0226] 1. Immunotherapy Several clinical cancer immunotherapies seek to disrupt the balance of immunosuppression against antitumor immunity. Strategies to stimulate immunity through direct administration of cytokines such as IL-2 and IFN-α have resulted in modest clinical responses in a minority of patients while simultaneously inducing severe systemic inflammation-associated toxicity (Sharma et al. (2011) Nat Rev Cancer 11:805-812). The immune system has evolved several checks and balances to limit autoimmunity, such as the upregulation of programmed cell death protein 1 (PD-1) on T cells and its binding to its cognate ligand, programmed death ligand 1 (PD-L1), expressed on both antigen-presenting cells (APCs) and tumor cells. Binding of PD-L1 to PD-1 interferes with the CD8+ T cell signaling pathway, impairing CD8+ T cell proliferation and effector function and inducing T cell tolerance. PD-1 and PD-L1 are two examples of numerous inhibitory "immune checkpoints," which function by downregulating immune responses. Other inhibitory immune checkpoints include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), signal-regulatory protein α (SIRPα), V-domain Ig suppressor of T-cell activation (VISTA), programmed death-ligand 2 (PD-L2), indoleamine 2,3-dioxygenase (IDO) 1 and 2, lymphocyte-activation gene 3 (LAG3), galectin-9, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and T-cell immunoglobulin and mucin domain-containing 3 (T These include IM-3, also known as hepatitis A virus cellular receptor 2 (HAVCR2), herpesvirus entry mediator (HVEM), CD39, CD73, B7-H3 (also known as CD276), B7-H4, CD47, CD48, CD80 (B7-1), CD86 (B7-2), CD155, CD160, CD244 (2B4), B- and T-lymphocyte attenuator (BTLA, or CD272), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, or CD66a).

[0227] Antibodies designed to block immune checkpoints, such as anti-PD-1 (e.g., pembrolizumab, nivolumab) and anti-PD-L1 (e.g., atezolizumab, avelumab, durvalumab), have been successful in preventing T cell anergy and permanently breaking immune tolerance. Clinical benefit has been demonstrated in only a small proportion of treated patients, and those who often exhibit autoimmune-related toxicity (see, e.g., Ribas (2015) N Engl J Med 373:1490-1492; Topalian et al. (2012) N Engl J Med 366:3443-3447). This is further evidence of the need for more effective and less toxic therapies provided herein.

[0228] Another checkpoint blockade strategy involves binding to and inhibiting costimulatory receptors on APCs, such as CD80 or CD86, and inhibiting the induction of CTLA-4 on T cells, which outcompetes costimulatory cluster differentiation 28 (CD28), which binds to the same receptor but with lower affinity. This blocks stimulatory signals from CD28 and simultaneously transmits inhibitory signals from CTLA-4, preventing T cell activation (see Phan et al. (2003) Proc. Natl. Acad. Sci. USA 100:8372-8377). Anti-CTLA-4 therapy (e.g., ipilimumab) has shown clinical success and durability in some patients, while also showing a higher incidence of severe immune-related adverse events (see, for example, Hodi et al. (2010) N. Engl. J. Med. 363:711-723; Schadendorf et al. (2015) J. Clin. Oncol. 33:1889-1894). Tumors have also been shown to develop resistance to anti-immune checkpoint antibodies, highlighting the need for more durable anti-cancer therapies, which are provided herein.

[0229] 2. Adoptive Immunotherapy In an attempt to revitalize cold tumors and make them more immunogenic, a type of immunotherapy known as adoptive cell therapy (ACT) involves various strategies that utilize immune cells and reprogram them to have anti-tumor activity (Hinrichs et al. (2011) Immunol. Rev. 240:40-51). Dendritic cell-based therapies introduce genetically engineered dendritic cells (DCs) with additional immune stimulatory properties. These therapies have been unsuccessful due to their inability to break immune tolerance to cancer (see, for example, Rosenberg et al. (2004) Nat. Med. 12:1279). A method known as GVAX, which uses whole-body irradiated tumor cells containing endogenous tumor antigens and granulocyte-macrophage colony-stimulating factor (GM-CSF) to stimulate DC recruitment, has similarly failed clinical application due to its inability to break tumor tolerance (Copier et al. (2010) Curr. Opin. Mol. Ther. 12:647-653). Sipuleucel-T (Provenge), a personalized autologous cell-based therapy, was FDA-approved in 2010 for castration-resistant prostate cancer. It utilizes APCs harvested from the patient, rearmed to express the prostatic acid phosphatase (PAP) antigen, stimulate T cell responses, and then reintroduced after lymphectomy. Unfortunately, its widespread adoption has been limited by the low observed objective response rates and high cost, limiting its use to early stages of prostate cancer (Anassi et al. (2011) PT. 36(4):197-202). Similarly, autologous T cell therapy (ATC) involves harvesting a patient's own T cells, reactivating them ex vivo to overcome tumor tolerance, and then reintroducing them into the patient after lymphectomy. ATC has had limited clinical success, primarily in melanoma, and has encountered significant safety and feasibility issues that limit its usefulness (Yee et al. (2013) Clin. Cancer Res. 19:1-3).

[0230] Chimeric antigen receptor T cell (CAR-T) therapy involves patient-derived T cells that are genetically engineered to express a fusion protein between the T cell receptor and an antibody Ig variable extracellular domain. This confers the antigen-recognition properties of an antibody with the cytolytic properties of an activated T cell (Sadelain (2015) Clin. Invest. 125:3392-4000). Success has been limited to B-cell and hematopoietic malignancies, at the cost of fatal immune-related adverse events (Jackson et al. (2016) Nat. Rev. Clin. Oncol. 13:370-383). Tumors may mutate to avoid recognition by target antigens, including CD19 (Ruella et al., (2016) Comput Struct Biotechnol J. 14: 357-362) and EGFRvIII (O'Rourke et al. (2017) Sci Transl Med. Jul 19; 9:399), thereby facilitating immune escape. Furthermore, CAR-T therapies, approved in the context of hematologic malignancies, face a significant hurdle for their feasibility for treating solid tumors: overcoming the highly immunosuppressive nature of the solid tumor microenvironment. Several additional modifications to existing CAR-T therapies will be essential and potentially offer feasibility for solid tumors (Kakarla, et al. (2014) Cancer J. Mar-Apr; 20(2): 151-155). If the safety of CAR-T improves significantly and their efficacy is extended to solid tumors, the feasibility and costs associated with these labor-intensive therapies will continue to limit their widespread adoption.

[0231] 3. Cancer vaccines and oncolytic viruses Cold tumors lack T cell and dendritic cell (DC) infiltration and are non-T cell inflammatory (Sharma et al. (2017) Cell 9; 168(4):707-723). In an attempt to reactivate cold tumors and make them more immunogenic, another type of immunotherapy utilizes microorganisms that can accumulate naturally or engineered in tumors. These include viruses designed to stimulate the immune system, express tumor antigens, and subsequently activate and reprogram the immune system to reject the tumor. Viral-based cancer vaccines have largely failed clinically due to several factors, including pre-existing or acquired immunity to the viral vector itself and a lack of sufficient immunogenicity to present tumor antigens (Larocca et al. (2011) Cancer J. 17(5):359-371). The lack of adequate adjuvant activation of APCs also hampers other non-viral vector cancer vaccines, such as DNA vaccines. In contrast, oncolytic viruses preferentially replicate in dividing tumor cells rather than healthy tissue, and subsequent tumor cell lysis leads to immunogenic tumor cell death and further viral dissemination. The oncolytic virus talimogene laherparepvec (T-VEC), which uses a modified herpes simplex virus in combination with the DC-recruiting cytokine GM-CSF, is FDA-approved for metastatic melanoma (Bastin et al. (2016) Biomedicines 4(3):21). While it has demonstrated clinical benefit in some melanoma patients and less immunotoxicity than other immunotherapies, it suffers from limited intratumoral administration routes and manufacturing requirements, as well as a lack of distal tumor efficacy and broad applicability to other tumor types. Other oncolytic virus (OV)-based vaccines, including those utilizing paramyxoviruses, reoviruses, and picornaviruses, among others, have similar limitations in inducing systemic anti-tumor immunity (Chiocca et al. (2014) Cancer Immunol. Res. 2(4):295-300). Systemic administration of oncolytic viruses presents unique challenges.When administered intravenously, the virus is rapidly diluted, requiring high titers that can lead to liver toxicity. Furthermore, if pre-existing immunity is present, the virus is rapidly neutralized in the blood, and acquired immunity limits repeated dosing (Maroun et al. (2017) Future Virol. 12(4):193-213).

[0232] Among the limitations of virus-based vaccine vectors and oncolytic viruses, the greatest limitation may be the virus itself. Viral antigens have significantly higher affinity for human T cell receptors (TCRs) than tumor antigens (Aleksic et al. (2012) Eur J Immunol. 42(12):3174-3179). Furthermore, tumor antigens simultaneously presented by MHC-1 on the surface of highly active APCs with viral vector antigens will outcompete for binding to TCRs, resulting in very low antigen-specific antitumor immunity. The tumor-targeting immunostimulatory vectors provided herein, which do not themselves confer high-affinity T cell epitopes, can circumvent these limitations.

[0233] D. Bacterial cancer immunotherapy 1. Bacterial therapy The recognition that bacteria have anti-cancer activity dates back to the 1800s, when several physicians observed tumor regression in patients infected with Streptococcus pyogenes. William Coley first began researching the use of bacteria for the treatment of advanced cancers and developed a vaccine composed of S. pyogenes and Serratia marcescens that was successfully used to treat a variety of cancers, including sarcoma, carcinoma, lymphoma, and melanoma. Subsequently, several bacteria, including species of Clostridium, Mycobacterium, Bifidobacterium, Listeria such as L. monocytogenes, and Escherichia, have been investigated as sources of anti-cancer vaccines (see, e.g., WO 1999 / 013053; WO 2001 / 025399; Bermudes et al. (2002) Curr. Opin. Drug Discov. Devel. 5:194-199; Patyar et al. (2010) Journal of Biomedical Science 17:21; Pawelek et al. (2003) Lancet Oncol. 4:548-556).

[0234] Bacteria can infect animal and human cells, and some have the natural ability to deliver DNA into the cytosol of cells, making them candidate vectors for gene therapy. Bacteria are also suitable for therapeutic use because they can be administered orally, are easily propagated in vitro and in vivo, and can be stored and transported in lyophilized form. Bacterial genetic traits are easily manipulated, and the complete genomes of numerous strains have been fully characterized (Felgner et al. (2016) mbio 7(5):e01220-16). Consequently, bacteria have been used to deliver and express a variety of genes, including those encoding cytokines, angiogenesis inhibitors, toxins, and prodrug-converting enzymes. For example, Salmonella has been used to express immune stimulatory molecules like IL-18 (Loeffler et al. (2008) Cancer Gene Ther. 15(12):787-794), LIGHT (Loeffler et al. (2007) Proc. Natl. Acad. Sci. USA 104(31):12879-12883), and Fas ligand (Loeffler et al. (2008) J. Natl. Cancer Inst. 100:1113-1116) in tumors. Bacterial vectors are also less expensive, easier to produce, and can be rapidly cleared by antibiotics if necessary, making bacterial delivery a preferable and safer alternative to viral delivery.

[0235] However, to be useful, the bacterial strain itself must not be pathogenic, or it must not become pathogenic after being modified for use as a therapeutic agent. For example, in cancer treatment, therapeutic bacterial strains must be attenuated or rendered sufficiently non-virulent so that they do not cause systemic disease and / or septic shock, but still maintain some level of infectivity and effectively colonize tumors. Genetically modified bacteria have been described for use as antitumor agents to induce direct tumorigenic effects and / or to deliver tumorigenic molecules (Clairmont, et al. (2000) J. Infect. Dis. 181:1996-2002; Bermudez, D. et al. (2002) Curr. Opin. Drug Discov. Devel. 5:194-199; Zhao, M. et al. (2005) Proc. Natl. Acad. Sci. USA 102:755-760; Zhao, M. et al. (2006) Cancer Res. 66:7647-7652). One of these is a bioengineered strain of Salmonella enterica serovar Typhimurium (S. Typhimurium). These bacteria preferentially accumulate in tumors at over 1,000-fold higher abundance than in healthy tissues and are homogeneously distributed in tumor tissues (Pawelek, J. et al. (1997) Cancer Res. 57:4537-4544; Low, KB et al. (1999) Nat. Biotechnol. 17:37-41). Preferential replication allows the bacteria to produce and deliver high concentrations of various anticancer therapeutic agents directly into tumors, while minimizing toxicity to healthy tissues.These attenuated bacteria are safe in mice, pigs, and monkeys when administered intravenously (Zhao, M. et al. (2005) Proc Natl Acad Sci USA 102:755-760; Zhao, M. et al. (2006) Cancer Res 66:7647-7652; Tjuvajev J. et al. (2001) J. Control Release 74:313-315; Zheng, L. et al. (2000) Oncol. Res. 12:127-135), and certain live attenuated Salmonella strains have been shown to be well tolerated after oral administration in human clinical trials (Chatfield, SN et al. (1992) Biotechnology 10:888-892; DiPetrillo, MD et al. (1999) Vaccine 18:449-459; Hohmann, EL et al. (1996) J. Infect. Dis. 173:1408-1414; Sirard, JC et al. (1999) Immunol. Rev. 171:5-26). The S. typhimurium phoP / phoQ operon is a typical bacterial two-component regulatory system consisting of a membrane-associated sensor kinase (PhoQ) and a cytoplasmic transcriptional regulator (PhoP: Miller, SI et al. (1989) Proc Natl Acad Sci USA 86:5054-5058; Groisman, EA et al. (1989) Proc Natl Acad Sci USA 86: 7077-7081).PhoP / phoQ is essential for virulence, and its deletion results in poor survival of the bacterium in macrophages and marked attenuation in mice and humans (Miller, S. I. et al. (1989) Proc Natl Acad Sci USA 86:5054-5058; Groisman, E. A. et al. (1989) Proc Natl Acad Sci USA 86: 7077-7081; Galan, J. E. and Curtiss, R. III. (1989) Microb Pathog 6:433-443; Fields, P. I. et al. (1986) Proc Natl Acad Sci USA 83:189-193). PhoP / phoQ-deficient strains have been used as effective vaccine delivery vehicles (Galan, JE and Curtiss, R. III. (1989) Microb Pathog 6:433-443; Fields, PI et al. (1986) Proc Natl Acad Sci USA 83:189-193; Angelakopoulos, H. and Hohmann, EL (2000) Infect Immun 68:213-241). Attenuated Salmonella have been used for targeted delivery of oncolytic proteins (Bermudes, D. et al. (2002) Curr Opin Drug Discov Devel 5:194-199; Tjuvajev J. et al. (2001) J Control release 74:313-315).

[0236] Bacterial-based cancer therapies have demonstrated limited clinical benefit. Examples include Clostridium novyi (Dang et al. (2001) Proc. Natl. Acad. Sci. USA 98(26):15155-15160; U.S. Patent Application Publication Nos. 2017 / 0020931, 2015 / 0147315; U.S. Patent Nos. 7,344,710, and 3,936,354), Mycobacterium bovis (U.S. Patent Application Publication Nos. 2015 / 0224151 and 2015 / 0071873), and Bifidobacterium bifidum (Kimura et al. (1980) Cancer Res. 40:2061-2068), Lactobacillus casei (Yasutake et al. (1984) Med Microbiol Immunol. 173(3):113-125), Listeria monocytogenes (Le et al. (2012) Clin. Cancer Res. 18(3):858-868; Starks et al. (2004) J. Immunol. 173:420-427; U.S. Patent Application Publication No. 2006 / 0051380), and Escherichia coli (U.S. Patent No. 9,320,787) have been investigated as potential anticancer therapeutic agents.

[0237] For example, Bacillus Calmette-Guerin (BCG) strains have been approved for the treatment of bladder cancer in humans, are more effective than intravesical chemotherapy agents, and are often used as first-line treatment (Gardlik et al. (2011) Gene therapy 18:425-431). Another approach utilizes the attenuated intracellular bacterium Listeria monocytogenes, which can induce potent CD8+ T cell priming to express tumor antigens in mice (Le et al. (2012) Clin. Cancer Res. 18(3):858-868). In a clinical trial of a Listeria-based vaccine incorporating the tumor antigen mesothelin in a prime-boost approach with the allogeneic pancreatic cancer-based GVAX vaccine, a median survival of 6.1 months was observed in patients with advanced pancreatic cancer, compared with a median survival of 3.9 months for patients treated with the GVAX vaccine alone (Le et al. (2015) J. Clin. Oncol. 33(12):1325-1333). These results were not replicated in a large Phase 2b study, possibly demonstrating the difficulties of attempting to induce immunity to low-affinity self-antigens such as mesothelin.

[0238] Bacterial strains can be modified as described and exemplified herein to express inhibitory RNA (RNAi), such as shRNA and microRNA, that inhibit or disrupt TREX1 and / or PD-L1, and optionally one or more additional immune checkpoint genes. Strains can be attenuated by standard methods and / or by gene deletion or modification, and by altering or introducing genes that enable the bacteria to grow in vivo in primarily immune-privileged environments, such as the TME in tumor cells and solid tumors. Bacterial strains for modification as described herein can be selected from the genera Shigella, Listeria, E. coli, Bifidobacterium, and Salmonella, for example, Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, and Salmonella enteritidis. Other suitable bacterial species include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix.For example, Rickettsia Rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Franciesella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus sornnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, and Agrobacterium tumourfacium. Any known therapy involving immunostimulatory bacteria can be modified as described herein.

