Immunostimulatory bacteria for converting macrophages to a phenotype suitable for treatment and companion diagnostics for identifying subjects for treatment

Abstract

Provided are methods for treating cancer by converting tumor-resident macrophages to a hybrid M1 / M2 macrophage phenotype, which has attributes that are advantageous for cancer therapy. Hybrid markers (lower than M2 and higher than M1) include SPP1, CD209, and CD206, and inducible markers include MERTK, C1QC, IFNa, IFNb, CXCL10, 4-1BBL, and MYC. The method includes administering a therapeutic agent that achieves the phenotype conversion. The therapeutic agent, such as a delivery vehicle that includes an immunostimulatory bacterium with genomic modifications, is designed to inhibit type I IFN by not inducing or resulting in sufficient TLR2, TLR4, TLR5 responses. The therapeutic agent also encodes a payload that encodes an immunostimulatory protein, e.g., a cytokine, and a modified intracytoplasmic DNA / RNA sensor that constitutively induces type I IFN, such as a modified STING protein. The combination of the properties of the payload immunostimulatory protein and the therapeutic delivery vehicle, when administered, results in macrophages with a hybrid phenotype. The therapeutic agent is administered to a subject identified as having a tumor that contains proliferating M2 macrophages.

Description

[Technical Field]

[0001] Related Applications This application is the sole property of U.S. Provisional Application No. 63 / 378,853, filed October 7, 2022, entitled "IMMUNOSTIMULATORY BACTERIA FOR CONVERTING MACROPHAGES INTO A PHENOTYPE AMENABLE TO TREATMENT AND COMPANION DIAGNOSTIC," applicant Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Chingnam Cheung, Alexandre Charles Michel Iannello, Bret Nicholas Peterson, Nicholas Boyce Woodall, and Akshata Udyavar; and U.S. Provisional Application No. 63 / 311,424, filed February 17, 2022, entitled "IMMUNOSTIMULATORY BACTERIA FOR CONVERTING MACROPHAGES INTO A PHENOTYPE AMENABLE TO TREATMENT AND COMPANION DIAGNOSTIC," applicant Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Chingnam Cheung, Alexandre Charles Michel Iannello, Bret Nicholas Peterson, Nicholas Boyce Woodall, and Akshata Udyavar. Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Chingnam Cheung, Alexandre Charles Michel Iannello, Bret Nicholas Peterson, and Nicholas Boyce Woodall; and each of U.S. Provisional Application Nos. 63 / 277,601 and 63 / 278,076, filed November 9, 2021, and November 10, 2021, respectively (each entitled "IMMUNOSTIMULATORY BACTERIA-BASED VACCINES, THERAPEUTICS, AND RNA DELIVERY PLATFORMS"), each applicant Actym Therapeutics, Inc., and inventors Laura Hix Glickman, Chingnam Cheung, Alexandre Charles Michel Iannello, Bret Nicholas Peterson, and Christopher D.Thanos) claim priority.

[0002] This application is related to International PCT Application PCT / US2021 / 045832, filed August 12, 2021 (published February 17, 2022 as WO2022 / 036159) (entitled "IMMUNOSTIMULATORY BACTERIA-BASED VACCINES, THERAPEUTICS AND RNA DELIVERY PLATFORMS," applicant Actym Therapeutics, Inc., and inventors Laura Hix Glickman, Bret Nicholas Peterson, Haixing Kehoe, Alexandre Charles Michel Iannello, and Christopher D. Thanos).

[0003] This application is related to U.S. Provisional Application No. 63 / 064,869, filed August 12, 2020, entitled "IMMUNOSTIMULATORY BACTERIA DELIVERY PLATFORM," applicant Actym Therapeutics, Inc., and inventors Laura Hix Glickman, Christopher D. Thanos, Alexandre Charles Michel Iannello, Chris Rae, and Haixing Kehoe.

[0004] This application is related to U.S. Provisional Application No. 63 / 188,443, filed May 13, 2021, entitled "IMMUNOSTIMULATORY BACTERIA DELIVERY PLATFORM," applicant Actym Therapeutics, Inc., and inventors Laura Hix Glickman, Christopher D. Thanos, Alexandre Charles Michel Iannello, Chris Rae, and Haixing Kehoe.

[0005] This application is also related to U.S. Patent Application No. 17 / 320,200, filed May 13, 2021, entitled "IMMUNOSTIMULATORY BACTERIA DELIVERY PLATFORMS AND THEIR USE FOR DELIVERY OF THERAPEUTIC PRODUCTS," applicant Actym Therapeutics, Inc., and inventors Laura Hix Glickman, Christopher D. Thanos, Alexandre Charles Michel Iannello, Chris Rae, Haixing Kehoe, Bret Nicholas Peterson, and Chingnam Cheung.

[0006] This application is related to International Patent Application No. PCT / US2020 / 060307, ​​filed November 12, 2020 (published May 20, 2021 as WO 2021 / 097144), entitled "IMMUNOSTIMULATORY BACTERIA DELIVERY PLATFORMS AND THEIR USE FOR DELIVERY OF THERAPEUTIC PRODUCTS," applicant Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Alexandre Charles Michel Iannello, Chris Rae, Haixing Kehoe, Bret Nicholas Peterson, and Chingnam Cheung.

[0007] This application is a continuation of International Patent Application No. PCT / US2020 / 020240, filed February 27, 2020 (published September 3, 2020 as WO 2020 / 176809), and co-pending U.S. Patent Application No. 16 / 824,500, filed March 19, 2020 (published August 27, 2020 as U.S. Patent Application Publication No. 2020 / 0270613A1), each entitled "IMMUNOSTIMULATORY BACTERIA ENGINEERED TO COLONIZE TUMORS, TUMOR-RESIDENT IMMUNE CELLS, AND THE TUMOR MICROENVIRONMENT," filed February 27, 2020, and filed March 19, 2020, and published August 27, 2020, as U.S. Patent Application Publication No. 2020 / 0270613A1, each entitled "IMMUNOSTIMULATORY BACTERIA ENGINEERED TO COLONIZE TUMORS, TUMOR-RESIDENT IMMUNE CELLS, AND THE TUMOR MICROENVIRONMENT," filed February 27, 2020, and filed March 19, 2020, and filed March 19, 2020, and each entitled "IMMUNOSTIMULATORY BACTERIA ENGINEERED TO COLONIZE TUMORS, TUMOR-RESIDENT IMMUNE CELLS, AND THE TUMOR MICROENVIRONMENT," filed February 27, 2020, and filed March 19, 2020, and each entitled "IMMUNOSTIMULATORY BACTERIA ENGINEERED TO COLONIZE TUMORS, TUMOR-RESIDENT IMMUNE CELLS, AND THE TUMOR Kehoe).