[0239] 2. Comparison of immune responses to bacteria and viruses Bacteria, like viruses, have the advantage of being innately immune-stimulating. Bacteria and viruses are known to contain conserved structures known as pathogen-associated molecular patterns (PAMPs), which are sensed by host cell pattern recognition receptors (PRRs). Recognition of PAMPs by PRRs triggers downstream signaling cascades that result in the induction of cytokines and chemokines, and the initiation of immune responses that result in pathogen clearance (Iwasaki and Medzhitov (2010) Science 327(5963):291-295). The manner in which PAMPs engage the innate immune system and from what type of infectious agent determines the appropriate adaptive immune response to block invading pathogens.

[0240] One class of PRRs known as Toll-like receptors (TLRs) recognizes PAMPs of bacterial and viral origin and are located in various compartments within the cell. TLRs bind to a variety of ligands, including lipopolysaccharide (TLR4), lipoproteins (TLR2), flagellin (TLR5), unmethylated CpG motifs in DNA (TLR9), double-stranded RNA (TLR3), and single-stranded RNA (TLR7 and TLR8) (Akira et al. (2001) Nat. Immunol. 2(8):675-680; Kawai and Akira (2005) Curr. Opin. Immunol. 17(4):338-344). For example, S. typhimurium host surveillance is largely mediated through TLR2, TLR4, and TLR5 (Arpaia et al. (2011) Cell 144(5):675-688). These TLR signals, via MyD88 and TRIF adaptor molecules, to mediate the induction of NF-kB, depend on pro-inflammatory cytokines such as TNF-α, IL-6, and IFN-γ (Pandey et al. (2015) Cold Spring Harb Perspect Biol 7(1):a016246).

[0241] Another category of PRRs is the nod-like receptor (NLR) family. These receptors reside in the cytosol of host cells and recognize intracellular PAMPs. For example, S. typhimurium flagellin was shown to activate the NLRC4 / NAIP5 inflammasome pathway, leading to the cleavage of caspase-1 and the induction of the pro-inflammatory cytokines IL-1β and IL-18, which then triggered cell death by pyroptosis in infected macrophages (Fink et al. (2007) Cell Microbiol. 9(11):2562-2570).

[0242] Engagement of TLR2, TLR4, TLR5, and inflammasomes induces pro-inflammatory cytokines that mediate bacterial clearance and primarily activates NF-κB-driven signaling cascades, leading to the recruitment and activation of neutrophils, macrophages, and CD4+ T cells, but not DCs and CD8+ T cells, which are essential for antitumor immunity (Lui et al. (2017) Signal Transduct Target Ther. 2:17023). To activate CD8+ T cell-mediated antitumor immunity, IRF3 / IRF7-dependent type I interferon signaling is important for DC activation, cross-presentation of tumor antigens, and CD8+ T cell priming (Diamond et al. (2011) J. Exp. Med. 208(10):1989-2003; Fuertes et al. (2011) J. Exp. Med. 208(10):2005-2016). Type I interferons (IFN-α, IFN-β) are signature cytokines induced by two distinct TLR-dependent and TLR-independent signaling pathways. The TLR-dependent pathway for IFN-β induction occurs after pathogen endocytosis, and TLRs 3, 7, 8, and 9 detect pathogen-derived DNA and RNA elements within endosomes. TLR7 and 8 recognize viral nucleosides and nucleotides, and synthetic agonists such as resiquimod and imiquimod have been clinically validated (Chi et al. (2017) Frontiers in Pharmacology 8:304). Synthetic dsRNAs such as polyinosinic polycytidylic acid (poly(I:C)) and poly(ICLC), analogs formulated with poly-L-lysine to resist RNase digestion, are agonists of the TLR3 and MDA5 pathways and potent inducers of IFN-β (Caskey et al. (2011) J. Exp. Med. 208(12):2357-66). TLR9 detection of endosomal CpG motifs present in viral and bacterial DNA can also induce IFN-β via IRF3.Furthermore, TLR4 has been shown to induce IFN-β through MyD88-independent TRIF activation of IRF3 (Owen et al. (2016) mBio.7:1 e02051-15). It was subsequently shown that the ability of TLR4 to activate DCs via type I IFNs is likely biologically irrelevant, as TLR4 activation of DCs was independent of type I IFNs (Hu et al. (2015) Proc. Natl. Acad. Sci. USA 112:45). Furthermore, TLR4 signaling has not been shown to directly recruit or activate CD8+ T cells.

[0243] One TLR-independent type I IFN pathway is mediated by host recognition of single-stranded (ss) and double-stranded (ds) RNA in the cytosol. These are sensed by RNA helicases, including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), and through the adaptor protein-mediated phosphorylation of the IRF-3 transcription factor IFN-β promoter stimulator 1 (IPS-1; also known as mitochondrial antiviral signaling protein or MAVS), leading to the induction of IFN-β (Ireton and Gale (2011) Viruses 3(6):906-919). Synthetic RIG-I-binding elements, in the form of AA dinucleotide sequences in the U6 promoter transcription start site, have also been unintentionally found in common lentiviral shRNA vectors. Its subsequent deletion in the plasmid prevented confounding off-target type I IFN activation (Pebernard et al. (2004) Differentiation. 72:103-111).

[0244] The second type of TLR-independent type I interferon induction pathway is mediated through stimulator of interferon genes (STING), a cytosolic, ER-resident adaptor protein now recognized as a central mediator for sensing cytosolic dsDNA from infectious pathogens or aberrant host cell damage (Barber (2011) Immunol. Rev 243(1):99-108). STING signaling activates the TANK-binding kinase (TBK1) / IRF3 axis and the NF-kB signaling axis, leading to the induction of IFN-β and other pro-inflammatory cytokines and chemokines that potently activate innate and adaptive immunity (Burdette et al. (2011) Nature 478(7370):515-518). STING-mediated sensing of cytosolic dsDNA requires the host cell nucleotidyl transferase cyclic GMP-AMP synthase (cGAS), which directly binds to dsDNA and, in response, synthesizes the cyclic dinucleotide (CDN) second messenger cyclic GMP-AMP (cGAMP), which binds to and activates STING (Sun et al. (2013) Science 339(6121):786-791; Wu et al. (2013) Science 339(6121):826-830). Bacterial CDNs, such as c-di-AMP produced by intracellular Listeria monocytogenes, can also directly bind to murine STING, but only three of the five human STING alleles do. Unlike bacterially produced CDNs, in which two purine nucleosides are joined by a phosphate bridge using a 3'-3' linkage, the internucleotide phosphate bridge in cGAMP synthesized by mammalian cGAS is joined by a non-canonical 2'-3' linkage.These 2'-3' molecules bind to STING with 300-fold higher affinity than bacterial 3'-3' CDNs and are therefore more potent physiological ligands of human STING (see, e.g., Civilil et al. (2013) Nature 498(7454):332-337; Diner et al. (2013) Cell Rep. 3(5):1355-1361; Gao et al. (2013) Sci. Signal 6(269):pl1; Ablasser et al. (2013) Nature 503(7477):530-534).

[0245] The cGAS / STING signaling pathway in humans may have evolved over time to preferentially respond to viral pathogens over bacterial pathogens, which may explain why bacterial vaccines carrying host tumor antigens have been generated as insufficient CD8+ T cell priming vectors in humans. TLR-independent activation of CD8+ T cells by STING-dependent type I IFN signaling from conventional DCs is the primary mechanism by which viruses are detected, and the STING pathway involves TLR-dependent type I IFN production by plasmacytoid DCs, which is only operational when the virus is inactivated (Hervas-Stubbs et al. (2014) J Immunol. 193:1151-1161). Furthermore, for bacteria such as S. typhimurium, although TLR4-mediated induction of IFN-β is possible, CD8+ T cells are neither induced nor required for clearance or protective immunity (Lee et al. (2012) Immunol Lett. 148(2): 138-143). For many bacterial strains, including S. typhimurium, the lack of physiologically relevant CD8+ T epitopes, even from genetically identical strains, hinders both bacterial vaccine development and post-infection protective immunity (Lo et al. (1999) J Immunol. 162:5398-5406). Therefore, bacterial-based cancer immunotherapies are biologically limited in their ability to induce type I IFNs to recruit and activate CD8+ T cells, which are necessary for tumor antigen cross-presentation and durable antitumor immunity. Therefore, engineering the bacterial immunotherapy provided herein to induce viral-like TLR-independent type I IFN signaling rather than TLR-dependent bacterial immune signaling will preferentially induce CD8+ T cell-mediated antitumor immunity.

[0246] STING activates innate immunity in response to sensing nucleic acids in the cytosol. Downstream signaling is activated through the binding of CDNs synthesized by bacteria or the host enzyme cGAS in response to binding to cytosolic dsDNA. Bacterial and host-produced CDNs have distinct phosphate bridge structures that distinguish their ability to activate STING. IFN-β is the signature cytokine of active STING, and virus-induced type I IFN, rather than bacterial-induced IFN, is required for effective CD8+ T cell-mediated antitumor immunity. The immunostimulatory bacteria provided herein include those that are STING agonists.

[0247] 3. Treatment with Salmonella Salmonella is exemplary of a bacterial genus that can be used as a cancer therapeutic. The Salmonella exemplified herein are attenuated strains, or those modified for use as cancer therapeutics based on reduced virulence.

[0248] a. Tumor-tropic bacteria Several bacterial species have been demonstrated to replicate preferentially within solid tumors when injected from a distal site. These bacteria include, but are not limited to, species of Salmonella, Bifidobacterium, Clostridium, and Escherichia. The natural tumor-homing properties of bacteria, combined with the host's innate immune response to bacterial infection, are thought to mediate antitumor responses. This tumor tissue tropism has been shown to reduce tumor size to varying degrees. One factor contributing to the tumor tropism of these bacterial species is their ability to replicate in anoxic or hypoxic environments. Some of these naturally tumor-tropic bacteria have been further engineered to enhance the efficacy of antitumor responses (reviewed in Zu et al. (2014) Crit Rev Microbiol. 40(3):225-235; and Felgner et al. (2017) Microbial Biotechnology 10(5):1074-1078).

[0249] b. Salmonella enterica serovar vertifimurium Salmonella enterica serovar typhimurium (S. typhimurium) is an exemplary bacterial species for use as an anticancer therapeutic. One approach to using bacteria to stimulate host immunity against cancer is via the Gram-negative, facultative anaerobe S. typhimurium, which preferentially accumulates in hypoxic and necrotic areas of the body, including the tumor microenvironment. S. typhimurium accumulates in these environments because it can utilize nutrients from necrotic tissue, the tumor vasculature is leaky, and it is more likely to survive in the immune-evasive tumor microenvironment (Baban et al. (2010) Bioengineered Bugs 1(6):385-294). S. typhimurium can grow under both aerobic and anaerobic conditions; therefore, it can colonize small tumors with low levels of hypoxia and large tumors with high levels of hypoxia.

[0250] S. typhimurium is a Gram-negative facultative pathogen transmitted via the fecal-oral route. This pathogen causes a localized gastrointestinal infection, but after oral ingestion, it can invade the bloodstream and lymphatic system and infect systemic tissues such as the liver, spleen, and lungs. Systemic administration of wild-type S. typhimurium overstimulates TNF-α induction, leading to cytokine cascades and septic shock, which can be life-threatening if left untreated. Consequently, pathogenic bacterial strains such as S. typhimurium must be attenuated to prevent systemic infection without completely suppressing their ability to effectively colonize tumor tissue. Attenuation is often achieved by mutating cellular structures capable of inducing an immune response, such as the bacterial outer membrane, or by limiting their ability to replicate in the absence of supplemental nutrients.

[0251] S. typhimurium is an intracellular pathogen that can be rapidly internalized by myeloid cells such as macrophages or induce internalization in non-phagocytic cells such as epithelial cells. Once inside the cell, it can replicate within Salmonella-containing vacuoles (SCVs) and can also escape into the cytosol of some epithelial cells. Many of the molecular determinants of S. typhimurium virulence have been identified, and the genes are grouped into Salmonella pathogenicity islands (SPIs). The two best-characterized pathogenicity islands are SPI-1, which mediates bacterial invasion of non-phagocytic cells, and SPI-2, which is essential for replication within SCVs (Agbor and McCormick (2011) Cell Microbiol. 13(12): 1858-1869). Both of these pathogenicity islands encode a macromolecular structure termed a type III secretion system (T3SS) that is capable of translocating effector proteins across the host membrane (Galan and Wolf-Watz (2006) Nature 444:567-573).

[0252] C. Attenuation of the bacteria Therapeutic bacteria for cancer treatment should be attenuated. Various methods for attenuating bacterial pathogens are known in the art. For example, auxotrophic mutations render bacteria unable to synthesize essential nutrients, and deletions / mutations in genes such as aro, pur, gua, thy, nad, and asd (US Patent Application Publication No. 2012 / 0009153) are widely used. The nutrients produced by the biosynthetic pathways involved in these genes are often unavailable to host cells, making bacterial survival a challenge. For example, the attenuation of Salmonella and other bacterial species can be achieved by deleting the aroA gene, which is part of the shikimate pathway and is involved in glycolysis for aromatic amino acid biosynthesis (Felgner et al. (2016) MBio 7(5):e01220-16). Thus, deletion of aroA results in aromatic amino acid auxotrophy and subsequent attenuation of the bacterium (U.S. Patent Application Publication Nos. 2003 / 0170276, 2003 / 0175297, 2012 / 0009153, 2016 / 0369282, WO 2015 / 032165, and WO 2016 / 025582). Similarly, other enzymes involved in the biosynthetic pathway for aromatic amino acids, including aroC and aroD, have been deleted to achieve attenuation (U.S. Patent Application Publication Nos. 2016 / 0369282; WO 2016 / 025582). For example, S. typhimurium strain SL7207 is an aromatic amino acid auxotroph (aroA mutant); strains A1 and A1-R are leucine-arginine auxotrophs. VNP20009 is a purine auxotroph (purI mutant). As shown herein, it is also auxotrophic for the immunosuppressive nucleoside adenosine.

[0253] Mutations that attenuate bacteria include, but are not limited to, mutations in genes that alter lipopolysaccharide biosynthesis, such as rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; mutations that introduce suicide genes, such as sacB, nuk, hok, gef, kil, or phlA; mutations that introduce lysis genes, such as hly and cly; and mutations that introduce lysis genes, such as IsyA, pag, prg, iscA, virG, p There are also mutations in virulence factors, such as lc and act; mutations that alter stress responses, such as recA, htrA, htpR, hsp, and groEL; mutations that disrupt the cell cycle, such as min; and mutations that disrupt or inactivate regulatory functions, such as cya, crp, phoP / phoQ, and ompR (U.S. Patent Application Publication Nos. 2012 / 0009153, 2003 / 0170276, 2007 / 0298012; U.S. Patent No. 6,190,657; WO 2015 / 032165; Felgner et al. (2016) Gut microbes 7(2):171-177; Broadway et al. (2014) J. Biotechnology 192:177-178; Frahm et al. (2015) mBio 6(2):e00254-15; Kong et al. (2011) Infection and Immunity 79(12):5027-5038; Kong et al. (2012) Proc. Natl. Acad. Sci. USA 109(47):19414-19419). Ideally, genetic attenuation involves gene deletions rather than point mutations to prevent spontaneous compensatory mutations that may result in reversion to the virulent phenotype.

[0254] i.msbB-mutant The enzyme lipid A biosynthesis myristoyltransferase, encoded by the msbB gene in S. typhimurium, catalyzes the addition of a terminal myristyl group to the lipid A domain of lipopolysaccharide (LPS) (Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Deletion of msbB therefore alters the acyl composition of the lipid A domain of LPS, a major component of the outer membrane of Gram-negative bacteria. This modification significantly reduces the ability of LPS to induce septic shock, attenuates the bacterial strain, and reduces the potentially harmful production of TNFα, thus reducing systemic toxicity. S. typhimurium msbB mutants maintain their ability to preferentially colonize tumors across other tissues in mice, retain antitumor activity, and thus increase the therapeutic index of Salmonella-based immunotherapy (U.S. Patent Application Publication Nos. 2003 / 0170276, 2003 / 0109026, 2004 / 0229338, 2005 / 0225088, 2007 / 0298012).

[0255] For example, deletion of msbB in S. typhimurium strain VNP20009 results in the production of predominantly pentaacylated LPS, which is less toxic than native hexaacylated LPS and allows for systemic delivery without inducing toxic shock (Lee et al. (2000) International Journal of Toxicology 19:19-25). Other LPS mutations can be introduced into the bacterial strains provided herein, including Salmonella strains, that dramatically reduce virulence, thereby providing lower toxicity and allowing for the administration of higher doses.

[0256] ii.purI-mutant Immunostimulatory bacteria are used that can be attenuated by being made auxotrophic for one or more essential nutrients, such as purines (e.g., adenine), nucleosides (e.g., adenosine), or amino acids (e.g., arginine and leucine). In particular, in embodiments of the immunostimulatory bacteria provided herein, such as S. typhimurium, the bacteria are rendered auxotrophic for adenosine, which preferentially accumulates in the tumor microenvironment. Thus, the immunostimulatory bacterial strains described herein are attenuated because they require adenosine for growth and preferentially colonize the adenosine-rich TME, as discussed below.

[0257] The enzyme phosphoribosylaminoimidazole synthetase encoded by the purI gene (synonymous with the purM gene) is involved in the purine biosynthesis pathway. Therefore, disruption of the purI gene renders bacteria auxotrophic for purines. In addition to being attenuated, purI mutants are abundant in the tumor environment and have significant antitumor activity (Pawelek et al. (1997) Cancer Research 57:4537-4544). This colonization has been previously attributed to the high concentration of purines present in the interstitial fluid of tumors as a result of their rapid cell turnover. Because purI bacteria cannot synthesize purines, they require the external source adenine, which is thought to lead to their growth restriction in the purine-rich tumor microenvironment (Rosenberg et al. (2002) J. Immunotherapy 25(3):218-225). Although the VNP20009 strain was initially reported to contain a deletion of the purI gene (Low et al. (2003) Methods in Molecular Medicine Vol. 90, Suicide Gene Therapy:47-59), subsequent analysis of the entire VNP20009 genome demonstrated that the purI gene is not deleted but is disrupted by a chromosomal inversion (Broadway et al. (2014) Journal of Biotechnology 192:177-178). The entire gene is contained within two portions of the VNP20009 chromosome (one of which harbors an active transposase) flanking the insertion sequence.

[0258] It is shown herein that the purI mutant S. typhimurium strain is auxotrophic for the nucleoside adenosine, which is highly abundant in the tumor microenvironment. Therefore, when using VNP20009, it is not necessary to introduce any additional modifications to achieve adenosine auxotrophy. For other strains and bacteria, the purI gene may be disrupted as present in VNP20009, or may contain a deletion of all or part of the purI gene to prevent reversion to the wild-type gene.

[0259] iii. Combinations of attenuating mutations Bacteria with multiple genetic attenuations using gene deletions in different regions of the chromosome are desirable for bacterial immunotherapy because they increase attenuation and simultaneously reduce the likelihood of reversion to a virulent phenotype through gene acquisition by homologous recombination with wild-type genetic material. Restoration of virulence through homologous recombination would require two separate recombination events to occur within the same organism. Ideally, the combination of attenuating mutations selected for use in immunotherapy agents would increase tolerability without reducing efficacy, resulting in an increased therapeutic index. For example, disruption of the msbB and purI genes in S. typhimurium strain VNP20009 has been used to target tumors, inhibit their growth, and induce low virulence in animal models (Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002; Bermudez et al. (2000) Cancer Gene Therapy: Past Achievements and Future Challenges, edited by Habib Kluwer Academic / Plenum Publishers, New York, pp. 57-63; Low et al. (2003) Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy:47-59; Lee et al. (2000) International Journal of Toxicology 19:19-25; Rosenberg et al. (2002) J. Immunotherapy 25(3):218-225;Broadway et al. (2014) J. Biotechnology 192:177-178;Loeffler et al. (2007) Proc. Natl. Acad. Sci. USA 104(31):12879-12883;Luo et al. (2002) Oncology Research 12:501-508).When VNP20009 (msbB- / purI-) was administered to mice bearing syngeneic or human xenograft tumors, the bacteria preferentially accumulated within the extracellular components of tumors at ratios exceeding 300–1000:1, reduced TNFα induction, tumor regression, and prolonged survival compared with control mice (Clairmont et al. (2000) J. Infect. Dis. 181:1996–2002). However, results from a phase 1 human clinical trial demonstrated that VNP20009 was relatively safe and well tolerated, but insufficient accumulation was observed in human melanoma tumors, demonstrating very little antitumor activity (Toso et al. (2002) J. Clin. Oncol. 20(1):142–152). High doses, required to demonstrate any antitumor activity, were not possible due to toxicity.

[0260] Therefore, further improvements are needed. The immunostimulatory bacteria provided herein address this challenge.

[0261] iv. VNP20009 and other attenuated and wild-type S. typhimurium strains The starting strain can be a wild-type, non-attenuated strain, such as a strain that has all of the identifying characteristics of ATCC 14028. The strain is then modified to increase its specificity or targeting to the tumor microenvironment or to tumor cells and / or to tumor-resident immune cells. It can also be modified to be adenosine auxotrophic. The strain can be modified to be flagellin auxotrophic. - (fliC - / fljB - ) and msbB - , purI - / M - , and pagP - The strain may be one or more of: -The engineered strains may encode the therapeutic product under the control of a promoter recognized by the mammalian host, such as RNA polymerase II or III, on a plasmid typically present at low to medium copy number. Additional regulatory sequences may also be included to control expression in the tumor microenvironment and intracellular trafficking.

[0262] An exemplary therapeutic bacterium that can be modified as described herein is the strain designated VNP20009 (ATCC #202165, YS1646), derived from ATCC Accession No. 14028. The strain designated VNP20009 (ATCC #202165, YS1646) is a clinical candidate and has been attenuated at least 50,000-fold for safety reasons by deletion of the msbB and purI 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 are also contemplated. As mentioned above, deletion of msbB alters the composition of the lipid A domain of lipopolysaccharide, a major component of the Gram-negative bacterial outer membrane (Low et al. (1999) Nat. Biotechnol. 17(1):37-41). This prevents lipopolysaccharide-induced septic shock, attenuates the bacterial strain, reduces systemic toxicity, and simultaneously reduces the potentially harmful production of TNFα (Dinarello, CA (1997) Chest 112(6 Suppl):321S-329S; Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Deletion of the purI gene renders the bacterium auxotrophic for purines, further attenuating the bacterium and enriching it in the tumor microenvironment (Pawelek et al. (1997) Cancer Res. 57:4537-4544; Broadway et al. (2014) J. Biotechnology 192:177-178).

[0263] The accumulation of VNP20009 in tumors is due to a combination of factors, including the inherent invasiveness of the parent strain, ATCC Accession No. 14028, its ability to replicate in hypoxic environments, and its high requirement for purines present in tumor interstitial fluid. VNP20009 (Peter Vaupel and Arnulf Mayer, Oxygen Transport to Tissue XXXVII, Advances in Experimental Medicine and Biology 876 chapter 22, pp. 177-183), which can accumulate to pathologically high levels in the tumor microenvironment and may contribute to an immunosuppressive tumor microenvironment, is shown herein to also be auxotrophic for the nucleoside adenosine. When VNP20009 was administered to mice bearing syngeneic or human xenograft tumors, the bacteria preferentially accumulated within the extracellular components of the tumor at a ratio of over 300-1000 to 1, inhibiting tumor growth and demonstrating prolonged survival compared to control mice (Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002). Results from a phase 1 clinical trial demonstrated that VNP20009 was relatively safe and well tolerated, but insufficient accumulation was observed in human melanoma tumors, demonstrating very little antitumor activity (Toso et al. (2002) J. Clin. Oncol. 20(1):142-152). High doses, which would be necessary to affect any antitumor activity, were not possible due to toxicity correlated with high levels of proinflammatory cytokines. Flagellin-deficient (fliC) - / fljB - ), and optional pagP - and / or hilA -The modifications provided herein, including modifications, significantly increase the accumulation of immunostimulatory bacteria in tumors, in the tumor microenvironment, and / or in tumor-resident immune cells, such as myeloid cells. Other modifications that enhance targeting to immune cells and abolish infection of other cells, such as epithelial cells, increase bacterial accumulation in tumors and in the tumor microenvironment. An additional modification that renders wild-type bacteria adenosine auxotrophic further increases accumulation in the tumor microenvironment.

[0264] For example, the leucine-arginine auxotroph A-1 (Zhao et al. (2005) PNAS 102(3):755-760; Yu et al. (2012) Scientific Reports 2:436; U.S. Patent No. 8,822,194; U.S. Patent Application Publication No. 2014 / 0178341) and its derivative AR-1 (Yu et al. (2012) Scientific Reports 2:436; Kawagushi et al. (2017) Oncotarget 8(12):19065-19073; Zhao et al. (2006) Cancer Res. 66(15):7647-7652; Zhao et al. (2012) Cell Cycle 11(1):187-193; Tome et al. (2013) Anticancer Research 33:97-102; Murakami et al. (2017) Oncotarget 8(5):8035-8042; Liu et al. (2016) Oncotarget 7(16):22873-22882; Binder et al. (2013) Cancer Immunol Res. 1(2):123-133; aroA-mutant S. typhimurium strain SL7207 (Guo et al. (2011) Gene therapy 18:95-105; U.S. Patent Application Publication Nos. 2012 / 0009153, 2016 / 0369282, and 2016 / 0184456) and its obligately anaerobic derivative YB1 (Yu et al. (2012) Scientific Reports 2:436; Leschner et al. (2009) PLoS ONE 4(8): e6692; Yu et al. (2012) Scientific Reports 2:436); aroA- / aroD- mutant S. typhimurium strain BRD509, a derivative of the SL1344 (WT) strain (Yoon et al. (2017) European J. of Cancer 70:48-61); asd- / cya- / crp- mutant S. typhimurium strain χ4550 (Sorenson et al.(2010) Biology: Targets & Therapy 4:61-73) and other strains of S. typhimurium, such as phoP- / phoQ- S. typhimurium strain LH430 (WO 2008 / 091375), can be used for tumor-targeted delivery of therapeutic proteins and therapy.

[0265] Although strain VNP20009 failed to demonstrate clinical benefit in studies involving patients with advanced melanoma, treatment was safely administered to patients with advanced cancer. A maximum tolerated dose (MTD) was established. Therefore, this strain, as well as other similarly engineered bacterial strains, can be used as a starting material for tumor-targeting therapeutic delivery vehicles. The modifications provided herein offer a strategy for enhancing efficacy by increasing the antitumor efficacy and / or safety and tolerability of therapeutic agents.

[0266] v. S. typhimurium genetically engineered to deliver macromolecules S. typhimurium has also been engineered to deliver the tumor-associated antigen (TAA) survivin (SVN) to APCs to prime adaptive immunity (U.S. Patent Application Publication No. 2014 / 0186401; Xu et al. (2014) Cancer Res. 74(21):6260-6270). SVN is an inhibitor of apoptosis protein (IAP) that prolongs cell survival, provides cell cycle control, and is overexpressed in all solid tumors but underexpressed in healthy tissues. This technology utilizes Salmonella pathogenicity island 2 (SPI-2) and its type III secretion system (T3SS) to deliver the TAA into the cytosol of APCs, which then activate and induce TAA-specific CD8+ T cells and antitumor immunity (Xu et al. (2014) Cancer Res. 74(21):6260-6270). Similar to Listeria-based TAA vaccines, this approach has shown promise in mouse models, but effective tumor antigen-specific T cell priming in humans has yet to be demonstrated.

[0267] In addition to gene delivery, S. typhimurium has also been used to deliver small interfering RNA (siRNA) and short hairpin RNA (shRNA) for cancer therapy. For example, attenuated S. typhimurium has been engineered to express certain shRNAs, such as those targeting STAT3 and IDO1 (U.S. Patent Application Publication No. 2007 / 074272 and U.S. Patent No. 9,453,227). VNP20009 was transformed with an shRNA plasmid against the immunosuppressive gene indoramin deoxygenase (IDO), successfully silencing IDO expression in a murine melanoma model, resulting in tumor cell death and significant tumor infiltration by neutrophils (Blache et al. (2012) Cancer Res. 72(24):6447-6456). Combining this vector with co-administration of hyaluronidase, such as PEGylated soluble PH20 (PEGPH20; an enzyme that depletes extracellular hyaluronan), has shown beneficial results in the treatment of pancreatic ductal adenocarcinoma tumors (Manuel et al. (2015) Cancer Immunol. Res. 3(9):1096-1107; U.S. Patent Application Publication No. 2016 / 0184456). In another study, S. typhimurium strains attenuated by phoP / phoQ deletion and expressing signal transducer and activator of transcription 3 (STAT3)-specific shRNA were found to inhibit tumor growth, reduce the number of metastatic organs, and extend the lifespan of C57BL6 mice (Zhang et al. (2007) Cancer Res. 67(12):5859-5864).In another example, S. typhimurium strain SL7207 has been used to deliver shRNA targeting CTNNB1, the gene encoding β-catenin (Guo et al. (2011) Gene therapy 18:95-105; U.S. Patent Application Publication Nos. 2009 / 0123426, 2016 / 0369282), while S. typhimurium strain VNP20009 has been utilized in the delivery of shRNA targeting STAT3 (Manuel et al. (2011) Cancer Res. 71(12):4183-4191; U.S. Patent Application Publication Nos. 2009 / 0208534, 2014 / 0186401, and 2016 / 0184456; WO 2008 / 091375; and WO 2012 / 149364. siRNA targeting the autophagy genes Atg5 and Beclin 1 has been delivered to tumor cells using S. typhimurium strains A1-R and VNP20009 (Liu et al. (2016) Oncotarget 7(16):22873-22882). Improvements to such strains, such as the immunostimulatory bacteria provided herein, are needed to more effectively colonize tumors, the TME, and / or tumor-resident immune cells, stimulate immune responses, and possess other advantageous properties. Modifications of various bacteria are described in International PCT Application Publication No. WO2019 / 014398 and U.S. Patent Application Publication No. 2019 / 0017050A1. The bacteria described in each of these publications, which are also described herein, can be modified as described herein to further improve their immune stimulatory and tumor targeting properties.

[0268] Bacteria can be modified to have a reduced inflammatory effect as described herein, and thus be less virulent.As a result, for example, higher dosages can be administered.These strains of Salmonella, as well as other bacterial species known to those skilled in the art and / or listed above and herein, can be modified for immune checkpoint inhibition and other modifications as described herein, for example, by introducing adenosine auxotrophy into a plasmid encoding a therapeutic product, such as an immunostimulatory protein and / or RNAi, for example, miRNA or shRNA, or an antibody or a fragment thereof, as described herein.An exemplary example is the S. typhimurium species described herein.

[0269] The bacterial strains provided herein are genetically engineered to deliver therapeutic molecules. The strains herein deliver immunostimulatory proteins, such as cytokines, that promote anti-tumor immune responses in the tumor microenvironment. The strains can also contain genomic modifications that reduce phagocyte pyroptosis, resulting in a more robust immune response, and / or reduce or eliminate their ability to infect / invade epithelial cells but retain their ability to infect / invade phagocytes, thereby allowing them to more effectively accumulate in tumors and tumor-resident immune cells. The bacterial strains can also be engineered to encode therapeutic products, including, for example, RNAi, that are targeted and inhibitory to immune checkpoints and other such targets.

[0270] 4. Enhancing immunostimulatory bacteria to increase the therapeutic index Enhancements to immunostimulatory bacteria that reduce toxicity and improve antitumor activity are provided herein. Exemplary of such enhancements are the following, which are described with respect to Salmonella, particularly S. typhimurium; those skilled in the art will recognize that similar enhancements can be made in other bacterial species and other Salmonella strains.

[0271] ASD gene deletion The asd gene in bacteria encodes aspartate-semialdehyde dehydrogenase. S. typhimurium asd mutants have an obligate requirement for diaminopimelic acid (DAP), essential for cell wall synthesis, resulting in lysis in DAP-deficient environments. When the asd gene is complemented in trans on a plasmid, this DAP auxotrophy can be used in vivo to select for the plasmid and maintain its stability without antibiotics. An antibiotic-free plasmid selection system would be advantageous, allowing 1) the use of antibiotics administered as a rapid clearance mechanism in adverse symptomatic cases and 2) the scale-up of antibiotic-free production when such use is normally avoided. The asd gene complementation system provides such selection (Galan et al. (1990) Gene 28:29-35). Using the asd gene complementation system to maintain plasmids in the tumor microenvironment is expected to enhance the efficacy of S. typhimurium engineered to deliver plasmids encoding genes or interfering RNA.

[0272] An alternative use for asd mutants of S. typhimurium is to exploit the DAP auxotrophy to deliver macromolecules to infected cells without the ability to persistently colonize host tumors, producing autolytic (or suicidal) strains. Deletion of the asd gene renders the bacteria auxotrophic for DAP when grown in vitro or in vivo. The example described herein provides an asd deletion strain that is DAP auxotrophic and contains a plasmid suitable for delivering RNAi, such as shRNA or miRNA, resulting in a strain that does not contain the asd complementing gene and is defective for in vivo replication. This strain is propagated in vitro in the presence of DAP, grows normally, and is then administered as an immunotherapeutic agent to a mammalian host lacking DAP. The suicidal strain is able to invade host cells but is unable to replicate due to the absence of DAP in mammalian tissues, and automatically lyses, delivering its cytosolic contents (e.g., plasmids or proteins). In the example provided herein, the asd gene deletion strain of VNP20009 was further engineered to express the LLO protein without its endogenous periplasmic secretion signal sequence, allowing it to accumulate in the cytoplasm of Salmonella. LLO is a cholesterol-dependent pore-forming hemolysin from Listeria monocytogenes that mediates bacterial escape from phagocytosis. When the autolytic strain is introduced into tumor-bearing mice, the bacteria are engulfed by phagocytic immune cells and enter Salmonella-containing vacuoles (SCVs). In this environment, the absence of DAP prevents bacterial replication and leads to bacterial autolysis in the SCV. Subsequent lysis of the suicidal strain will release the plasmid, allowing the accumulated LLO to form pores in the cholesterol-containing SCV membrane, enabling delivery of the plasmid into the host cell cytosol.