[0008] This application is also related to International Patent Application PCT / US2018 / 041713, filed July 11, 2018 (published January 17, 2019 as WO 2019 / 014398), and U.S. Patent Application Serial No. 16 / 033,187, filed November 9, 2021 (filed July 11, 2018 as U.S. Patent No. 11,168,326 and published January 17, 2019 as U.S. Patent Application Publication No. 2019 / 0017050A1), each entitled "ENGINEERED IMMUNOSTIMULATORY BACTERIAL STRAINS AND USES THEREOF," to Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, and Justin Skoble.

[0009] This application is also related to International Patent Application PCT / US2019 / 041489, filed July 11, 2019 (published January 16, 2020 as WO 2020 / 014543), to Actym Therapeutics, Inc., entitled "ENGINEERED IMMUNOSTIMULATORY BACTERIAL STRAINS AND USES THEREOF," inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, and Alexandre Charles Michel Iannello.

[0010] The immunostimulatory bacteria and methods provided in each of these applications can be modified and used as described in the present application, and such bacteria are incorporated herein by reference. Where permitted, the subject matter of each of these applications is incorporated by reference in its entirety.

[0011] Incorporation by reference of electronically provided sequence listings An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. Electronic file created on November 8, 2022. Size is 2,127 kilobytes and titled 1710PCSEQ001.xml.

[0012] FIELD OF THE INVENTION Provided are attenuated immunostimulatory bacteria having genomes that have been modified to, for example, reduce unwanted inflammatory responses and toxicity, and improve antitumor and / or immunostimulatory activity by increasing resistance to complement inactivation, reducing immune cell death, promoting adaptive immunity, and enhancing T cell function. Increased phagocyte colonization for anticancer applications improves delivery of the encoded therapeutic product to the tumor microenvironment and tumor, allowing for systemic administration of the immunostimulatory bacteria, among other routes. The immunostimulatory bacteria provided herein infect proliferating macrophages, and upon expression of their encoded payload, result in a previously undescribed M1 / M2 hybrid phenotype. [Background technology]

[0013] The field of cancer immunotherapy has made great strides, as evidenced by the clinical success of anti-CTLA-4, 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 significantly immunosuppressive environment. Tumors initiate multiple mechanisms to evade immune surveillance, reprogram antitumor immune cells to suppress immunity, and continually mutate resistance to current cancer therapies [see, e.g., Mahoney et al. (2015) Nat. Rev. Drug Discov. 14(8): 561-584]. Overcoming immune tolerance and designing immunotherapies and cancer treatments that avoid but limit the autoimmune-related toxicities of current immunotherapies is a challenge in the field of immuno-oncology. Therefore, additional and innovative immunotherapies and other treatments are needed. Summary of the Invention [Problem to be solved by the invention]

[0014] Immunostimulatory bacteria include other Gram-negative Enterobacteriaceae and Gram-positive bacteria, such as Listeria and Shigella species. As described herein, bacteria can accumulate in immunoprivileged and / or immunosuppressed cells and tissues and have therefore been used for the delivery of active molecules, e.g., therapeutic molecules, to such cells and tissues, including tumors and the tumor microenvironment and immune cells, e.g., phagocytic cells, including macrophages. As described herein, genome modification can improve such properties of bacteria. For example, bacteria can be modified so that they do not infect epithelial cells but retain or enhance infection or uptake by phagocytic cells, such as macrophages. It is also shown herein that proliferative macrophages are advantageous for the delivery of active molecules, e.g., proteins and nucleic acids, particularly for the expression of nucleic acids that require entry across the nuclear membrane for nuclear transcription. Active molecules can be encoded on a plasmid under the control of transcriptional and / or translational regulatory sequences for expression. The encoding nucleic acid may be under the control of prokaryotic regulatory sequences for expression in bacteria for protein delivery; or may be under the control of a bacterial promoter for transcription, but the nucleic acid may be encoded such that the transcript is not translated in the bacterial host, resulting in RNA being delivered to cells and tissues; or the encoding nucleic acid may be under the control of eukaryotic regulatory sequences, e.g., a eukaryotic promoter, for plasmid delivery to cells and tissues. As a result, the immunostimulatory bacteria herein have a variety of applications, including, but not limited to, as anti-tumor therapeutics, anti-cancer vaccines, and vaccines, including vaccines against infectious agents, for RNA delivery, for protein delivery, and other applications that will be apparent from the description and examples herein.

[0015] Immunostimulating bacteria and proliferating macrophages Tumors have evolved a highly immunosuppressive environment. They initiate multiple mechanisms to evade immune surveillance, reprogram antitumor immune cells to suppress immunity, and continually mutate resistance to current cancer treatments [see, e.g., Mahoney et al. (2015) Nat. Rev. Drug Discov. 14(8):561-584]. Across the spectrum of solid tumors, only inflammatory tumors rich in exhausted T cells respond well to checkpoint immunotherapy [e.g., non-small cell lung cancer (NSCLC) and melanoma]. Tumors with T cells excluded from the tumor stroma (T cell-excluded) and tumors lacking T cells (immune desert) do not respond to existing immunotherapies, thus creating an unmet need for therapeutics across solid tumors. Immunotherapeutic efficacy, for example, immune checkpoint inhibitor-mediated antitumor responses, depends on the infiltration of T cells capable of recognizing and killing tumor cells. These immunotherapies are not effective in so-called "cold" tumors, or immune-desert or T-cell-negative tumors, characterized by a lack of T-cell infiltration. The tumor core of T-cell-negative and immune-desert tumors is enriched in myeloid cells. T-cell-negative tumors have abundant tumor-associated macrophages (TAMs), which are immunosuppressive and form a barrier that keeps out T cells [Keren et al. (2018) Cell 174:1373-1387; Bindea et al. (2013) Immunity 39:782-795].