[0273] B adenosine auxotrophy Tryptophan and metabolites derived from the ATP / adenosine pathway are key drivers of the immunosuppressive environment within tumors. Adenosine, present in free form both inside and outside cells, is an effector of immune function. Adenosine reduces T cell receptor-induced activation of NF-κB and inhibits IL-2, IL-4, and IFN-γ. Adenosine reduces T cell cytotoxicity, increases T cell anergy, and enhances T cell differentiation into Foxp3+ or Lag-3+ regulatory (T-reg) T cells. Regarding NK cells, adenosine reduces IFN-γ production and suppresses NK cell cytotoxicity. Adenosine blocks neutrophil adhesion and extravasation, reduces phagocytosis, and attenuates superoxide and nitric oxide levels. Adenosine also reduces the expression of TNF-α, IL-12, and MIP-1α on macrophages, attenuates MHC class II expression, and increases the levels of IL-10 and IL-6. Adenosine immunomodulatory activity occurs after its release into the extracellular space of tumors and activation of adenosine receptors (ADRs) on the surface of target immune cells, cancer cells, or endothelial cells. High adenosine levels in the tumor microenvironment result in localized immunosuppression, which limits the immune system's ability to eliminate cancer cells.

[0274] Extracellular adenosine is produced by the continuous activity of the membrane-bound ectoenzymes CD39 and CD73 expressed on tumor stromal cells, which phosphohydrolyze ATP or ADP from dead or dying cells, along with adenosine production. CD39 converts extracellular ATP (or ADP) to 5'AMP, which is then converted to adenosine by 5'AMP. Expression of CD39 and CD73 on endothelial cells is increased under hypoxic conditions in the tumor microenvironment, resulting in increased adenosine levels. Tumor hypoxia may result from insufficient blood supply and a disrupted tumor vasculature, impairing oxygen delivery (Carroll and Ashcroft (2005) Expert. Rev. Mol. Med. 7(6):1-16). Hypoxia in the tumor microenvironment also inhibits adenylate kinase (AK), which converts adenosine to AMP and leads to extremely high extracellular adenosine concentrations. Extracellular concentrations of adenosine in the hypoxic tumor microenvironment have been measured at 10–100 μM, which is up to approximately 100–1000 times higher than the typical extracellular adenosine concentration of approximately 0.1 μM (Vaupel et al. (2016) Adv Exp Med Biol. 876:177–183; Antonioli et al. (2013) Nat. Rev. Can. 13:842–857). Because hypoxic regions in tumors are distal to microvasculature, localized concentrations of adenosine in some areas of the tumor may be higher than others.

[0275] By directly acting and inhibiting the immune system, adenosine can also control cancer cell growth and dissemination by acting on cancer cell proliferation, apoptosis, and angiogenesis. For example, adenosine can initially promote angiogenesis through stimulation of A2A and A2B receptors. Stimulating receptors on endothelial cells can regulate intercellular adhesion molecule 1 (ICAM-1) and E-selectin expression on endothelial cells, maintaining vascular integrity and promoting vascular growth (Antonioli et al. (2013 Nat. Rev. Can. 13:842-857)). Activating one or more of A2A, A2B, or A3 on various cells by adenosine can stimulate the production of angiogenic factors such as vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), or angiopoietin 2 (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857).

[0276] Adenosine can also directly regulate tumor cell proliferation, apoptosis, and metastasis through interactions with receptors on cancer cells. For example, studies have shown that activating A1 and A2A receptors promotes tumor cell proliferation in some breast cancer cell lines, while activating A2B receptors has cancer growth-promoting properties in colon carcinoma cells (Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Adenosine can also induce apoptosis in cancer cells, and various studies have correlated this activation with activation of the A3-mediated extrinsic apoptosis pathway or the A2A and A2B-mediated intrinsic apoptosis pathway (Antonioli et al. (2013)). Adenosine can promote tumor cell migration and metastasis by increasing cell motility, adhesion to the extracellular matrix, and the expression of cell-binding proteins and receptors, promoting cell migration and motility.

[0277] The extracellular release of adenosine triphosphate (ATP) occurs from stimulated immune cells and damaged, dead, or stressed cells. When stimulated by this extracellular release of ATP, the NLR family pyrin domain-containing 3 (NLRP3) inflammasome activates caspase-1, leading to the secretion of cytokines IL-1β and IL-18, activating innate and adaptive immune responses (Stagg and Smyth (2010) Oncogene 29:5346-5358). ATP is catabolized to adenosine by the enzymes CD39 and CD73. Activated adenosine acts as a highly immunosuppressive metabolite through a negative feedback mechanism and has pleiotropic effects on multiple immune cell types in the hypoxic tumor microenvironment (Stagg and Smyth (2010) Oncogene 29:5346-5358). The adenosine receptors A2A and A2B are expressed on various immune cells and stimulated by adenosine, promoting cAMP-mediated signaling changes that result in an immunosuppressive phenotype in T cells, B cells, NK cells, dendritic cells, mast cells, macrophages, neutrophils, and NKT cells. As a result, adenosine levels can accumulate in pathological tissues, such as solid tumors, exceeding their normal concentrations by 100 times, leading to overexpression of ectonucleotidases such as CD73. Adenosine has also been shown to promote tumor angiogenesis and progression. Therefore, genetically engineered bacteria that are adenosine auxotrophic exhibit enhanced tumor targeting and colonization.

[0278] Immunostimulatory bacteria such as Salmonella typhimurium can be rendered adenosine auxotrophic by deletion of the tsx genes (Bucarey et al. (2005) Infection and Immunity 73(10):6210-6219) or purD (Husseiny (2005) Infection and Immunity 73(3):1598-1605). In the Gram-negative bacterium Xanthomonas oryzae, knockout of the purD gene has been shown to result in adenosine auxotrophy (Park et al. (2007) FEMS Microbiol Lett 276:55-59). As exemplified herein, S. typhimurium strain VNP20009 is adenosine auxotrophic due to its purI deletion, and therefore, further modification to render it adenosine auxotrophic is not necessary. Thus, embodiments of the immunostimulatory bacterial strains provided herein are adenosine auxotrophic. Such auxotrophic bacteria selectively replicate in the tumor microenvironment, further increasing the accumulation and replication of the administered bacteria in the tumor and reducing adenosine levels in and around the tumor, thereby reducing or eliminating the immunosuppression caused by adenosine accumulation. Exemplary of such bacteria provided herein are engineered strains of S. typhimurium that contain purI / msbB mutations to provide adenosine auxotrophy.

[0279] C. flagellin-deficient strain Flagella are organelles on the surface of bacteria that consist of long filaments attached via hooks to rotary motors that can rotate in a clockwise or counterclockwise manner, providing a means for locomotion. Flagella in S. typhimurium are important for chemotaxis and for establishing infection via the oral route due to their ability to mediate motility across mucosal layers in the gastrointestinal tract. Flagella have been demonstrated to be essential for chemotaxis to and colonization of cylindrical tumors in vitro (Kasinskas and Forbes (2007) Cancer Res. 67(7):3201-3209), and motility has been shown to be important for tumor penetration (Toley and Forbes (2012) Integr Biol (Camb). 4(2):165-176). However, flagella are not essential for tumor colonization in animals when the bacteria are administered intravenously (Stritzker et al. (2010) International Journal of Medical Microbiology 300:449-456). Each flagellar filament consists of tens of thousands of flagellin subunits. The S. typhimurium chromosome contains two genes, fliC and fljB, which encode antigenically distinct flagellin monomers. Mutants defective in both fliC and fljB are nonmotile and virulent when administered via the oral route of infection but maintain virulence when administered parenterally.

[0280] Flagellin is the major pro-inflammatory determinant of Salmonella (Zeng et al. (2003) J Immunol 171:3668-3674) and is directly recognized by TLR5 on the surface of cells and by NLCR4 in the cytosol (Lightfield et al. (2008) Nat Immunol. 9(10):1171-1178). Both pathways lead to a pro-inflammatory response, resulting in the secretion of cytokines including IL-1β, IL-18, TNF-α, and IL-6. Attempts are being made to create more potent Salmonella-based cancer immunotherapy by increasing the pro-inflammatory response to flagellin by genetically engineering bacteria to secrete Vibrio vulnificus flagellin B, which induces greater inflammation than the flagellin-encoded fliC and fljB (Zheng et al. (2017) Sci. Transl. Med. 9(376):eaak9537).

[0281] With this in mind, Salmonella bacteria S. typhimurium have been genetically engineered to lack both flagellin subunits fliC and fljB, reducing pro-inflammatory signaling. For example, as shown herein, a Salmonella strain lacking msbB, which results in reduced TNF-alpha induction, is combined with a fliC and fljB knockout. This results in a Salmonella strain with a combination of reduced TNF-alpha induction and reduced TLR5 recognition. These modifications, combined with the msbB - , fliC - and fljB - These variants can be transformed with immunostimulatory plasmids that may contain CpGs and therapeutic molecules, such as antibodies or RNAi molecule(s) that target immune checkpoints, such as TREX1, PD-L1, VISTA, SIRP-alpha, TGF-beta, beta-catenin, CD47, VEGF, and combinations thereof. The resulting bacteria have reduced pro-inflammatory signaling yet robust anti-tumor activity.

[0282] For example, provided herein, a fliC and fljB double mutant was constructed in an asd deletion strain of S. typhimurium VNP20009. VNP20009, whose virulence was attenuated by disrupting purI / purM, was also engineered to contain an msbB deletion, which results in the production of a lipid A subunit that is less toxigenic than wild-type lipid A. This results in reduced TNF-α production in a mouse model after intravenous administration compared to strains with wild-type lipid A. The resulting strain is exemplary of a strain in which bacterial inflammation has been attenuated by modifying lipid A to reduce TLR2 / 4 signaling and deleting flagellin subunits to reduce TLR5 recognition and inflammasome induction. Deletion of flagellin subunits, combined with LPS modification, allows for excellent tolerability within the host and directs an immunostimulatory response to the delivery of RNA interference to desired targets in the TME, inducing anti-tumor responses and promoting adaptive immune responses against tumors.

[0283] d. Salmonella genetically engineered to escape Salmonella-containing vacuoles (SCVs) Salmonella, including S. typhimurium, are intracellular pathogens that primarily replicate in membrane-bound compartments called Salmonella-containing vacuoles (SCVs). It has been shown that S. typhimurium can escape into the cytosol and replicate in some epithelial cell lines at low frequencies. Because the lipid bilayer of the SCV is a potential barrier, Salmonella engineered to escape SCVs with high efficiency may be more efficient in delivering macromolecules, such as plasmids. Provided herein are Salmonella and methods that enhance the frequency of escape from SCVs. This is achieved by deleting a gene essential for Salmonella that induces SIF (Single Insect Filament) formation. These mutants exhibit increased frequency of escape from SCVs and are able to replicate in the cytosol.

[0284] For example, enhanced plasmid delivery has been demonstrated using a sifA mutant of S. typhimurium. The sifA gene encodes SPI-2, a T3SS-2 secreted effector protein that mimics or activates the RhoA family of host GTPases (Ohlson et al. (2008) Cell Host & Microbe 4:434-446). Other genes encoding secreted effectors involved in SIF formation can be targeted. These include, for example, sseJ, sseL, sopD2, pipB2, sseF, sseG, spvB, and steA. Enhancing escape of S. typhimurium by preventing SIF formation allows viable bacteria to be released into the cytosol, where they can replicate.

[0285] Another approach to enhance S. typhimurium escape from SCVs and increase the delivery of macromolecules such as plasmids is to express a heterologous hemolysin that leads to pore formation or disruption of the SCV membrane. One such hemolysin is the listeriolysin O protein (LLO) from Listeria monocytogenes, encoded by the hlyA gene. LLO is a cholesterol-dependent pore-forming cytolysin secreted from L. monocytogenes and primarily responsible for escape from phagocytosis and entry into the host cell cytosol. Secretion of LLO from S. typhimurium can result in bacterial escape and replication in the cytosol. To prevent intact S. typhimurium from escaping from the SCV and replicating in the cytosol, the nucleotides encoding the signal sequence can be removed from the gene. In this way, active LLO is contained within the cytoplasm of S. typhimurium, and LLO is released only upon bacterial lysis. Provided herein, VNP20009, engineered to express cytoLLO and enhance delivery of plasmids for expressing interfering RNA against targets such as TREX1, can enhance the therapeutic efficacy of immunostimulatory bacteria.

[0286] e. Deletion in a Salmonella gene required for biofilm formation Bacteria and fungi can form multicellular structures called biofilms. Bacterial biofilms are encapsulated in a mixture of secreted cell wall-associated polysaccharides, glycoproteins, and glycolipids, as well as extracellular DNA, collectively referred to as extracellular polymeric substances (ECPS). These EPS protect bacteria from multiple insults, including detergents, antibiotics, and antimicrobial peptides. Bacterial biofilms colonize surfaces and are a significant cause of infection in prosthetic devices, such as injection ports and catheters. Biofilms form in tissues with ongoing infection, increasing bacterial persistence and shedding and potentially limiting the effectiveness of antibiotic therapy. Chronic persistence of bacteria in biofilms is associated with increased tumorigenesis, for example, in S. typhi infections of the gallbladder (Di Domenico et al. (2017) Int. J. Mol. Sci. 18:1887).

[0287] S. typhimurium biofilm formation is regulated by CsgD, which activates the csgBAC operon and increases the production of the curli subunits CsgA and CsgB (Zakikhani et al. (2010) Molecular Microbiology 77(3):771-786). CsgA is recognized as a PAMP by TLR2 and induces IL-8 production from human macrophages (Tukel et al. (2005) Molecular Microbiology 58(1):289-304). Furthermore, CsgD indirectly increases cellulose production by activating the adrA gene, which encodes diguanylate cyclase. The small molecule cyclic di-guanosine monophosphate (c-di-GMP) produced by AdrA is a ubiquitous second messenger found in almost all bacterial species. The AdrA-mediated increase in c-di-GMP enhances expression of the cellulose synthase gene bcsA, which in turn increases cellulose production via stimulation of the bcsABZC and bcsEFG operons. A reduction in the ability of immunostimulatory bacteria such as S. typhimurium to form biofilms can be achieved through deletion of genes involved in biofilm formation, such as csgD, csgA, csgB, adrA, bcsA, bcsB, bcsZ, bcsE, bcsF, bcsG, dsbA, or dsbB (Anwar et al. (2014) Plos One 9(8):e106095).

[0288] S. typhimurium can form biofilms in solid tumors as a defense against phagocytosis by host immune cells. Salmonella mutants unable to form biofilms are more rapidly taken up by host phagocytes and removed from infected tumors (Crull et al. (2011) Cellular Microbiology 13(8):1223-1233). This increase in intracellular localization within phagocytes can reduce the persistence of extracellular bacteria, enhancing the effectiveness of plasmid delivery and gene knockdown by RNA interference, as described herein. Immunostimulatory bacteria engineered to reduce biofilm formation will increase the rate of clearance from tumors / tissues, thereby increasing the tolerability of therapy and preventing the colonization of prosthetic devices in patients, thereby enhancing the therapeutic benefit of these strains. Adenosine mimetics can inhibit S. typhimurium biofilm formation, indicating that high adenosine concentrations in the tumor microenvironment may contribute to tumor-associated biofilm formation (Koopman et al. (2015) Antimicrob Agents Chemother 59:76-84). Live attenuated bacterial strains provided herein, such as S. typhimurium, that contain a purI disruption (and thus colonize adenosine-rich tumors) and that have biofilm formation blocked by deletion of one or more genes essential for biofilm formation, are engineered to deliver a plasmid encoding an interfering RNA that stimulates a robust antitumor immune response.

[0289] The adrA gene encodes a diguanylate cyclase that produces c-di-GMP, essential for S. typhimurium biofilm formation. c-di-GMP binds to and is an agonist for the host cytosolic protein STING. STING agonists, such as those described above, have been pursued as vaccine adjuvants for anti-cancer treatments, and bacteria have been engineered to secrete cyclic dinucleotides for use in immunotherapy (Libanova 2012, Synlogic 2018 AACR poster). Although immunostimulatory bacteria with reduced c-di-GMP production via deletion of adrA may seem counterintuitive, bacterial mutants, such as S. typhimurium mutants (including adrA mutants) that are unable to form biofilms, have demonstrated reduced therapeutic potential in mouse tumor models (Crull et al. (2011) Cellular Microbiology 13(8):1223-1233). Some human alleles of STING are insensitive to bacterially produced 3'3' CDN binding (Corrales et al. (2015) Cell Reports 11:1022-1023).

[0290] Bacterial strains described herein, such as S. typhimurium strains that have been genetically engineered to be adenosine auxotrophic, have their ability to induce pro-inflammatory cytokines reduced by modifying LPS and / or deleting flagellin and / or deleting genes essential for biofilm formation, and have been further engineered to deliver interfering RNA, promote robust anti-tumor immune responses.

[0291] f. Deletion in a gene in the LPS biosynthetic pathway LPS of Gram-negative bacteria is a major component of the outer leaflet of the bacterial membrane. It consists of three major parts: lipid A, a nonrepeating core oligosaccharide, and the O antigen (or O polysaccharide). The O antigen is the outermost part of LPS and serves as a protective layer against bacterial permeability, but the sugar composition of the O antigen varies widely among bacterial strains. Lipid A and the core oligosaccharide are very scarce and are more typically conserved within strains of the same bacterial species. Lipid A is the portion of LPS that contains endotoxin activity. It is typically a disaccharide modified with multiple fatty acids. These hydrophobic fatty acid chains anchor LPS to the bacterial membrane, while the rest of the LPS protrudes from the cell surface. The lipid A domain is responsible for much of the toxicity of Gram-negative bacteria. Typically, LPS in the blood is recognized as a severe pathogen-associated molecular pattern (PAMP) and induces a severe pro-inflammatory response. LPS is a ligand for a membrane-bound receptor complex containing CD14, MD2, and TLR4. TLR4 can signal through the MyD88 and TRIF pathways to stimulate the transporter membrane protein NFκB pathway, leading to the production of proinflammatory cytokines such as TNF-α and IL-1β, resulting in endotoxic shock, which can be life-threatening. LPS in the cytosol of mammalian cells can directly bind to the CARD domains of caspases 4, 5, and 11, causing autoactivation and pyroptotic cell death (Hagar et al. (2015) Cell Research 25:149-150). The composition of lipid A and the toxigenicity of lipid A variants are well documented. For example, monophosphorylated lipid A is less inflammatory than lipid A with multiple phosphate groups. The number and length of acyl chains on lipid A can also have a profound effect on the degree of toxicity. Standard lipid A from E. coli has six acyl chains, and this hexaacylation is highly toxic. S. typhimurium lipid A is similar to that of E. coli; it is a glucosamine disaccharide bearing four primary and two secondary hydroxyacyl chains (Raetz and Whitfield (2002) Annu Rev Biochem. 71:635-700).

[0292] The msbB mutant of S. typhimurium, as described above, is unable to undergo terminal myristoylation of its LPS and primarily produces penta-acylated LPS, which is significantly less toxic than hexa-acylated lipid A. The modification of lipid A with palmitate is catalyzed by palmitoyltransferase (PagP). Transcription of the pagP gene is under the control of the PhoP / PhoQ system, which is activated, for example, by low concentrations of magnesium within the SCV. Therefore, the acyl content of S. typhimurium is variable and can be hexa- or penta-acylated in wild-type bacteria. The ability of S. typhimurium to palmitate its lipid A increases its resistance to antimicrobial peptides secreted into the phagolysosome.

[0293] In wild-type S. typhimurium, expression of pagP results in heptaacylated lipid A. In msbB mutants (which cannot add the terminal acyl chain of lipid A), induction of pagP results in hexaacylated LPS (Kong et al. (2011) Infection and Immunity 79(12):5027-5038). Hexaacylated LPS has been shown to be the most pro-inflammatory. Other groups have sought to exploit this pro-inflammatory signal, for example by deleting pagP so that only hexaacylated LPS is produced (Felgner et al. (2016) Gut Microbes 7(2):171-177; Felgner et al. (2018) Oncommunelogy 7(2): e1382791), but this can lead to poor tolerability due to the TNF-α-mediated pro-inflammatory nature of LPS and, paradoxically, poor adaptive immunity (Kocijancic et al. (2017) Oncotarget 8(30):49988-50001).

[0294] LPS is a potent TLR-4 agonist that induces TNF-α and IL-6. In the IVVNP20009 clinical trial (Toso et al. (2002) J. Clin. Oncol. 20(1):142-152), 1 × 10 9 CFU / m 2 Dose-limiting toxicity in VNP20009 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 the msbB deletion in VNP20009 and its reduced pyrogenicity, LPS can still be toxic at high doses, which may be due to the presence of hexaacylated LPS. Therefore, pagP - / msbB - The strain does not produce hexaacylated LPS and is therefore better tolerated at higher doses, reaching 1 × 10 in humans. 9 CFU / m 2 Higher doses can lead to increased tumor colonization and thus enhance the therapeutic efficacy of the immunostimulatory bacteria.

[0295] Provided herein are live, attenuated Salmonella strains, such as exemplary strains of S. typhimurium, capable of producing only pentaacylated LPS and containing a deletion of the msbB gene (which prevents terminal myristoylation of lipid A, as described above) and further modified by a deletion of pagP (which prevents palmitoylation). When further modified to express an interfering RNA against an immune checkpoint, such as TREX1, strains engineered to produce pentaacylated LPS exhibit reduced levels of pro-inflammatory cytokines, increased susceptibility to and tolerance of antimicrobial peptides, and increased anti-tumor immunity.

[0296] g. Deletion of the SPI-1 and SPI-2 genes As mentioned above, pathogenesis in certain bacterial species, including Salmonella species such as S. typhimurium, involves a cluster of genes called the Salmonella pathogenicity island (SPI; see Figure 22). The SPI, designated SPI-1, mediates epithelial cell invasion. The operon and genes and their functions are shown in Figure 22. SPI-1 genes include, but are 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. Deletion of one or more of these genes reduces or eliminates the ability of bacteria to infect epithelial cells but does not affect their ability to infect or invade phagocytes, including immune phagocytes.

[0297] Salmonella invade non-phagocytic intestinal epithelial cells using a type 3 secretion system (T3SS) encoded by Salmonella pathogenicity island 1, which forms a needle-like structure that injects effector proteins directly into the cytosol of the host cell. These effector proteins result in rearrangements of the eukaryotic cytoskeleton to facilitate invasion of the intestinal epithelium and also induce proinflammatory cytokines. The SPI-1 locus contains 39 genes encoding components of this invasion apparatus (see, e.g., Figure 22, reproduced from Kimbrough and Miller (2002) Microbes Infect. 4(1):75-82).

[0298] SPI-1 encodes a type 3 secretion system (T3SS) that contributes to the translocation of effector proteins into the host cell cytosol and may contribute actin, leading to Salmonella uptake. The SPI-1 T3SS is essential for crossing the gastrointestinal epithelial layer but is not critical for infection when bacteria are injected parenterally. The injection of some proteins and the needle complex itself can also induce inflammasome activation and phagocyte pyroptosis. This pro-inflammatory cell death can limit the initiation of robust adaptive immune responses by directly inducing antigen-presenting cell (APC) death, as well as altering the cytokine environment and preventing the generation of memory T cells. The SPI-1 genes comprise several operons, including sitABCD, sprB, avrA, hilC, orgABC, prgKJIH, hilD, hilA, iagB, sptP, sicC, iacP, sipADCB, sicA, spaOPQRS, invFGEABCIJ, and invH.

[0299] T3SSs are complexes that play a major role in the infectivity of Gram-negative bacteria by injecting bacterial protein effectors directly into host cells in an ATP-dependent manner. The T3SS complex traverses the bacterial inner and outer membranes and creates a pore in the eukaryotic plasma membrane upon contact with the host cell. They consist of an efflux mechanism, a needle complex, and a translocon at the top of the needle (Figure 22). The needle complex contains the needle protein PrgI, a basal body that anchors the complex within the bacterial membrane and is composed of proteins PrgH, PrgK, and InvG, and other proteins, including InvH, PrgJ (a rod protein), and InvJ. The translocon, which forms the pore in the host cell, is a complex of proteins SipB, SipC, and SipD. The efflux mechanism, which enables translocation of effector proteins, is composed of proteins SpaP, SpaQ, SpaR, SpaS, InvA, InvC, and OrgB. The cytoplasmic sorting platform that establishes the specific order of protein secretion is composed of the proteins SpaO, OrgA, and OrgB (Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0300] Effectors translocated into host cells by T3SS-1 include SipA, SipC, SopB, SopD, SopE, SopE2, and SptP, which are essential for cell invasion. For example, S. typhimurium sipA mutants exhibit a 60-80% reduction in invasion, sipC deletion results in a 95% reduction in invasion, and sopB deletion results in a 50% reduction in invasion (Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364). Other effectors include AvrA, which regulates Salmonella-induced inflammation. Chaperones that bind to secreted proteins and maintain them in a secretion-competent conformation include SicA, InvB, and SicP. Transcriptional regulators include HilA, HilD, InvF, SirC, and SprB. Unclassified T3SS SPI-1 proteins that play various roles in type III secretion include OrgC, InvE, InvI, IacP, and IagB (see Figure 22 in Kimbrough et al. (2002) Microbes Infect. 4(1):75-82).

[0301] Thus, inactivation of SPI-1-dependent invasion by inactivating or knocking out one or more genes involved in the SPI-1 pathway, including but not limited to, 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, eliminates the ability of bacteria to infect epithelial cells.

[0302] Salmonella mutants lacking T3SS-1 have been shown to invade numerous cell lines and types via a T3SS-1-dependent invasion mechanism involving several proteins, including the invasins Rck, PagN, and HlyE. The rck operon contains six open reading frames: pefI, srgD, srgA, srgB, rck, and srgC. pefI encodes a transcriptional regulator of the pef operon, involved in the biogenesis of Pef pili. These pili are involved in biofilm formation, adhesion to the mouse small intestine, and fluid accumulation in suckling mice. SrgA oxidizes the disulfide bond of PefA, the major structural subunit of Pef pili. srgD encodes a putative transcriptional regulator; SrgD acts with PefI to induce a synergistic negative regulator of flagellar gene expression. srgB encodes a putative outer membrane protein and srgC encodes a putative transcriptional regulator (Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0303] Rck is a 17-kDa outer membrane protein encoded by the large virulence plasmids of S. Enteritidis and S. Typhimurium that induces epithelial cell adhesion and invasion and confers high levels of resistance to complement neutralization by preventing the formation of the membrane attack complex. While rck mutants exhibit a two- to three-fold reduction in epithelial cell invasion compared to wild-type strains, Rck overexpression results in increased invasion. Rck induces cell invasion through a receptor-mediated process that promotes localized actin remodeling and extension of weakly tight membranes. Thus, Salmonella can invade cells by two distinct mechanisms: a T3SS-1-mediated trigger mechanism and an Rck-induced zipper mechanism (Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0304] The invasin PagN is an outer membrane protein that has also been shown to be involved in Salmonella invasion. pagN expression is regulated by phoP. Certain stimuli, such as the acidified macrophage phagosomal environment or low Mg, mediate the growth of Salmonella. 2+ The concentration of Salmonella is sensed by PhoQ, which then activates PhoP to regulate specific genes. Deletion of pagN in S. typhimurium has been shown to result in a three-fold decrease in invasion of enterocytes without altering cell adhesion. The mechanism of PagN-mediated invasion is not fully understood, but actin polymerization has been shown to be necessary for invasion. Studies have shown that PagN is required for Salmonella survival in BALB / c mice, and that pagN mutants are less competitive than the parent strain for colonizing the mouse spleen. Because pagN is activated by PhoP, it is primarily expressed in cells where the SPI-1 island, which encodes T3SS-1, is downregulated. Thus, bacteria exiting epithelial cells or macrophages may have optimal levels of PagN expression but low levels of T3SS-1 expression that may mediate subsequent interactions with other cells encountered after host cell destruction, indicating a role for PagN in Salmonella pathogenesis (Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0305] hlyE shares greater than 90% sequence identity with the E. coli HlyE (ClyA) hemolysin. The HlyE protein lyses epithelial cells upon extrusion from bacterial cells by outer membrane vesicle shedding and is involved in epithelial cell invasion. HlyE is also involved in the establishment of systemic Salmonella infections (Manon et al. (2012), Salmonella, Chapter 17, eds. Annous and Gurtler, Rijeka, pp. 339-364).

[0306] Loss of the ability to infect epithelial cells can also be achieved by genetically engineering the immunostimulatory bacteria herein to contain a knockout or deletion of a gene encoding a protein involved in SPI-1-independent entry, such as one or more of the genes rck, pagN, hlyE, pefI, srgD, srgA, srgB and srgC.

[0307] Immunostimulatory bacteria as described herein are provided that are modified so that they do not infect epithelial cells but retain the ability to infect phagocytes, including tumor-resident immune cells, thereby effectively targeting the immunostimulatory bacteria to the tumor microenvironment. This is achieved by deleting or knocking out any of the proteins in SPI-1, including, but not limited to, deleting 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, and one or more of rck, pagN, hlyE, pefI, srgD, srgA, srgB, rck and srgC.

[0308] Immunostimulatory bacteria that do not infect epithelial cells can be further modified as described herein to encode products that stimulate the immune stimulatory system, including, for example, cytokines. The bacteria generally have an asd deletion that renders them unable to replicate in a mammalian host.

[0309] For example, provided are strains of S. typhimurium that have been modified by deletion of one or more SPI-1 genes, and also by one or more of purI, msbB, and asd deletions, and by delivery of a plasmid encoding a protein that stimulates the immune system, such as a gene encoding an immunostimulatory cytokine. For example, provided are bacteria that have deletions of regulatory genes (e.g., hilA or invF), T3SS-1 structural genes (e.g., invG or prgH), and / or T3SS-1 effector genes (e.g., sipA or avrA) required for expression of the SPI-1-associated type 3 secretion system (T3SS-1). As discussed above, this secretion system is involved in injecting effector proteins into the cytosol of host non-phagocytic cells, such as epithelial cells, resulting in bacterial uptake, and deletion of one or more of these genes abolishes infection / invasion of epithelial cells. Deletion of one or more of these genes, such as hilA, results in immunostimulatory bacteria that can infect phagocytes by intravenous or intratumoral administration, do not require the SPI-1 T3SS for uptake, and extend the lifespan of these phagocytes. hilA mutations also reduce the amount of pro-inflammatory cytokines, increasing the tolerability of therapy as well as the quality of the adaptive immune response.

[0310] Salmonella also possesses Salmonella pathogenicity island 2 (SPI-2), which encodes another T3SS that is activated after bacterial entry into host cells and interferes with phagosome maturation, resulting in the generation of specialized Salmonella-containing vacuoles (SCVs) in which Salmonella reside during intracellular survival and replication. SPI-2 T3SS effectors include SseB, SseC, SseD, and SpiC, which are involved in the assembly of an F-actin coat around intracellular bacteria. This actin coat promotes fusion of SCVs with actin-containing or actin-propelled vesicles and prevents fusion with undesirable counterparts. SifA is involved in the formation of Salmonella-induced filaments (SIFs), tubules that connect individual SCVs within infected cells. sifA is essential for maintaining SCV integrity, and sifA mutants are released into the host cell cytosol. SseF and SseG are components of the SPI-2 T3SS involved in SCV positioning and the cellular transport process that directs materials necessary for bacterial survival and replication to the SCV. 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 maintaining vacuolar integrity; SpvC, SseL, and SspH1, which are involved in host immune signaling; and SteC, SspH2, SrfH / SseI, and SpvB, which are involved in SCV F-actin meshwork formation, migration of infected phagocytes, inhibition of actin polymerization, and P-body disassembly within infected cells (Coburn et al. (2007) Clinical Microbiology Reviews 20(4):535-549; Figueira and Holden (2012) Microbiology 158:1147-1161).

[0311] The immunostimulatory bacteria herein can contain deletions or modifications of any of the SPI-2 T3SS genes that affect the formation or integrity of SCVs and related structures, such as SIFs. These mutants have increased SCV escape frequency and are able to replicate in the cytosol. For example, because the lipid bilayer of the SCV is a potential barrier, immunostimulatory bacteria, such as Salmonella species, engineered to escape SCVs are more efficient at delivering macromolecules, such as plasmids. This can be achieved, for example, by deletion or mutation of genes required for Salmonella-induced filament (SIF) formation, including sifA, sseJ, sseL, sopD2, pipB2, sseF, sseG, spvB, and steA.

[0312] Immunostimulatory bacteria capable of evading SCV can be further modified as described herein to encode products that stimulate the immune system, including, for example, cytokines. These bacteria generally have an asd deletion that renders them unable to replicate in a mammalian host.

[0313] h. Endonuclease (endA) mutation to increase plasmid delivery The endA gene (e.g., SEQ ID NO: 250) encodes an endonuclease (e.g., SEQ ID NO: 251) that mediates degradation of double-stranded DNA in the periplasm of Gram-negative bacteria. Mutations in the endA gene enable high yields of plasmid DNA, so the most common strains of laboratory E. coli are endA-. This gene is conserved with the bacterial species. To facilitate intact plasmid DNA delivery, the endA gene of genetically engineered immunostimulatory bacteria is deleted or mutated to prevent its endonuclease activity. An exemplary such mutation is the E208K amino acid substitution (Durfee, et al. (2008) J. Bacteriol. 190(7):2597-2606) or a corresponding mutation in the bacterial species of interest. The E208-containing endA is conserved with bacterial species, including Salmonella. Therefore, the E208K mutation can be used to eliminate endonuclease activity in other bacterial species, including Salmonella species. Those skilled in the art can introduce other mutations or deletions to eliminate endA activity. This mutation, deletion, or perturbation of the gene to eliminate endA activity in the immunostimulatory bacteria herein, such as Salmonella, increases the efficiency of intact plasmid DNA delivery, resulting in increased expression of RNAs, such as shRNAs and / or miRNAs, that target one or more of the immune checkpoints encoded in the plasmid, thereby increasing RNAi-mediated knockdown of checkpoint genes and enhancing anti-tumor efficacy.

[0314] i. RIG-I inhibition One of the TLR-independent type I IFN pathways is mediated by host recognition of single-stranded (ss) and double-stranded (ds) RNA in the cytosol. These are sensed by RNA helicases, including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), which induce type I IFN induction through the IFN-β promoter stimulator 1 (IPS-1) adaptor protein-mediated phosphorylation of the IRF-3 transcription factor (Ireton and Gale (2011) Viruses 3(6):906-919). RIG-I recognizes dsRNA and ssRNA with 5'-triphosphates. This moiety can bind directly to RIG-I or can be synthesized by poly(dA-dT)-dependent RNA polymerase III (Pol III) from a poly(dA-dT) template (Chiu, YH et al. (2009) Cell 138(3):576-91). A poly(dA-dT) template containing two AA dinucleotide sequences occurs at the U6 promoter transcription start site in a common lentiviral shRNA cloning vector. Its subsequent deletion in the plasmid blocks type I IFN activation (Pebernard et al. (2004) Differentiation. 72:103-111). The RIG-I binding sequence can be included in the plasmids provided herein; its inclusion can increase immune stimulation, which increases the antitumor activity of the immunostimulatory bacteria herein.