[0016] Successful attenuated vaccinia virus vaccines, such as the modified vaccinia Ankara virus (MVA) for smallpox and the oral poliovirus vaccine (Sabin), can induce appropriate T cell-mediated antiviral immune responses. These vaccines initiate by targeting epithelial or fibroblast cells, inducing apoptosis rather than the lytic cell death characteristic of pathogenic wild-type strains. These apoptotic cells then recruit monocyte-derived and tissue-resident macrophages through caspase-dependent secretion of chemoattractant factors, such as ATP [Elliott et al. (2009) Nature 461:282-286]. Virus-infected apoptotic cells are then phagocytosed by macrophages, which sense the presence of viral intracytoplasmic DNA or RNA and induce type I interferon (IFN), leading to an antiviral signaling cascade that recruits and activates CD8+ T cells [Royo et al. (2014) J Virol 88:5511-5523]. Virus-infected macrophages then migrate to lymph nodes, where they prime CD4+ helper T cells to promote germinal center B cell antibody production and also prime CD8+ T cells, which then transport to infected tissues and remain as long-lived, tissue-resident memory CD8+ T cells. These immune cells are important for recognizing early infection of tissue-resident cells and eliminating them by FasL-induced (Fas ligand or CD95L or CD178, a type II transmembrane protein in the TNF family) apoptosis, often before the virus is detected by the immune system [Hobbs et al. (2018) Curr Opin Virol. 28:12-19 El-Jesr et al. (01 October 2020) Front Immunol doi.org / 10.3389 / immun.2020.568412; Wahid et al. (2005) J Virol 79:401-409].

[0017] Macrophages are the primary immune cells that induce interferon-beta (IFNβ) and CD8+ T cell activation, whereas dendritic cells primarily produce IFNα and activate CD4+ T cells [Corrales et al. (2015) Cell Reports 11:1018-1030; Wahid et al. (2005) J Virol 79:401-409]. The intersection of apoptotic cells, macrophage phagocytosis, and induction of type I IFNs that prime CD8+ T cells is a hallmark of a vaccine's ability to generate lifelong humoral and cellular immunity. Similarly, deficiencies in cytoplasmic nucleases (e.g., DNase II, TREX1) can enable leaked or phagocytosed nuclear DNA to trigger cytoplasmic DNA sensing via cyclic GA synthase (cGAS), which generates cyclic dinucleotide amphiphile (cGAMP), which activates the stimulator of IFN genes (STING) pathway. This results in type I IFN production and induction of CD8+ T cells directed against either self-antigens to induce autoimmunity or tumor antigens to induce anti-tumor immunity [Barber et al. (2015) Nat Rev Immunol 15:760-770; Ahn et al. (2018) Cancer Cell 33:862-873].

[0018] Addressing T cell exclusion and immune desert tumors requires the design of cancer immunotherapies that convert immunosuppressive tumor-associated macrophages (TAMs) into type I IFN-producing macrophages capable of in situ priming of CD8+ T cells against tumor antigens and inducing durable antitumor immunity. The immunostimulatory bacteria provided herein address this need. The immunostimulatory bacteria provided herein, as described herein, are genomically modified to eliminate unwanted bacterial sensing from Toll-like receptor 2 (TLR2), TLR4, and / or TLR5. As described herein, sensing from these TLRs, particularly TLR2, as well as TLR4 and TLR5, directly suppresses macrophages' ability to produce type I IFNs and induces proinflammatory cytokines that impair CD8+ T cell priming. The immunostimulatory bacteria are also modified to contain purine auxotrophy, which provides tumor targeting after systemic, e.g., IV, administration. Immunostimulatory bacteria can also be modified by genomic modification, which eliminates flagella that can only be taken up by phagocytic tumor-resident myeloid cells, including macrophages. Among the immunostimulatory bacteria described and provided herein, genomic modification allows bacteria to infect phagocytes at least to the same extent or at an increased level compared to bacteria that do not contain modifications. Included are bacteria that do not infect epithelial and / or endothelial cells. Macrophages then destroy these attenuated bacteria, providing plasmid transfer and expression of payloads as described herein. Of particular interest are combinatorially encoded payloads, e.g., cytokines, such as IL-15, particularly in an engineered IL-15 / IL-15R alpha chain complex (human IL-15 cytokine fused with the IL-15 receptor alpha chain (IL-15Rα-IL-15sc)), in combination with mutants of cytoplasmic DNA / RNA sensors that render them constitutive, e.g., the STING (eSTING) proteins provided herein that are constitutively active and can also be engineered or selected to favor antiviral type I IFN signaling and limit the generation of immunosuppressive NF-kB signaling.An exemplary cytoplasmic DNA / RNA sensor is a chimeric STING containing a gain-of-function mutation(s) and aSTING protein with reduced NF-κB signaling activity, such as the CTT derived from eSTING (engineered STING) designated huSTING tazCTT N154S / R284G. As shown herein, the combined expression of such cytokines and cytoplasmic DNA / RNA sensors, such as eSTING, in the tumor microenvironment and in macrophages overcomes the immunosuppressive tumor microenvironment in T cell-desert / depleted tumors. As demonstrated in the Examples, the provided immunostimulatory bacteria colonize the tumor microenvironment, deliver their payload to phagocytic APCs, and induce durable antitumor responses. This was observed after a single intravenous dose. For example, the exemplary strain YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD containing a plasmid encoding huIL-15Rα-IL-15sc+huSTING N154S / R284G tazCTT results in IL-15 secretion and IFN-beta expression in cell lines and primary M2 macrophages, respectively. The bacteria are selectively internalized by phagocytic APCs in vitro and by tumor-resident APCs in vivo. Primary M2 macrophages are polarized to a costimulatory and phagocytic hybrid M1 / M2 phenotype. The bacteria induce immune reprogramming and tumor microenvironment remodeling through myeloid and CD8+ T cell infiltration and activation. Furthermore, as illustrated, synergistic antitumor activity was observed in vivo using combination therapy with immunotherapy, e.g., anti-PD-1 antibodies.