[0315] j.DNase II inhibition Another nuclease responsible for exogenous and self-DNA degradation is the endonuclease DNase II, which resides in endosomal compartments and degrades DNA after apoptosis. The absence of DNase II (Dnase2a in mice) leads to the accumulation of endosomal DNA, which escapes into the cytosol and activates cGAS / STING signaling (Lan YY et al. (2014) Cell Rep. 9(1):180-192). Similar to TREX1, DNase II deficiency in humans results in autoimmune type I interferonopathy. In cancer, dead tumor cells engulfed by tumor-resident macrophages prevent cGAS / STING activation and potential autoimmunity via DNase II digestion of DNA in the endosomal compartment (Ahn et al. (2018) Cancer Cell 33:862-873). Thus, embodiments of the immunostimulatory bacterial strains provided herein encode RNAi, such as shRNA or miRNA, that can inhibit, suppress, or disrupt the expression of DNase II and inhibit DNase II in the tumor microenvironment, resulting in the accumulation of endocytosed apoptotic tumor DNA in the cytosol and can act as potent cGAS / STING agonists.

[0316] k.RNase H2 inhibition While TREX1 and DNase II function to remove abnormal DNA accumulation, RNase H2 similarly functions to eliminate pathogenic accumulation of RNA:DNA hybrids in the cytosol. Similar to TREX1, RNase H2 deficiency also contributes to the autoimmune phenotype of Aicardi-Goutières syndrome (Rabe, B. (2013) J Mol Med. 91:1235-1240). Notably, loss of RNase H2 and the subsequent accumulation of RNA:DNA hybrids or genome-embedded ribonucleotide substrates have been shown to activate cGAS / STING signaling (MacKenzie et al. (2016) EMBO J. Apr15;35(8):831-44). Thus, embodiments of the immunostimulatory bacterial strains provided herein encode RNAi, such as shRNA or miRNA, that inhibit, suppress, or disrupt the expression of RNAse H2, thereby inhibiting RNase H2 and resulting in tumor-derived RNA:DNA hybrids and their derivatives, which activate cGAS / STING signaling and anti-tumor immunity.

[0317] l. Stabilin-1 / CLEVER-1 inhibition Another molecule that is primarily expressed on monocytes and is involved in immunoregulation is stabilin-1 (also known by the gene names STAB1, CLEVER-1, and FEEL-1). Stabilin-1 is a type I transport membrane protein that is upregulated on endothelial cells and macrophages, particularly tumor-associated macrophages, after inflammation (Kzhyshkowska et al. (2006) J. Cell. Mol. Med. 10(3):635-649). When inflammation is activated, stabilin-1 acts as a scavenger, assisting wound healing and apoptotic body clearance and preventing tissue damage such as liver fibrosis (Rantakari et al. (2016) PNAS 113(33):9298-9303). Upregulation of stabilin-1 has been shown to directly inhibit antigen-specific T cell responses, and knockdown by siRNA in monocytes enhances their pro-inflammatory function (Palani, S. et al. (2016) J Immunol. 196:115-123). Accordingly, embodiments of immunostimulatory bacterial strains provided herein encode RNAi, such as shRNA or miRNA, that inhibit, suppress, or disrupt stabilin-1 / CLEVER-1 expression in the tumor microenvironment, thereby enhancing the pro-inflammatory function of tumor-resident macrophages.

[0318] 5. Immunostimulatory proteins The immunostimulatory bacteria herein can be modified to encode an immunostimulatory protein that promotes, induces, or strengthens an anti-tumor response. The immunostimulatory protein can be encoded on a plasmid in the bacteria under the control of a eukaryotic promoter, such as a promoter recognized by RNA polymerase II, for expression in a eukaryotic subject, particularly a subject such as a human to whom the immunostimulatory bacteria will be administered. In addition to the eukaryotic promoter, the nucleic acid encoding the immunostimulatory protein can include other regulatory signals for expression or transport in cells, for example, for secretion or expression on the surface of the cell.

[0319] Immunostimulatory proteins are those that can promote, participate in, or enhance anti-tumor responses by subjects to which the immunostimulatory bacteria are administered in an appropriate environment, such as the tumor microenvironment (TME). Immunostimulatory proteins include, but are not limited to, cytokines, chemokines, and costimulatory molecules. These include, but are not limited to, cytokines such as IL-2, IL-7, IL-12, IL-15, and IL-18; chemokines such as, but are not limited to, CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11; and / or costimulatory molecules such as, but are not limited to, CD40, CD40L, OX40, OX40L, 4-1BB, 4-1BBL, members of the TNF / TNFR superfamily, and members of the B7-CD28 family. Other such immunostimulatory proteins known to those of skill in the art that are used in the treatment of tumors or that can promote, enhance, or otherwise enhance or induce an anti-tumor response are contemplated for encoding in the immunostimulatory bacteria provided herein.

[0320] The genome of the immunostimulatory bacteria provided herein can also be modified to increase or promote infection of immune cells, particularly immune cells in tumor microenvironments, such as phagocytes. Bacteria can also be modified to reduce pyroptosis in immune cells. Immunostimulatory bacteria include those with modifications that disrupt / inhibit the SPI-1 pathway, such as disruption or deletion of hilA and / or disruption / deletion of flagellin genes, rod proteins, needle proteins, and / or pagP, as described and exemplified elsewhere herein.

[0321] Immunostimulatory bacteria encoding cytokines and chemokines In some embodiments, the immunostimulatory bacteria herein are genetically engineered to express cytokines to stimulate the immune system, including, but not limited to, IL-2, IL-7, IL-12 (IL-12p70 (IL12p40 + IL-12p35)), IL-15 (and IL-15:IL-15R alpha chain complex), and IL-18. Cytokines stimulate immune effector cells and stromal cells at the tumor site and enhance tumor cell recognition by cytotoxic cells. In some embodiments, the immunostimulatory bacteria can be genetically engineered to express chemokines, such as CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11.

[0322] IL-2 Interleukin-2 (IL-2), the first cytokine approved for the treatment of cancer, is involved in activating the immune system through several mechanisms, including activating and promoting CTL growth, lymphokine-activated killer (LAK) cell development, promoting Treg cell growth and proliferation, stimulating TILs, and promoting T cell, B cell, and NK cell proliferation and differentiation. Recombinant IL-2 (rIL-2) has been approved by the FDA for the treatment of metastatic renal cell carcinoma (RCC) and metastatic melanoma (Sheikhi et al. (2016) Iran J. Immunol. 13(3):148-166).

[0323] IL-7 IL-7, a member of the IL-2 superfamily, is involved in T cell survival, proliferation, and homeostasis. Mutations in the IL-7 receptor have been shown to result in T cell loss and the development of severe combined immunodeficiency (SCID), highlighting the important role IL-7 plays in T cell development. IL-7 is a homeostatic cytokine that provides continuous signals to resting naive and memory T cells and accumulates during lymphopenic states, leading to an increase in both T cell proliferation and T cell repertoire diversity. Compared to IL-2, IL-7 inhibits CD4 + FOXP3 + CD8 rather than regulatory T cells +It is selective for T cell expansion. Recombinant IL-7 has been shown to enhance antigen-specific T cell responses after vaccination and adoptive cell therapy in mice. IL-7 may also be involved in promoting T cell recovery after chemotherapy for hematopoietic stem cell transplantation. Early clinical trials in patients with advanced malignancies have demonstrated that recombinant IL-7 is well tolerated and has limited toxicity at bioactive doses (i.e., circulating CD4 + and CD8 + IL-7 has been shown to have antitumor effects in tumors such as glioma, melanoma, lymphoma, leukemia, prostate cancer, and glioblastoma, and in vivo administration of IL-7 in mouse models resulted in reduced cancer cell growth. IL-7 has also been shown to enhance the antitumor effects of IFN-γ in rat glioma tumors and induce the production of IL-1α, IL-1β, and TNF-α by monocytes, resulting in the inhibition of melanoma growth. In addition, administration of recombinant IL-7 after treatment of pediatric sarcoma promoted immune recovery (Lin et al. (2017) Anticancer Research 37:963-968).

[0324] IL-12(IL-12p70(IL-12p40+IL-12p35) Bioactive IL-12 (IL-12p70), which promotes immune-mediated immunity, is a heterodimer composed of p35 and p40 subunits, whereas IL-12p40 monomers and homodimers act as IL-12 antagonists. IL-12 secreted by antigen-presenting cells promotes IFN-γ secretion from NK and T cells, inhibits tumor angiogenesis, and stimulates NK cells, CD8 + T cells and CD4 + Activation and proliferation of T cells, CD4 +IL-12 promotes the differentiation of Th0 cells into Th1 cells and promotes antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells. IL-12 has been shown to have antitumor effects in mouse models of melanoma, colon cancer, breast cancer, and sarcoma (Kalinski et al. (2001) Blood 97:3466-3469; Sheikhi et al. (2016) Iran J. Immunol.13(3):148-166; Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893).

[0325] IL-15 and IL-15:IL-15Rα IL-15 is structurally similar to IL-2, and although both IL-2 and IL-15 provide the initial stimulus for T cell proliferation and activation, IL-15 blocks IL-2-induced apoptosis, a process that leads to the loss of stimulated T cells and the induction of T cell tolerance, potentially limiting memory T cell responses and limiting the therapeutic effectiveness of IL-2 alone. IL-15 also mediates the activation of memory CD8 T cells to maintain long-term antitumor immunity. + It also supports T cell survival and inhibits CD8 in an antigen-independent manner in preclinical mouse models. + We demonstrated significant antitumor activity through direct activation of effector T cells. + In addition to T cells, IL-15 is involved in the development, proliferation, and activation of effector natural killer (NK) cells (Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893; Han et al. (2011) Cytokine 56(3):804-810).

[0326] IL-15 and IL-15 receptor alpha (IL-15Rα) are coordinately expressed by antigen-presenting cells such as monocytes and dendritic cells, and IL-15 is expressed by CD8 +IL-15 is presented in trans by IL-15Rα to the IL-15Rβγc receptor complex expressed on the surface of T cells and NK cells. Soluble IL-15:IL15-Rα complexes have been shown to modulate immune responses via the IL-15Rβγc complex, and the biological activity of IL-15 has been shown to be increased 50-fold by administering it as a preformed complex of IL-15 and soluble IL-15Rα, which has an increased half-life compared to IL-15 alone. This significant increase in the therapeutic efficacy of IL-15 due to pre-association with IL-15Rα has been demonstrated in mouse tumor models (Han et al. (2011) Cytokine 56(3):804-810).

[0327] IL-18 IL-18 stimulates NK and CD8 + IL-18 induces the secretion of IFN-γ by T cells, enhancing their virulence. IL-18 also activates macrophages and promotes the expression of Th1 helper CD4 + IL-18 has shown promising antitumor activity in several preclinical mouse models. For example, administration of recombinant IL-18 (rIL-18) inhibited CD4 T cell proliferation in allogeneic mice. +Activation of T cell- and / or NK cell-mediated responses has resulted in regression of melanoma or sarcoma. Other studies have shown that the antitumor effects of IL-18 are mediated by IFN-γ and involve angiogenic mechanisms. Combining IL-18 with other cytokines, such as IL-12, or with costimulatory molecules, such as CD80, enhances the IL-18-mediated antitumor effects. Phase I clinical trials in patients with advanced solid tumors and lymphomas have shown that IL-18 administration is safe and that it exerts immunomodulatory activity, resulting in increased serum IFN-γ and GM-CSF levels and some clinical responses in patients. Clinical trials have shown that IL-18 can be combined with other anti-cancer therapeutic agents, such as monoclonal antibodies, cytotoxic drugs, or vaccines (Fabbi et al. (2015) J. Leukoc. Biol.97:665-675; Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893).

[0328] An attenuated strain of Salmonella typhimurium engineered to express IL-18 was found to inhibit the growth of sc tumors or lung metastasis after systemic administration in syngeneic mice without any toxic effects. Treatment with this engineered bacterium induced the accumulation of T cells, NK cells, and granulocytes in the tumor, resulting in intratumoral production of cytokines (Fabbi et al. (2015) J. Leukoc. Biol.97:665-675).

[0329] chemokines Chemokines are a family of small cytokines that mediate leukocyte migration to sites of injury or inflammation and are involved in mediating immune and inflammatory responses. Chemokines are classified into four subfamilies based on the location of cysteine residues in their sequences: XC chemokine ligands, CC chemokine ligands, CXC chemokine ligands, and CX3C chemokine ligands, or XCLs, CCLs, CXCLs, and CX3CLs. Chemokine ligands bind to their cognate receptors and regulate the circulation, homing, and retention of immune cells, and each chemokine ligand-receptor pair selectively regulates a specific type of immune cell. Different chemokines attract different leukocyte populations, forming a concentration gradient in vivo, which induces migration of attracted immune cells toward higher chemokine concentrations (Argyle D. and Kitamura, T. (2018) Front. Immunol. 9:2629; Dubinett et al. (2010) Cancer J. 16(4):325-335). Chemokines can improve antitumor immune responses by increasing immune cell infiltration into tumors and promoting the migration of antigen-presenting cells (APCs) to tumor-draining lymph nodes, where they stimulate naive T and B cells (Lechner et al. (2011) Immunotherapy 3(11):1317-1340). The immunostimulatory bacteria herein can be genetically engineered to encode chemokines, including but not limited to CCL3, CCL4, CCL5, CXCL9, CXCL10 and CXCL11.

[0330] CCL3, CCL4, CCL5 CCL3, CCL4, and CCL5 share a high degree of homology and bind to CCR5 (CCL3, CCL4, and CCL5) and CCR1 (CCL3 and CCL5) on several cell types, including mature DCs and T cells, in both humans and mice. Therapeutic T cells have been shown to induce chemotaxis of innate immune cells to tumor sites through tumor-specific secretion of CCL3, CCL4, and CCL5 (Dubinett et al. (2010) Cancer J. 16(4):325-335).

[0331] CCL3 is released upon induction of T helper cell type 1 (Th1) responses. In vivo and in vitro studies in mice have demonstrated that both neutrophils and monocytes exhibit chemotaxis toward CCL3. Specifically, CCL3 can mediate myeloid progenitor cell (MPC) mobilization from the bone marrow, exerting MPC regulatory and stimulatory effects. CCL3-transfected human ovarian cancer cells demonstrated enhanced T cell infiltration and macrophage infiltration within tumors, leading to improved antitumor responses. Furthermore, CCL3-mediated chemotaxis of neutrophils was shown to suppress tumor growth. DCs transfected with the tumor antigen human melanoma-associated gene (MAGE)-1, recruited by CCL3, demonstrated superior antitumor efficacy in a mouse model of melanoma, including increased lymphocyte proliferation, cytolytic capacity, survival, and reduced tumor growth. The combination of an antigen-specific platform for MAGE-1 with CCL3 has also been used to treat gastric cancer. CCL3 production by CT26, a highly immunogenic murine colon tumor, has been shown to signal tumor growth in vivo, a process driven by the CCL3-dependent accumulation of natural killer (NK) cells and, in turn, IFNγ, resulting in the production of CXCL9 and CXLC10 (Allen et al. (2017) Oncoimmunology 7(3):e1393598; Schaller et al. (2017) Expert Rev. Clin. Immunol.13(11):1049-1060).

[0332] CCL3 has been used as an adjuvant in cancer treatment. Administration of the active CCL3 mutant ECI301 after radiofrequency ablation in murine hepatocellular carcinoma enhanced tumor-specific responses, a mechanism further shown to be dependent on CCR1 expression. CCL3 has also shown success as an adjuvant in systemic cancers; accordingly, mice vaccinated with CCL3 and IL-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF) in leukemia / lymphoma models showed increased survival (Schaller et al. (2017) Expert Rev. Clin. Immunol. 13(11): 1049-1060).

[0333] CCL3 and CCL4 upregulate CD8 in melanoma and colon cancer + It is involved in directing T cell infiltration to the primary tumor site. Tumor production of CCL4 is associated with CD103 + Suppression of CCL4 by a WNT / β-catenin-dependent pathway leads to the accumulation of DCs and the uptake of CD103 into melanoma tumors. + CCL3 inhibited DC infiltration into primary tumor sites in a mouse model of colon cancer (Spranger et al. (2015) Nature 523(7559):231-235). + and CD8 + It has also been shown to enhance T cell infiltration (Allen et al. (2017) Oncoimmunology 7(3):e1393598).

[0334] Binding of CCL3 or CCL5 to their receptors (CCR1 and CCR5, respectively) induces the migration of immature DCs, monocytes, and memory and effector T cells from the circulation to sites of inflammation or infection. For example, CCL5 expression in colorectal tumors contributes to T lymphocyte chemoattraction and survival. CCL3 and CCL5 have been used alone or in combination therapy to induce tumor regression and immunity in several preclinical models. For example, studies have shown that subcutaneous injection of Chinese hamster ovary cells genetically modified to express CCL3 resulted in tumor inhibition and neutrophil infiltration. In another study, a recombinant oncolytic adenovirus expressing CCL5 (Ad-RANTES-E1A) resulted in primary tumor regression and blocked metastasis in a mouse model of breast cancer (Lechner et al. (2011) Immunotherapy 3(11):1317-1340).

[0335] In translational studies of colorectal cancer, CCL5 induced an "antiviral response pattern" in macrophages. CCL5 is produced as a result of CXCR3-mediated migration of lymphocytes at the invasive front of liver metastases in colorectal cancer. Blockade of the CCL5 receptor, CCR5, resulted in tumor death, which was driven by macrophages producing IFN and reactive oxygen species. Macrophages reside in the tumor microenvironment, and CCR5 inhibition induced a phenotypic shift from an M2 to an M1 phenotype. CCR5 blockade also resulted in clinical responses in colorectal cancer patients (Halama et al. (2016) Cancer Cell 29(4):587-601).

[0336] CCL3, CCL4 and CCL5 can be used to treat conditions including lymphoid tumors, bladder cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, ovarian cancer, cervical cancer or liver cancer (U.S. Patent Application Publication No. 2015 / 0232880; International Patent Publication Nos. WO2015 / 059303, WO2017 / 043815, WO2017 / 156349 and WO2018 / 191654).

[0337] CXCL9, CXCL10, CXCL11 CXCL9 (MIG), CXCL10 (IP10), and CXCL11 (ITAC) are induced by IFN-γ production. These chemokines bind to CXCR3, which is preferentially expressed on activated T cells, and function both as angiogenesis inhibitors and in the recruitment and activation of leukocytes. The prognosis of colorectal cancer is influenced by the level of tumor-infiltrating T cells, particularly Th1 and CD8. + High intratumoral expression of CXCL9, CXCL10, and CXCL11, which strongly correlate with effector T cells, indicates a favorable prognosis. For example, in samples from 163 patients with colon cancer, those with high CXCL9 or CXCL11 levels showed increased postoperative survival, and patients with high CXC expression had significantly more CD3 + T cells, CD4 + Helper T cells and CD8 + In liver metastases from colorectal cancer patients, CXCL9 and CXCL10 levels were increased at the invasive front and correlated with effector T cell density. Stimulation of lymphocyte migration by the action of CXCL9 and CXCL10 on CXCR3 resulted in the production of CCL5 at the invasive front (Halama et al. (2016) Cancer Cell 29(4):587-601; Kistner et al. (2017) Oncotarget 8(52):89998-90012).

[0338] In vivo, CXCL9 functions as a chemoattractant for tumor-infiltrating lymphocytes, activated peripheral blood lymphocytes, natural killer (NK) cells, and Th1 lymphocytes. CXCL9 is important for T cell-mediated suppression of skin tumors. For example, when combined with systemic IL-2, CXCL9 binds CXCR3 + It has been shown to inhibit tumor growth by increasing intratumoral infiltration of mononuclear cells. In a mouse model of colon cancer, the combination of huKS1 / 4-IL-2 fusion protein and CXCL9 gene therapy inhibited CD8 + and CD4 +Chemoattraction and activation of T lymphocytes achieved excellent antitumor effects and extended lifespan (Dubinett et al. (2010) Cancer J. 16(4):325-335; Ruelmann et al. (2001) Cancer Res. 61(23):8498-8503).

[0339] Activated T cells exhibit chemotaxis toward CXCL10, which is produced by activated monocytes, fibroblasts, epithelial cells, and keratinocytes, and this CXCL10 can act as an inhibitor of angiogenesis in vivo. Expression of CXCL10 in colorectal tumors has been shown to contribute to the chemoattraction of cytotoxic T lymphocytes and prolonged survival. Administration of immunostimulatory cytokines, such as IL-12, has been shown to enhance the antitumor effects induced by CXCL10. DC vaccines primed with tumor cell lysates and transfected with CXCL10 increased immunological protection and efficacy in mice, and animals exhibited resistance to tumor challenge, slowed tumor growth, and longer survival. In vivo and in vitro studies in mice using a CXCL10-mucin-GPI fusion protein showed that tumors recruited higher levels of NK cells compared with tumors not treated with the fusion protein. Interferon (which can be produced by plasmacytoid dendritic cells, which are associated with primary melanoma lesions and can be recruited to tumor sites by CCL20) has been shown to produce CXCL9 / 10 in mouse melanoma models and is associated with CXCL9 / 10 in human disease by tumor DC subsets, such as CD103. +It can act on DCs. CXCL10 also shows higher expression in human metastatic melanoma samples compared to primary melanoma tumors. Therapeutically, adjuvant IFN-α melanoma therapy upregulates CXCL10 production, whereas the chemotherapy drug cisplatin induces CXCL9 and CXCL10 (Dubinett et al. (2010) Cancer J. 16(4):325-335; Kuo et al. (2018) Front. Med. (Lausanne) 5:271; Li et al. (2007) Scand. J. Immunol. 65(1):8-13; Muenchmeier et al. (2013) PLoS One 8(8):e72749).

[0340] CXCL10 / 11 and CXCR3 expression have been confirmed in human keratinocytes derived from basal cell carcinoma (BCC). CXCL11 can also promote immunosuppressive indoleamine 2,3-dioxygenase (IDO) expression in human basal cell carcinoma and enhance keratinocyte proliferation, which may be related to any infiltrating CXCR3 + This may reduce the antitumor activity of effector T cells (Kuo et al. (2018) Front. Med. (Lausanne) 5:271).

[0341] CXCL9, CXCL10, and CXCL11 can be encoded into oncolytic viruses to treat cancer (U.S. Patent Application Publication No. 2015 / 0232880, International Patent Publication No. WO2015 / 059303). Pseudotyped oncolytic viruses or genetically engineered bacteria encoding the gene for CXCL10 can also be used to treat cancer (International Patent Publication Nos. WO2018 / 006005 and WO2018 / 129404).

[0342] costimulatory molecules Costimulatory molecules enhance immune responses to tumor cells, and costimulatory pathways are inhibited by tumor cells to promote tumorigenesis. The immunostimulatory bacteria herein can be genetically engineered to express costimulatory molecules, such as CD40, CD40L, 4-1BB, 4-1BBL, OX40 (CD134), OX40L (CD252), other members of the TNFR superfamily (e.g., CD27, GITR, CD30, Fas receptor, TRAIL-R, TNF-R, HVEM, RANK), B7, and CD28. The immunostimulatory bacteria herein can also be genetically engineered to express agonistic antibodies against costimulatory molecules to enhance anti-tumor immune responses.

[0343] TNF receptor superfamily TNF superfamily ligands (TNFSFs) and their receptors (TNFRSFs) are involved in the proliferation, differentiation, activation, and survival of tumor and immune effector cells. Members of this family include CD30, Fas-L, TRAIL-R, and TNF-R, which induce apoptosis, and CD27, OX40L, CD40L, GITR-L, and 4-1BBL, which regulate B and T cell immune responses. Other members include herpesvirus entry mediator (HVEM) and CD27. Expression of TNFSFs and TNFRSFs by immunostimulatory bacteria herein can enhance antitumor immune responses. For example, it has been shown that expression of 4-1BBL in mouse tumors enhances immunogenicity, and intratumoral injection of dendritic cells (DCs) with increased expression of OX40L can result in tumor rejection in a mouse model. Studies have also shown that injection of adenoviruses expressing recombinant GITR into B16 melanoma cells promotes T cell infiltration and reduces tumor volume. Stimulatory antibodies against molecules such as 4-1BB, OX40, and GITR can also be coded into immunostimulatory bacteria to stimulate the immune system. For example, agonistic anti-4-1BB monoclonal antibodies have been shown to enhance anti-tumor CTL responses, and agonistic anti-OX40 antibodies have been shown to increase anti-tumor activity in transplantable tumor models. In addition, agonistic anti-GITR antibodies have been shown to enhance anti-tumor responses and immunity (Lechner et al. (2011) Immunotherapy 3(11):1317-1340; Peggs et al. (2009) Clinical and Experimental Immunology 157:9-19).

[0344] CD40 and CD40L CD40, a member of the TNF receptor superfamily, is expressed by APCs and B cells, whereas its ligand, CD40L (CD154), is expressed by activated T cells. The interaction between CD40 and CD40L stimulates B cells to produce cytokines, resulting in T cell activation and tumor cell death. Studies have shown that antitumor immune responses are impaired by reduced expression of CD40L on T cells or reduced expression of CD40 on dendritic cells. CD40 is expressed on the surface of several B cell tumors, such as follicular lymphoma, Burkitt lymphoma, lymphoblastic leukemia, and chronic lymphocytic leukemia, and its interaction with CD40L reduces the expression of CD40. + It has been shown to increase the expression of B7.1 / CD80, B7.2 / CD86, and HLA class II molecules on tumor cells, improving their antigen-presenting capacity. Transgenic expression of CD40L in a mouse model of multiple myeloma enhances CD4 + and CD8 + This resulted in the induction of T cell, localized, and systemic antitumor immune responses and reduced tumor growth. Anti-CD40 agonist antibodies also induced antitumor T cell responses (Marin-Acevedo et al. (2018) Journal of Hematology & Oncology 11:39; Dotti et al. (2002) Blood 100(1):200-207; Murugaiyan et al. (2007) J Immunol. 178:2047-2055).

[0345] 4-1BB and 4-1BBL 4-1BB (CD137) is an inducible costimulatory receptor expressed by T cells, NK cells, and APCs (including DCs, B cells, and monocytes) that binds to its ligand, 4-1BBL, to induce immune cell proliferation and activation. 4-1BB results in longer and broader responses of activated T cells. Anti-4-1BB agonists and 4-1BBL fusion proteins inhibit CD4 + and CDs +It has been shown to increase T cell-mediated immune-mediated antitumor activity, for example against sarcomas and mastocytoma, as well as tumor-specific CTL activity (Lechner et al. (2011) Immunotherapy 3(11):1317-1340; Marin-Acevedo et al. (2018) Journal of Hematology & Oncology 11:39).

[0346] OX40 and OX40L OX40 (CD134) is a member of the TNF receptor superfamily expressed on activated effector T cells, whereas its ligand, OX40L, is expressed on APCs, including DCs, B cells, and macrophages, after activation by TLR agonists and CD40-CD40L signaling. OX40-OX40L signaling leads to T cell activation, enhancement, proliferation, and survival, as well as modulation of NK cell function and inhibition of Treg suppressive activity. OX40 signaling also leads to the secretion of cytokines (IL-2, IL-4, IL-5, and IFN-γ), resulting in enhanced Th1 and Th2 cell responses. Recognition of tumor antigens by TILs leads to increased expression of OX40 by TILs, and this increased expression has been shown to correlate with improved prognosis. Studies have shown that treatment with anti-OX40 agonist antibodies or Fc-OX40L fusion proteins significantly increased tumor-specific CD4 expression in mouse models of melanoma, sarcoma, colon cancer, and breast cancer. + While resulting in enhanced T cell responses and increased survival, Fc-OX40L incorporated into tumor cell vaccines has been demonstrated to protect mice from subsequent challenge with breast cancer cells (Lechner et al. (2011) Immunotherapy 3(11):1317-1340; Marin-Acevedo et al. (2018) Journal of Hematology & Oncology 11:39).

[0347] B7-CD28 family CD28 is a costimulatory molecule expressed on the surface of T cells that acts as a receptor for B7-1 (CD80) and B7-2 (CD86), which are costimulatory molecules expressed on antigen-presenting cells. CD28-B7 signaling is required for T cell activation and survival and for preventing T cell anergy, leading to the production of interleukins such as IL-6.

[0348] Optimal T cell priming requires two signals: (1) T cell receptor (TCR) recognition of MHC-presented antigen and (2) a costimulatory signal resulting from ligation of T cell CD28 with B7-1 (CD80) or B7-2 (CD86) expressed on antigen-specific receptors (APCs). Following T cell activation, the CTLA-4 receptor is induced, which then outcompetes CD28 for binding to B7-1 and B7-2 ligands. Antigen presentation by tumor cells is poor because these cells are unable to activate the T cell receptor complex due to the lack of expression of costimulatory molecules such as B7-1 / CD80 and B7-2 / CD86. Consequently, upregulation of these molecules on the surface of tumor cells can enhance their immunogenicity. Immunotherapy for solid tumors and hematologic malignancies has been successfully induced by B7, for example, via tumor cell expression of B7 or via soluble B7-immunoglobulin fusion proteins. Viral-mediated tumor expression of B7 in combination with other costimulatory ligands, such as ICAM-3 and LFA-3, has been successful in preclinical and clinical trials for the treatment of chronic lymphocytic leukemia and metastatic melanoma. In addition, soluble B7 fusion proteins have demonstrated promising results as single-agent immunotherapies in the immunotherapy of solid tumors (Lechner et al. (2011) Immunotherapy 3(11):1317-1340; Dotti et al. (2002) Blood 100(1):200-207).

[0349] 6. Modifications that increase immune cell uptake of Gram-negative bacteria such as Salmonella and reduce immune cell death The genome of the immunostimulatory bacteria provided herein can be modified to increase or promote infection of immune cells, particularly immune cells in the tumor microenvironment, such as phagocytes. This includes reducing infection of non-immune cells, such as epithelial cells, or increasing infection of immune cells. Bacteria can also be modified to reduce pyroptosis in immune cells. Numerous modifications of bacterial genomes can increase infection of immune cells and / or reduce pyroptosis. The immunostimulatory bacteria provided herein include such modifications, for example, deletion and / or disruption of genes involved in the SPI-1 T3SS pathway, such as disruption or deletion of hilA, and / or disruption / deletion of genes encoding flagellin, rod protein, and needle protein.

[0350] The invasive phenotype of Gram-negative bacteria, such as Salmonella, can result from the activity of genes encoded in pathways that facilitate invasion of host cells. The Salmonella invasion-associated Salmonella pathogenicity island-1 (SPI-1) is a prime example. SPI-1 contains a type 3 secretion system (T3SS) that contributes to the translocation of effector proteins into the host cell cytosol. These proteins can trigger actin rearrangements that lead to the uptake of Salmonella. T3SS effectors mediate the uptake of S. typhimurium into non-phagocytic host cells, such as epithelial cells. The SPI-1 T3SS has been shown to be essential for crossing the gastrointestinal epithelial layer but is not critical for infection, for example, when bacteria are parenterally injected. SPI-1 mutants are defective in epithelial cell invasion, thus dramatically reducing oral virulence, but are normally taken up by phagocytes such as macrophages (Kong et al. (2012) Proc. Natl. Acad. Sci. USA 109(47):19414-19419). The immunostimulatory S. typhimurium strains provided herein can be engineered with mutations in SPI-1 T3SS genes that prevent their uptake by epithelial cells and concentrate them in immune cells such as macrophages, enhancing anti-tumor immune responses.

[0351] T3SS effectors can also activate the NLRC4 inflammasome in macrophages, resulting in activation of caspase-1 and cell death by pyroptosis. Pyroptosis is a highly inflammatory, programmed cell death that occurs most frequently after infection with intracellular pathogens and is involved in antibacterial responses. Proinflammatory cell death can limit the initiation of robust adaptive immune responses by directly inducing antigen-presenting cell (APC) death and by altering the cytokine environment to prevent the development of memory T cells. SPI-1 induces pyroptosis by injecting flagellin, needle, and rod protein (PrgI / J), while extracellular flagellin stimulates TLR5 signaling. Therefore, genetically engineering the immunostimulatory bacteria herein to contain mutations in genes involved in pyroptosis can enhance antitumor immune effects by reducing cell death of immune cells such as macrophages.

[0352] Macrophage pyroptosis The macrophage NLRC4 inflammasome, involved in innate immunity and antibacterial responses, is a large multiprotein complex that recognizes cytosolic pathogens and leads to the autocatalytic activation of caspase-1. Activation of caspase-1 induces the maturation and release of the pro-inflammatory cytokines IL-1β and IL-18, triggering pyroptosis, a rapid inflammatory form of macrophage cell death. Infection with certain Gram-negative bacteria, such as Salmonella typhimurium and Pseudomonas aeruginosa, which encode type 3 or 4 secretion systems, triggers activation of the NLRC4 inflammasome through recognition of bacterial ligands, such as needle protein, rod protein, and flagellin, after translocation of the Stm pathogenicity island-1 into the host cell cytosol by the type III secretion system (SPI-1 T3SS). Pyroptosis is not limited to macrophages; caspase-1-dependent death has been observed in dendritic cells after infection with Salmonella (Li et al. (2016) Scientific Reports 6:37447; Chen et al. (2014) Cell Reports 8:570-582; (Fink and Cookson (2007) Cellular Microbiology 9(11):2562-2570)). As shown herein, knocking out genes in the Salmonella genome involved in inducing pyroptosis enhances antitumor immune responses. This prevents the loss of immune cells, including macrophages, after bacterial infection. For example, genes encoding hilA, rod protein (PrgJ), needle protein (PrgI), flagellin, and / or QseC can be knocked out / disrupted in the immunostimulatory bacteria provided herein.

[0353] hilA The invasion-associated Salmonella pathogenicity island-1 (SPI-1), which contains a type 3 secretion system (T3SS), is responsible for the translocation of effector proteins into the host cell cytosol, triggering actin rearrangements that lead to Salmonella uptake. hilA is a transcriptional activator of the SPI-1 gene, and its expression is regulated by environmental signals, such as oxygen, osmolality, pH, and growth phase. Suboptimal conditions suppress the bacterial invasive phenotype by repressing hilA expression (Kong et al. (2012) Proc. Natl. Acad. Sci. USA 109(47):19414-19419). T3SS effectors mediate the uptake of S. typhimurium into non-phagocytic host cells, such as eukaryotic cells. The SPI-1 T3SS is essential for crossing the gastrointestinal epithelial layer but is not essential for infection, such as when bacteria are parenterally injected. SPI-1 mutants are defective in epithelial cell invasion, thus reducing oral virulence, but are normally taken up by phagocytes such as macrophages. The immunostimulatory bacteria provided herein include those with deletion or disruption of the hilA gene and / or other genes in the T3SS pathway. When these bacteria are administered, for example, intravenously or intratumorally, infection is concentrated in phagocytes 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. They prevent invasion of off-target cells and prevent fecal-oral infection.