[0019] Some of the immunostimulatory bacteria provided herein target tumor-resident tumor-associated macrophages (TAMs) systemically, e.g., after IV administration. Their activity is primarily or exclusively restricted to phagocytic and proliferative TAMs, where they translocate DNA plasmids to the nuclei of dividing cells, where the encoded payload is expressed. Of interest herein are payload combinations, e.g., a cytokine, e.g., IL-15, particularly the IL-15 / IL-15R alpha chain complex, with a STING protein, particularly one that has constitutive activity and may also have reduced NF-κB signaling activity compared to wild-type human STING, e.g., wild-type human STING, e.g., any of SEQ ID NOS: 305-309, which set forth the sequences of allelic variants of human STING protein with substantially the same activity and properties. Also of interest are immunostimulatory bacteria encoding type I interferon as a single payload or in combination with one or more other immunostimulatory proteins, e.g., the IL-15 / IL-15R alpha chain complex and / or modified STING, e.g., constitutive STING as described herein.

[0020] Herein, we demonstrate that the production of combined payloads, e.g., huIL-15Rα-IL-15sc and chimeric STING, e.g., huSTING N154S / R284G tazCTT, by immunostimulatory bacteria, as well as other alternative immunostimulatory payloads, induces a hybrid macrophage phenotype that is particularly advantageous for antitumor therapy. The hybrid macrophage phenotype possesses the immunostimulatory and antitumor properties of M1 or M1-like macrophages while retaining the phagocytic properties of M2 or M2-like tumor-associated macrophages (TAMs). Infected macrophages as described herein also possess a hybrid SPP1+ and C1QC+ (expressing both SPP1 and C1QC) macrophage phenotype, which can be identified as having enhanced phagocytic and proliferation properties. The result is a hybrid macrophage phenotype that can phagocytose apoptotic tumor cells, induce constitutive type I IFN, recruit and prime tumor antigen-specific CD8+ T cells, and induce sustained antitumor immunity.

[0021] The uptake of immunostimulatory bacteria modified as described herein to lack flagella and possess modified LPS and other modifications is specific to phagocytes, particularly human M2 macrophages. These bacteria contain genome modifications that remove inflammatory surface components, including flagella, curli pili, and inflammatory LPS. They also contain adenosine auxotrophy, providing an inevitable dependency on nutrients stored in the TME, such as adenosine, ATP, AMP, and purines, which are immunosuppressive.

[0022] For embodiments in which the encoded payload is expressed by the eukaryotic host cell transcription machinery, expression of the payload occurs in proliferating macrophages, allowing the plasmid to enter the nucleus of the proliferating macrophages. It is also shown and described herein that the immunostimulatory bacteria provided herein are internalized by M2 macrophages but not by HUVECs, whereas VNP20009 is internalized by HUVECs and M2 macrophages, indicating that the immunostimulatory bacteria provided herein, lacking flagella and having pentaacylated LPS, are more specific and better able to colonize target tissues for antitumor treatment and as a vaccine. The bacteria are internalized and transported to the acidic lysosomes and nucleus in human macrophages.

[0023] The bacteria and therapeutic agents described and / or provided herein induce a hybrid M1 / M2 phenotype, and the resulting hybrid M1 / M2 phenotype is shown herein to induce anti-tumor immunity. The M1 / M2 phenotype can be identified by markers indicative of the M1 and / or M2 phenotype, which are up- and / or down-regulated after treatment. Markers detailed herein include costimulatory markers such as CD80 and CD86 and phagocytic markers such as CD206. Thus, therapeutic agents that can deliver non-integrated (non-genomically integrated) therapeutic payloads to macrophages and convert, generate, or result in macrophages with this phenotype can induce durable anti-tumor immunity. Durable anti-tumor immunity is evidenced by a dose-dependent anti-tumor response upon tumor rechallenge. Therapeutics, including immunostimulatory bacteria, reverse the immunosuppressive tumor microenvironment. Bacteria have also been shown herein to convert tumors refractory to immunotherapy, e.g., anti-PD-1, into tumors responsive to immunotherapy. For example, synergistic activity with anti-PD-1 was observed.

[0024] The presence of proliferating macrophages in tumors has been shown to be predictive of the efficacy of therapies that deliver non-integrated nucleic acid payloads. DNA introduced from immunostimulatory bacteria, for example, can be transcribed and translated in proliferating macrophages. Proliferating macrophages in tumors can be identified from biopsies by predictive biomarkers. Proliferating macrophages can be identified by some or all of the following markers: G2M module (>14 genes in the set), oncogene expression of stathmin 1 (STMN1); Biopsy surface markers: CD68+KI67 and / or PCNA, MERTK, Some tumor types: SPP1 in lung, stomach, and / or Some tumor types: C1QC in colon and breast. [Means for solving the problem]

[0025] Provided herein are methods for identifying proliferative macrophages in human tumors and for identifying subjects who may be responsive to treatment with immunostimulatory bacteria, particularly those with payloads containing cytokines and STING. Also provided are methods for therapeutically inducing optimal tumor macrophage phenotypes prior to treatment by administering apoptosis-inducing therapies.

[0026] Immunostimulatory bacteria, therapeutic agents and methods Provided are methods for treating cancer or tumors by administering a therapeutic agent that, upon administration, results in macrophages in the tumor with a hybrid M1 / M2 phenotype. The macrophages that undergo this phenotypic change are proliferative macrophages. Therapeutic agents include any designed or prepared to achieve this phenotypic change. In embodiments in which the delivery vehicle, e.g., an immunostimulatory bacterium, encodes an immunostimulatory protein, it is shown herein that the nucleic acid must enter the nucleus for transcription. This occurs in proliferative macrophages, e.g., immunostimulatory bacterium. The combination of the encoded payload, e.g., an immunostimulatory protein, e.g., a protein that is part of a cytoplasmic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), and also a cytokine, e.g., the IL-15 / IL-15R alpha chain complex, results in a phenotypic change in the macrophage. Proteins that are part of the DNA / RNA sensor pathway include, for example, STING, MDA5, IRF-3, IRF-7, IRF-5, IRF8, and RIG-I, particularly modified forms thereof that result in constitutive expression of type I interferon (IFN). The delivery vehicle can also deliver or encode agonists of these proteins, for example, one or more agonists of STING, MDA5, IRF-3, IRF-5, IRF-7, IRF-8, and / or RIG-I, resulting in type I interferon (IFN) expression.