[0354] In addition to reducing Salmonella uptake by non-phagocytic cells, such as epithelial cells, deletion or disruption of hilA and / or other genes in this pathway also extends phagocyte lifespan by preventing pyroptosis in macrophages and, therefore, inducing less cell death in human macrophages compared to bacteria lacking the hilA deletion. For example, HilA-deficient Salmonella strains prevent pyroptosis by preventing inflammasome activation, but maintain TLR5 signaling. HilA deletion / disruption also allows for sustained secretion of cytokines, such as those encoded by the immunostimulatory bacteria provided herein, and macrophage trafficking to tumors, thereby improving the efficacy of the immunostimulatory bacteria. For example, compared to S. typhimurium containing an intact hilA, such as VNP20009, the hilA deletion mutants presented herein further reduce the amount of pro-inflammatory cytokines, such as IL-6, resulting in improved treatment tolerability and a higher quality of adaptive immune responses.

[0355] Flagellin In addition to the SPI-1 T3SS, flagellin from bacteria such as Salmonella is required for the induction of pyroptosis in macrophages and can be detected by the macrophage NLRC4 inflammasome. Flagellin, a major component of flagella, is recognized by TLR5. Salmonella encodes two flagellin genes, fliC and fljB, and loss of flagellin subunits reduces pyroptosis in macrophages. For example, S. typhimurium with deletions of fliC and fljB results in significantly reduced IL-1β secretion compared to wild-type strains, yet bacterial cellular uptake and intracellular replication remain unaffected. This demonstrates that flagellin plays a significant role in inflammasome activation. Additionally, S. typhimurium strains genetically engineered to constitutively express FliC have been found to induce macrophage pyroptosis (Li et al. (2016) Scientific Reports 6:37447; Fink and Cookson (2007) Cellular Microbiology 9(11):2562-2570; Winter et al. (2015) Infect. Immun.83(4):1546-1555). The genome of the immunostimulatory bacteria herein can be modified to delete or mutate the flagellin genes fliC and fljB in S. typhimurium, resulting in reduced cell death of tumor-resident immune cells, such as macrophages, and enhancing the anti-tumor immune response of the immunostimulatory bacteria.

[0356] Rod protein (PrgJ) NLRC4 also detects flagellated S. typhimurium. A flagellin-independent response was discovered due to the deletion of PrgJ, an SPI-1 T3SS rod protein, in S. typhimurium. Delivery of purified PrgJ protein into macrophage cytosol resulted in rapid NLRC4-dependent caspase-1 activation and IL-1β secretion, similar to the effects induced by flagellin (Miao et al. (2010) PNAS 107(7):3076-3080). Thus, mutation or knockout of the gene encoding PrgJ in S. typhimurium can reduce macrophage pyroptosis, which enhances the antitumor immune effects of immunostimulatory bacteria by protecting immune cells susceptible to killing by these bacteria.

[0357] Needle protein (PrgI) PrgI, the SPI-1 T3SS needle protein in S. typhimurium, is also recognized by and activates NLRC4. Delivery of S. typhimurium PrgI into the cytosol of human primary monocyte-derived macrophages resulted in IL-1β secretion and subsequent cell death, while a Salmonella mutant expressing PrgI but not flagellin was shown to activate inflammasomes at a later time point in primary monocyte-derived macrophages than a strain expressing flagellin (Kortmann et al. (2015) J. Immunol. 195:815-819). The immunostimulatory bacteria provided herein can be modified to mutate or delete the gene encoding the needle protein in S. typhimurium, thereby preventing immune cell pyroptosis and enhancing anti-tumor immune effects.

[0358] QseC 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 virulence factors, including the flhDC gene, which encodes the master regulator of flagellum biosynthesis in S. typhimurium; the sopB gene, which encodes a protein involved in the entry of non-phagocytic cells, regulating the early maturation and transport of Salmonella-containing vacuoles (SCVs), and inhibiting SCV-lysosome fusion; and the sifA gene, which is required for SCV maintenance and membrane integrity. Selective inhibition of QseC by LED209 has been shown to inhibit bacterial virulence by blocking QseC-mediated activation of virulence-related gene expression (e.g., flhDC, sifA, and sopB) without suppressing S. typhimurium growth, and to provide some protection to mice against death after infection with S. typhimurium or Francisella tularensis. Blockade of QseC was found to inhibit caspase-1 activation, IL-1β release, and S. typhimurium-induced pyroptosis of macrophages by inhibiting excessive inflammasome activation in infected macrophages. Inhibition of QseC also suppressed flagellar gene expression and motility, and inhibited S. typhimurium's ability to invade and replicate in epithelial cells (Li et al. (2016) Scientific Reports 6:37447). Therefore, modifying the immunostimulatory bacteria herein to mutate or knock out the gene encoding QseC can enhance antitumor immune responses by focusing S. typhimurium infection on non-epithelial cells and reducing immune cell death, for example, by blocking pyroptosis in macrophages.

[0359] 7. Bacterial culture conditions Culture conditions for bacteria can affect their gene expression. It has been reported that S. typhimurium can induce rapid, proinflammatory, caspase-dependent cell death in macrophages, but not epithelial cells, within 30–60 minutes of infection through a mechanism involving SPI-1 and its associated T3SS-1 (Lundberg et al. (1999) Journal of Bacteriology 181(11):3433–3437). This cell death is now known to be mediated by activation of the inflammasome, which activates caspase-1, which then promotes the maturation and release of IL-1β and IL-18, initiating a novel form of cell death termed pyroptosis (Broz and Monack (2011) Immunol Rev. 243(1):174–190). This pyroptotic activity can be induced using log-phase bacteria, whereas stationary-phase bacteria do not induce this rapid cell death in macrophages. The SPI-1 gene is induced during logarithmic phase growth. Therefore, by harvesting S. typhimurium to be used therapeutically at stationary phase, rapid pyroptosis of macrophages can be prevented. Macrophages are important mediators of the innate immune system, and they can act to secrete cytokines that are important for establishing an appropriate anti-tumor response. Furthermore, limiting the secretion of pro-inflammatory cytokines such as IL-1β and IL-18 will improve the tolerability of the administered S. typhimurium therapy. The immunostimulatory S. typhimurium harvested at stationary phase provided herein will be used to induce anti-tumor responses.

[0360] E. Bacterial attenuation and colonization 1. Deletion of flagellin (fliC) - / fljB - ) Immunostimulatory bacteria, such as Salmonella species S. typhimurium, are provided that are genetically engineered to lack both flagellin subunits fliC and fljB to reduce pro-inflammatory signaling. For example, as shown herein, a Salmonella strain lacking msbB is combined with a fliC and fljB knockout, resulting in reduced TNF-alpha induction. The resulting Salmonella strain combines reduced TNF-alpha induction with reduced TLR5 recognition. These modifications, msbB, - , fliC - and fljB - can be combined with a bacterial plasmid, which may contain CpGs and may also contain a cDNA expression cassette under the control of a eukaryotic promoter to provide for the expression of a therapeutic protein, such as, for example, an immunostimulatory protein, e.g., a cytokine or chemokine, e.g., IL-2, and / or an inhibitory molecule, e.g., an antibody, including an antibody fragment such as a nanobody, and / or an RNAi molecule(s) targeting an immune checkpoint, such as TREX1, PD-L1, VISTA, SIRP-alpha, TGF-beta, beta-catenin, CD47, VEGF, and combinations thereof. The resulting bacteria have reduced pro-inflammatory signaling and robust anti-tumor activity.

[0361] For example, the fliC exemplified herein - and fljB -A double mutant was constructed in an asd deletion strain of S. typhimurium strain VNP20009 or in wild-type Salmonella typhimurium, such as one that has all of the identifying characteristics of the strain deposited under ATCC accession number 14028. VNP20009, a derivative of ATCC 14028, was attenuated for virulence by disruption of purI / purM and also engineered to contain an msbB deletion, which results in production of a lipid A subunit of LPS that is less toxigenic than wild-type lipid A. This resulted in reduced TNF-α production after intravenous administration in a mouse model compared to strains with wild-type lipid A.

[0362] fliC - and fljB - The double mutant was constructed using a wild-type strain of S. typhimurium and also genetically engineered to contain the asd, purI / purM, and msbB deletions. - The resulting strain is an example of a strain in which bacterial inflammation is attenuated by lipid A modification to reduce TLR2 / 4 signaling and deletion of flagellin subunits to reduce TLR5 recognition and inflammasome induction. The deletion of flagellin subunits combined with LPS modification allows for greater tolerance in the host and directs the immunostimulatory response toward the production of immunostimulatory proteins. Delivery of RNA interference by the engineered bacteria to desired targets in the TME elicits antitumor responses and promotes adaptive immune responses against tumors.

[0363] 2. Detection of genes in the LPS biosynthetic pathway The LPS of Gram-negative bacteria is a major component of the outer leaflet of the bacterial membrane. It is composed of three main moieties: lipid A, a nonrepeating core oligosaccharide, and the O antigen (or O polysaccharide). The O antigen is the outermost part of LPS and serves as a protective layer against bacterial permeability, but the sugar composition of the O antigen varies widely among bacterial strains. Lipid A and the core oligosaccharide are less distinct and are more typically conserved within strains of the same bacterial species. Lipid A is the portion of LPS that contains endotoxin activity. It is typically a disacchar...

Claims

1. 1. An immunostimulatory bacterium for use in the treatment of cancer, comprising a plasmid encoding one or more therapeutic products, wherein the therapeutic products are anti-cancer therapeutic products, and wherein the genome of said immunostimulatory bacterium is modified such that the bacterium does not produce flagellin and lacks flagella, and wherein the wild-type bacterium comprises flagella.

2. An immunostimulatory bacterium comprising a plasmid encoding one or more therapeutic products, wherein the genome of said immunostimulatory bacterium has been modified so that the bacterium lacks flagella and is pagP- / msbB-, and the wild-type bacterium contains flagella.

3. An immunostimulatory bacterium for use in the treatment of cancer, said bacterium comprising a plasmid encoding a therapeutic product under the control of a eukaryotic promoter, and said bacterium comprising a genomic modification such that the bacterium expresses pagP. － / msbB － , immunostimulatory bacteria.

4. 4. The immunostimulatory bacterium of claim 1, wherein the anti-cancer therapeutic product is an immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment.

5. The immunostimulatory bacterium according to claim 4, wherein the immunostimulatory protein that confers or contributes to anti-tumor immunity in the tumor microenvironment is a cytokine or a chemokine.

6. Immunostimulatory proteins that confer or contribute to an anti-tumor immune response in the tumor microenvironment include IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-36 gamma, IL-2 that has reduced binding to IL-2Ra, IL-15 / IL-15R alpha chain complex, IL-18, IL-21, IL-23, IL-2 modified to have reduced or no binding to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, interferon-β, and IL-2.

6. The immunostimulatory bacterium of claim 4 or 5, selected from one or more of feron-γ, CCL3, CCL4, CCL5, proteins involved in or causing or promoting T-cell recruitment / persistence, CD40, CD40 ligand, CD28, OX40, OX40 ligand, 4-1BB, 4-1BB ligand, members of the B7-CD28 family, CD47 antagonists, TGF-beta polypeptide antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily.

7. The immunostimulatory bacterium of any one of claims 1 to 5, wherein the therapeutic product is a TGF-beta antagonist polypeptide.

8. The immunostimulatory bacterium of any one of claims 1 to 7, wherein the therapeutic product is or further comprises IL-15 or an IL-15 / IL-15R alpha chain complex.

9. 9. The immunostimulatory bacterium of claim 1, wherein the plasmid encodes a therapeutic product that is or further comprises an antibody or an antigen-binding fragment thereof.

10. 10. The immunostimulatory bacterium of any one of claims 1 to 9, wherein the therapeutic product is an antibody or fragment thereof that is a tandem scFv that is a dual affinity retargeting (DART) antibody or a bispecific T cell-engaging antibody (BiTE).

11. The immunostimulatory bacterium of claim 9, wherein the antibody or antigen-binding fragment thereof is an antagonist of PD-1, PD-L1, CTLA-4, VEGF, VEGFR2, or IL-6.

12. The immunostimulatory bacteria contain a genome modification such that the bacteria expresses csgD. － 12. The immunostimulatory bacterium according to any one of claims 1 to 11, wherein

13. 13. The immunostimulatory bacterium of claim 1, wherein a nucleic acid encoding a therapeutic product is operably linked for expression to a nucleic acid encoding a secretion signal, such that the therapeutic product is secreted upon expression.

14. 14. The immunostimulatory bacterium of any one of claims 1 to 13, which is adenosine auxotrophic or adenosine and adenine auxotrophic.

15. Aspartate-semialdehyde dehydrogenase － (asd － 15. The immunostimulatory bacterium according to any one of claims 1 to 14, wherein

16. Aspartate-semialdehyde dehydrogenase － (asd － ) in which all or part of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (ASD) is disrupted or deleted, so that endogenous ASD is not expressed, resulting in ASD － 16. The immunostimulatory bacterium of claim 1 , wherein

17. 17. The immunostimulatory bacterium of any one of claims 1 to 16, wherein aspartate-semialdehyde dehydrogenase (asd) is encoded on a plasmid.

18. genomic modifications that cause the bacterium to － , purI － , msbB － , flagellin － and pagP － 18. The immunostimulatory bacterium of claim 1 , wherein

19. 19. The immunostimulatory bacterium of any one of claims 1 to 18, which encodes one or more therapeutic products on a plasmid in the bacterium, the expression of said products being under the control of eukaryotic regulatory signals.

20. 20. The immunostimulatory bacterium of claim 19, wherein the regulatory signal comprises a eukaryotic promoter that is an RNA polymerase II promoter.

21. 21. The immunostimulatory bacterium of any one of claims 4 to 12 and 14 to 20, wherein an immunostimulatory protein that confers or contributes to anti-tumor immunity in the tumor microenvironment is encoded on a plasmid such that when expressed it is membrane-anchored.

22. 22. The immunostimulatory bacterium of any one of claims 1 to 21, wherein the plasmid encodes a therapeutic product that is a cytotoxin.

23. 23. The immunostimulatory bacterium of claim 22, wherein the therapeutic product is a cytotoxin selected from among ricin, saporin, and shiga toxin.

24. 24. The immunostimulatory bacterium of any one of claims 1 to 23, which encodes a tumor antigen.

25. 25. The immunostimulatory bacterium of any one of claims 1 to 24, wherein the bacterium encodes an inhibitory RNA (RNAi) or CRISPR cassette, wherein the RNAi or CRISPR cassette results in the inhibition, perturbation, and / or suppression of an immune checkpoint.

26. 26. The immunostimulatory bacterium of any one of claims 1 to 25, comprising nucleic acids encoding two of the therapeutic proteins.

27. 27. The immunostimulatory bacterium of any one of claims 1 to 26, which has been treated with polymyxin to reduce pyrogenicity.

28. 28. A pharmaceutical composition comprising the immunostimulatory bacteria of any one of claims 1 to 27 in a pharmaceutically acceptable vehicle.

29. 29. The immunostimulatory bacterium of any one of claims 1 to 27 or the pharmaceutical composition of claim 28 for use in treating cancer in a subject.

30. 30. The immunostimulatory bacterium or pharmaceutical composition of claim 29, wherein the cancer comprises a solid tumor or a hematological malignancy.

31. 31. The immunostimulatory bacterium or pharmaceutical composition of any one of claims 28 to 30, wherein the treatment comprises a combination therapy regimen including a second anti-cancer agent or treatment.

32. 32. The immunostimulatory bacterium or pharmaceutical composition of claim 31, wherein the second anti-cancer agent or treatment is immunotherapy, radiation or chemotherapy.

33. 33. The immunostimulatory bacterium or pharmaceutical composition of any one of claims 1 to 32, wherein the cancer is selected from leukemia, lymphoma, gastric cancer, and cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectum, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix and liver.

34. 28. An isolated or cultured cell comprising an immunostimulatory bacterium according to any one of claims 1 to 27.

35. 35. The isolated or cultured cell of claim 34, which is or comprises an immune cell, a stem cell, a tumor cell or a primary cell line.

36. 36. The isolated or cultured cell of claim 35, which is or comprises a hematopoietic cell or a T cell.

37. 37. The immunostimulatory bacterium, pharmaceutical composition or cell of any one of claims 1 to 36, wherein the bacterium is a strain of the genera Salmonella, Shigella, E. coli, Bifidobacterium, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, and Erysipelothrix, or an attenuated or modified strain thereof of any of the preceding lists of strains.

38. 38. The immunostimulatory bacterium, pharmaceutical composition or cell of any one of claims 1 to 37, wherein the immunostimulatory bacterium is Salmonella, Shigella, Listeria, or E. coli.

39. 39. The immunostimulatory bacterium, pharmaceutical composition or cell of any one of claims 1 to 38, wherein the immunostimulatory bacterium is a strain of the genus Salmonella.

40. genome modification, so that the bacterium － / msbB － , csgD － 28. The immunostimulatory bacterium of any one of claims 1 to 27, wherein said bacterium is a bacterium having a phenotype of β-actin and lacks flagella.

41. 41. The immunostimulatory bacterium, pharmaceutical composition or cell of claim 39 or 40, wherein the immunostimulatory bacterium is a Salmonella typhimurium strain or serotype.

Images