[0027] Also provided are isolated macrophages that have been treated by introducing a therapeutic agent, e.g., the immunostimulatory bacteria provided herein, which convert the macrophages to an M1 / M2 phenotype. Such macrophages can phagocytose apoptotic tumor cells, induce constitutive type I IFN, recruit and prime tumor antigen-specific CD8+ T cells, and induce durable antitumor immunity. Macrophages or immune cells comprising macrophages can be isolated from a subject, or they can be allogeneic, as appropriate, from subjects with tumor(s), particularly immune desert tumors or T-cell-negative tumors, containing suppressor myeloid cells, and for whom treatments such as immunotherapy have not been effective, or who have been treated with but failed to respond to immunotherapy, e.g., checkpoint inhibitors. Macrophages or cells comprising macrophages can be treated in vitro with a therapeutic agent, e.g., infected with immunostimulatory bacteria, cultured or preserved as needed, and then introduced into a subject, as in cell therapy protocols, thereby providing macrophages with antitumor activity. Macrophages or cells containing macrophages that have been treated in vitro to convert them to M1 / M2 hybrids can be administered systemically, intratumorally, intraperitoneally, or by other routes to produce an anti-tumor response in a subject. Any of the immunostimulatory bacteria, delivery vehicles, and other therapeutic agents provided herein that achieve this phenotype conversion can be introduced into cells, including macrophages. These macrophages can reprogram the tumor microenvironment to produce anti-tumor immunity, including bone marrow repolarization. Provided are compositions and cell cultures containing macrophages with an M1 / M2 phenotype, including compositions formulated for cell therapy. The compositions contain macrophages infected in vitro with any of the bacteria provided herein that induce type I interferon (IFN), such as bacteria encoding DNA / RNA sensor proteins and / or cytokines and bacteria encoding type I interferon (IFN) or multiple such interferons.

[0028] Exemplary therapeutic agents include the immunostimulatory bacteria described and provided herein. Additionally, cancers and / or tumors susceptible to treatment with the immunostimulatory bacteria provided herein include those with elevated adenosine, elevated TGF-beta compared to non-tumor tissue, and / or tumors that are hypoxic. Exemplary cancers include chronic lymphocytic leukemia (CLL) or myeloid malignancies. The immunostimulatory bacteria described and provided herein colonize solid tumors.

[0029] Provided and described herein are immunostimulatory bacteria that are cancer therapeutics due to their ability to effectively colonize tumors, particularly tumor-resident immune cells, and due to the encoded payload that confer an anti-tumor immune response. As described below, upon administration, these bacteria infect or are taken up by phagocytic cells, such as macrophages, in tumors. As described herein, the bacteria and therapeutics described and provided herein can convert macrophage phenotypes to M1 / M2 hybrid phenotypes. Due to their ability to colonize tumors and tumor microenvironments, the immunostimulatory bacteria provided herein can be used to treat immune desert (immune-negative, T-cell-negative, or cold) tumors, which have low or absent T-cell infiltration in the tumor microenvironment and tumor. These include tumors with a stromal barrier. The immunostimulatory bacteria provided herein can convert so-called "cold" tumors, which are resistant or unresponsive to immunotherapy, into "hot" tumors.

[0030] Immunostimulatory bacteria contain the genome modifications described herein, such that they are attenuated in TLR2 / 4 / 5, e.g., by elimination of flagella, have an msbB- / pagP- phenotype, and have additional mutations, such as elimination of curli pili, and other mutations, such as the ansB- phenotype described herein. These bacteria can grow in vivo. The payload encoded in the bacterial plasmid(s) is part of the cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN). In particular, these products include one or more mutations, e.g., gain-of-function mutations, that cause constitutive expression of type I IFN. These immunostimulatory bacteria can also encode cytokine(s), such as IL-15, particularly the IL-15 / IL-15R alpha chain complex, and can encode tumor-associated antigens and / or bispecific T cell engager antibodies. They can also encode type I interferon (IFN) and / or other immunostimulatory proteins. For cancer treatment, bacteria can be administered systemically.

[0031] Described herein are immunostimulatory bacteria, e.g., bacteria with genomic modifications as described herein that target or accumulate in macrophages, e.g., bacteria called STACT (S. Typhimurium-Attenuated Cancer Therapy). STACT bacteria contain specific genomic modifications, such as flagella-free, modified LPS, and other properties. STACT bacteria contain a plasmid. The plasmid encodes a payload of interest, e.g., one or more immunostimulatory proteins, e.g., cytokines, and a cytoplasmic DNA / RNA sensor, e.g., modified STING, which generally has constitutive activity such that type I interferon is constitutively expressed upon infection of macrophages and expression of the encoded STING. For example, the bacteria contain a plasmid encoding a cytokine, e.g., the IL-15 / IL-15R alpha chain complex, and modified STING that constitutively induces type I IFN. These bacteria specifically target immunosuppressive tumor-associated macrophages (TAMs) and reprogram them into phagocytic, T cell-priming macrophages of a hybrid M1 / M2 phenotype.

[0032] Included are bacteria, as described below, that encode a payload under the control of a prokaryotic promoter. In some embodiments, the nucleic acid contains or encodes a signal that prevents or disables translation in the bacteria, thereby delivering the RNA to the tumor microenvironment and tumors and immune cells, including macrophages, within the tumor. In other embodiments, the RNA is transcribed by bacterial ribosomes and the protein is delivered.

[0033] When administered to a subject, the immunostimulatory bacteria provided herein convert immunosuppressive tumor-associated macrophages (TAMs) into tumor antigen-presenting cells (APCs) capable of inducing type I interferon (IFN)-mediated recruitment and in situ priming of CD8+ T cells. When administered to a cancer-bearing subject, the immunostimulatory bacteria achieve phenotypic transformation and induce durable antitumor immunity in T cell-negative solid tumors and immune-desert solid tumors. Immunostimulatory bacteria, for example, bacteria engineered to target macrophages and encoding a combination of cytokines and proteins that constitutively induce type I interferon expression in macrophages, can achieve phenotypic changes. These changes can convert T cell-negative / desert tumors into hot tumors that are sensitive to treatment with immunotherapies such as anti-checkpoint antibodies. Furthermore, the immunostimulatory bacteria provided herein that encode one or more type I interferons (IFNs) can be used to convert T cell-negative and desert tumors into hot tumors that are responsive to immunotherapy, such as therapy with immune checkpoint inhibitors.

[0034] The immunostimulatory bacteria herein have genome modifications that make them auxotrophic for purines and purine metabolites, particularly adenosine. Purines and metabolites accumulate to pathological concentrations in tumors but not in healthy tissues, and among purine metabolites is adenosine, which is immunosuppressive. Thus, the immunostimulatory bacteria that accumulate in the tumor microenvironment and tumors reduce the concentration and reverse or prevent the immunosuppressive effect of the accumulation of metabolites such as adenosine.

[0035] The immunostimulatory bacteria provided herein are genomically modified to reduce bacterial component recognition by TLR2, TLR4, and TLR5. These modifications reduce or prevent TLR-mediated signaling pathways that impair the production of inflammatory cytokines that suppress CD8+ T cell priming and macrophage induction of type I IFN. Type I IFN induction can be constitutively mediated by expression of a bacterially encoded modified cytoplasmic DNA / RNA sensor, such as STING, particularly a modified STING that constitutively regulates type I interferon expression in macrophages. Following phagocytosis of the administered bacteria, such as by intravenous administration, the bacteria are rapidly eliminated, and the immunomodulatory payload encoded by the plasmid is ectopically expressed. As shown herein, because the prerequisite for bacterial uptake and DNA plasmid transfer to the nucleus is that macrophages are phagocytic and proliferative, payload delivery is restricted to immunosuppressive tumor antigens (TAMs) in the tumor microenvironment and not to phagocytic macrophages in the liver or other tissues.

[0036] The immunostimulatory bacteria provided herein, and the methods and uses therein, address an unmet need for treating tumors that are rich in macrophages but lack T cells and are unresponsive to existing immunotherapies. The bacteria provided herein are taken up by phagocytes and achieve conversion to an M1 / M2 hybrid phenotype. Proliferating macrophages are shown herein to be able to translocate bacterial plasmids into the nucleus and transcribe the encoded payload, which is then translated to produce immunostimulatory or immunomodulatory proteins, such as modified STING and cytokines. An exemplary example of these bacteria is called STACT. STACT contains a plasmid-encoded human IL-15 cytokine fused to the IL-15 receptor alpha chain (IL-15plex) and engineered constitutive STING (eSTING). Examples herein demonstrate that such bacteria promote CD8+ T cell-mediated tumor clearance in T cell-deficient tumors, induce durable antitumor immunity, and have a highly favorable safety profile after IV administration in primates. The combination of IL-15plex and eSTING delivered by STACT to immunosuppressive TAMs induces a previously unknown hybrid M1 / M2 macrophage phenotype. Macrophages with this phenotype exhibit the immune stimulatory and T cell priming properties of M1-like macrophages while retaining the tumor cell phagocytic properties of M2-like TAMs. As shown herein, infected macrophages possess a hybrid SPP1+C1QC+ phenotype with enhanced phagocytic and proliferation properties. The resulting macrophages with this phenotype phagocytose apoptotic tumor cells, induce type I IFNs, produce IL-15, and recruit, prime, and maintain tumor antigen-specific CD8+ T cells, promoting durable antitumor immunity.

[0037] As noted above, in exemplary embodiments, some of the immunostimulatory bacteria provided herein are referred to by the acronym STACT (S. Typhimurium-Attenuated Cancer Therapy). These are exemplary of the immunostimulatory bacteria described and provided herein. The immunostimulatory bacteria, including the exemplary bacterium designated STACT, contain genomic modifications that confer advantageous properties, including, but not limited to, (1) enhanced tolerability after IV administration compared to the unmodified parent strain VNP20009 (also referred to as YS1456), (2) tumor-specific enrichment, (3) phagocytosis by tumor-resident antigen-presenting cells (APCs) that lack epithelial cell infectivity, (4) multigene cargo delivery, and (5) attenuation of bacterial pathways that impair CD8+ T cell function. Specific examples of exemplary STACT immunostimulatory bacteria are those that encode immunomodulatory molecules including immunostimulatory proteins, e.g., cytokines, e.g., IL-15, e.g., the IL-15 / IL-15R alpha chain complex, and modified STING proteins that constitutively induce type I IFN. Exemplary of a STACT immunostimulatory bacteria is the strain designated YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD.

[0038] It is shown herein that the presence of proliferating macrophages in tumors can be predictive of the efficacy of a therapy that delivers a non-integrated nucleic acid payload. Proliferating macrophages in tumors can be identified from biopsies by predictive biomarkers. Proliferating macrophages can be identified by the following markers: G2M module (>14 genes in the set), oncogene expression of stathmin 1 (STMN1); Biopsy surface markers: CD68+KI67 and / or PCNA, MERTK, Some tumor types: SPP1 in lung, stomach, Some tumor types: C1QC in colon and breast.

[0039] Provided herein are methods for identifying proliferative macrophages in human tumors and for identifying subjects who will be responsive to treatment with immunostimulatory bacteria, for example, bacteria encoding a payload, wherein the expressed payload comprises cytokines and STING. Also provided are methods for therapeutically inducing optimal tumor macrophage phenotypes prior to treatment by administering apoptosis-inducing therapy.

[0040] Immunostimulatory bacteria encoding other combinations of encoded immune stimulatory proteins are provided. These include cytokines, type I interferon (IFN) inducers, costimulatory receptors, checkpoint antibodies, and TGFβR-Fc decoys. The engineered STING (eSTING) proteins described herein may have constitutive type I IFN-inducing activity and may have reduced NF-κB signaling activity compared to wild-type human STING. Combinations of eSTING and immune stimulatory proteins were evaluated in primary human APCs using in vitro functional assays. Many combinations had the desired activity. Among these, bacteria encoding IL-15Ra-IL-15 (IL-15) + eSTING were evaluated in mouse tumor models for therapeutic efficacy and mechanism after systemic administration, and in rodents and primates for tolerability. Treatment with bacteria encoding the modified STING (eSTING) protein and cytokine combinations demonstrated a high degree of complete tumor response that was entirely CD8+ T cell dependent. In an autochthonous breast cancer model lacking significant lymphocytic infiltrates, these bacteria uniformly enriched to high levels in each autochthonous lesion after IV administration, resulting in significant CD8+ T cell infiltration. In primates, these bacteria were well tolerated after IV administration, were rapidly cleared, and induced a minimal cytokine response. Thus, these engineered bacteria deliver a combined payload of cytokines, such as IL-15 (IL-15 / IL-15R alpha chain complex) and eSTING, to phagocytosis-mediated APCs in the solid tumor microenvironment after systemic administration. The bacteria promote CD8+ T cell-mediated tumor clearance in T cell-negative tumors, induce durable antitumor immunity, and are well tolerated in primates.

[0041] Tumors enriched in adenosine (AD) or ADO pathway metabolites (ATP, AMP, adenine, guanine, and adenosine) myeloid signatures are described herein as being particularly sensitive to treatment. These include, for example, tumors with elevated adenosine and / or TGF-beta compared to non-tumor tissue and / or hypoxic tumors or cancers. These include lymphoid leukemia and myeloid malignancies. Also included are tumors with these signatures, including renal clear cell carcinoma, mesothelioma, breast, pancreatic, NSCLC adenocarcinoma, sarcoma, ovarian, cervical, endocervical, head and neck squamous cell, esophageal adenocarcinoma, gastric cancer, NSCLC squamous cell tumor, and some thyroid tumors. These tumors can be treated with the therapeutic agents provided herein to transform them into hot tumors.

[0042] Provided are immunostimulatory bacteria containing genomic modifications and plasmids or related treatments encoding one or more therapeutic products, such as anti-cancer therapeutics. The genomic modifications result in the immunostimulatory bacteria accumulating within tumor-resident immune cells within the tumor microenvironment, where the encoded therapeutic products are expressed.

[0043] The immunostimulatory bacteria provided herein encode one or more complementary products that stimulate, induce, or result in a robust anti-cancer response in a subject.As demonstrated in the examples, the immunostimulatory bacteria provided and described herein reprogram the immunosuppressive tumor microenvironment into an anti-tumor phenotype, which leads to T cell infiltration and activation, B cell infiltration, macrophage repolarization and activation, dendritic cell activity, and induces a strong antigen-specific CD8+ T cell response.The immunostimulatory payload(s), such as engineered STING and cytokines, leads to the expression and production of cytokines and other factors, leading to anti-tumor immunity, including MHC upregulation.

[0044] Provided are methods for treating tumors, comprising administering a therapeutic agent that, upon administration, results in tumor macrophages with a hybrid M1 / M2 phenotype. Exemplary methods include those in which the resulting M1 / M2 macrophages are capable of phagocytosing apoptotic tumor cells and / or a delivery vehicle. Provided are methods for treating tumors with a therapeutic agent by identifying a subject whose tumor contains proliferative macrophages and administering a therapeutic agent that delivers a payload into the proliferative macrophages and converts them to macrophages with an M1 / M2 hybrid phenotype. Provided are therapeutic agents for use in treating tumors in a subject, wherein the therapeutic agent converts proliferative macrophages to M1 / M2 hybrid phenotype macrophages, and a tumor in the subject has been identified as containing proliferative macrophages; the therapeutic agent comprises a delivery vehicle with attenuated TLR2, TLR4, and / or TLR5 activity, thereby not inhibiting type I IFN production by macrophages containing the therapeutic agent. TLR2, TLR4 and TLR5 activity or responses are shown herein to inhibit type I IFN expression in macrophages, etc. Thus, the therapeutic agent has attenuated TLR2 or TLR2, TLR4 and TLR5 activity.

[0045] Provided are therapeutic agents that are effective in converting macrophages into M1 / M2 hybrid macrophages. The therapeutic agent comprises a delivery vehicle with attenuated TLR2, TLR4, and / or TLR5 activity, thereby not inhibiting type I IFN production by macrophages containing the therapeutic agent, and nucleic acids encoding at least two different immunostimulatory proteins, one of which induces type I IFN upon introduction into macrophages and the other of which stimulates an antiviral or anticancer immune response. The nucleic acid is generally provided in a form, such as a non-integrated plasmid, that is not integrated into the host cell genome, such as by integration into a chromosome of the genome.

[0046] Provided is a method for converting immune-negative or immune-desert tumors into T cell-infiltrated tumors, comprising administering into the tumor a therapeutic agent, such as those described above and herein, containing an immune-stimulating bacterium that has attenuated TLR2 / 4 / 5 activity and encodes an immune-stimulating protein. Thus, the therapeutic agent can be used to convert immune-desert tumors into T cell-infiltrated tumors. The encoded immune-stimulating protein includes, for example, a cytokine, such as the IL-15 / IL-15R alpha chain complex and / or a type I interferon (IFN), such as interferon-alpha or interferon-beta, and a combination of the cytokine.

[0047] Provided is a method for detecting subjects who are likely to respond or are predicted to respond to treatment with a therapeutic agent comprising a delivery vehicle and a non-integrated nucleic acid encoding one or more immunostimulatory proteins, comprising detecting proliferating macrophages and / or detecting specific markers in a tumor or body fluid sample. Thus, the method identifies a subset of subjects who are likely to respond to the therapeutic agent and excludes subjects who are not likely to respond to the therapeutic agent. The method may include detecting CD68, PCNA, and / or Ki67 to identify subjects who are likely to respond to treatment with a therapeutic agent comprising a delivery vehicle and a non-integrated nucleic acid encoding one or more immunostimulatory proteins.

[0048] These methods, therapeutic agents, and uses, as well as methods for identifying subjects likely to respond to treatment with therapeutic agents that convert macrophages to an M1 / M2 hybrid phenotype, include those in which responses to treatment and / or proliferating macrophages are identified by a combination of markers detectable by immunohistochemistry (IHC) and genetic markers for a particular tumor type. These methods can be achieved by obtaining a tumor biopsy or body fluid sample and detecting proliferating macrophages in the biopsy or sample, or by detecting a combination of a marker detectable by IHC and a genetic marker for the tumor type in subjects with a particular cancer or tumor. For example, markers that can be detected by IHC markers C1QC+ or SPP1+. These markers, as shown in the examples, correlate with specific cancer types, and their use as predictive markers varies depending on the cancer and is shown herein. It is also shown herein that C1QC+ or SPP1+ can be combined with genetic markers, depending on the tumor or cancer type, to identify subjects whose tumors are likely to respond to the therapeutic agents described and provided herein. As described herein, these therapeutic agents have been identified as those that convert macrophages to M1 / M2 hybrid phenotype tumors that are responsive to these therapeutic agents. Thus, the markers and methods described herein for identifying proliferative macrophages and / or tumors using specific markers identify those in which the macrophages are converted to a hybrid M1 / M2 phenotype. Tumors containing such macrophages or bearing markers are susceptible to treatment with the therapeutic agents described and provided herein.

[0049] In some embodiments of the methods, therapeutic agents, and uses, a combination of immunohistochemical markers and genetic markers of tumor type are used to select subjects for treatment, and then the subjects are treated with a therapeutic agent herein that results in macrophages with an M1 / M2 hybrid phenotype. Exemplary combinations of markers and tumor type include, but are not limited to, the following: SPP1+ and NRF2 pathway alterations in tumor biopsies or body fluid samples from subjects with squamous cell carcinoma, such as squamous cell carcinoma selected from among lung (LUSC), head and neck (HNSC), cervix (CESC), esophagus (ESCA), bladder (BLCA), and kidney renal papillary (KIRP) cancer; SPP1+ and TP53 mutations in breast cancer (BRCA), SPP1+ and PI3K mutations in prostate cancer (PRAD), SPP1+ and BRAF mutations in cutaneous melanoma (SKCM), C1QC+ and HIPPO pathway mutations, C1QC+ in uterine corpus endometrial cancer (UCEC), C1QC+ and KMT2A mutations in bladder cancer (BLCA), and C1QC+ and TP53 pathway mutations in breast cancer (BRCA) Includes:

[0050] The therapeutic agents provided and used in the methods and uses are tumor-targeted therapies that require or mediate nucleic acid transfer into immune cells for non-integrated ectopic gene expression, where the tumor-targeted therapy is directed to, accumulates in, or is taken up by tumors, the tumor microenvironment, and / or tumor-resident immune cells. For example, the therapeutic agent comprises a delivery vehicle and a nucleic acid encoding an immunostimulatory protein, and the therapeutic agent has attenuated TLR2 activity, thereby preventing type I IFN inhibition in macrophages containing the therapeutic agent or the encoded nucleic acid. For example, provided are therapeutic agents, methods, and uses in which the therapeutic agent comprises a delivery vehicle and a nucleic acid encoding an immunostimulatory protein, and the therapeutic agent has attenuated TLR2 and TLR4 or TLR2 / 4 / 5 activity, thereby preventing type I IFN inhibition in macrophages containing the therapeutic agent or the encoded nucleic acid.

[0051] Provided are methods for identifying therapeutic agents that convert macrophages to an M1 / M2 phenotype, the methods comprising: a) preparing one or more candidate therapeutic agents comprising a delivery vehicle and a nucleic acid encoding an immunostimulatory protein, one of which induces an anti-viral or anti-cancer immune response and another of which induces type I IFN, and wherein TLR2 or TLR4, or TLR2 and TLR4 or 5, or TLR2 / 4 / 5 of the delivery vehicle are attenuated, thereby preventing the therapeutic agent from inhibiting type I IFN in macrophages when introduced into or infecting macrophages; b) introducing the candidate therapeutic agent(s) into proliferating macrophages; c) determining the phenotype of the resulting macrophages; and d) selecting the candidate therapeutic agent(s) if the resulting macrophages have an M1 / M2 hybrid phenotype.

[0052] Provided are methods for treating cancer, comprising administering a therapeutic agent identified by the above methods to a subject identified as having proliferating macrophages and / or the markers and genetic markers described herein as predictive of the effectiveness of the therapeutic agents provided herein. Exemplary methods, therapeutic agents, and uses provided herein include those in which the therapeutic agent comprises a delivery vehicle and a nucleic acid encoding at least two immunostimulatory proteins, one of which induces or results in the expression of type I IFN in proliferating macrophages, and another of which induces or results in the expression of an anti-cancer or anti-viral cytokine or chemokine or other anti-cancer or anti-viral immunostimulatory effector. Other exemplary methods, therapeutic agents, and uses include those in which the therapeutic agent comprises a delivery vehicle containing a nucleic acid encoding an immunostimulatory protein that constitutively induces type I IFN in macrophages, and the vehicle does not induce or has reduced TLR2 or reduced TLR2 / 4 / 5 induction / response, resulting in uninhibited type I IFN and transcription and translation of the nucleic acid encoding the immunostimulatory protein in macrophages. In other examples, the therapeutic agent includes a delivery vehicle, and the delivery vehicle is a bacterium, a nanoparticle, a virus, or an exosome. For example, the therapeutic agent may include a delivery vehicle selected from among a nanoparticle, a virus, an exosome, a cell, and a bacterium, provided that, where appropriate, the delivery vehicle is not a bacterium, is not a Salmonella species, or is not a STACT species. In other examples, the therapeutic agent includes a delivery vehicle and a nucleic acid, and the delivery vehicle is a lipid nanoparticle, an attenuated bacterium, an immune cell, or an oncolytic virus, and the delivery vehicle is not TLR2 active or attenuates TLR2 activity, thereby not inhibiting type I IFN expression in macrophages containing the therapeutic agent or delivery vehicle.

[0053] Provided are methods, therapeutic agents, and uses in which the M1 / M2 phenotype markers include: a) at least two of the following hybrid markers (lower than M2, higher than M1): SPP1, CD209, CD206, and inducible markers: MERTK, C1QC, IFN-α2a, IFN-β1, CXCL10, 4-1BBL (TNFSF9), and MYC; and / or b) uptake of the therapeutic agent by M2 macrophages induces a hybrid M1 / M2 phenotype that retains M2 phagocytic ability, upregulates M1-like costimulatory receptors (CD80 / 86) and lymph node chemotactic receptors (CCR7), and produces type I IFN-mediated cytokines and chemokines. For example, macrophage M1 / M2 hybrid phenotype markers include CD209 and CD206 at lower levels in the resulting macrophages than in M2 macrophages, and at higher levels in M1 macrophages. Another combination of markers for identifying macrophages with an M1 / M2 hybrid phenotype is when the phenotypic markers are: Hybrid markers (lower than M2, higher than M1): SPP1, CD209, CD206 and / or Two or more inducible markers: MERTK, C1QC, IFN-α2a, IFN-β1, CXCL10, 4-1BBL (TNFSF9), MYC or Phenotypic markers are Hybrid markers (lower than M2, higher than M1): SPP1, CD209, CD206 and / or Inducible markers: MERTK, C1QC, IFN-α2a, IFNβ1, CXCL10, 4-1BBL (TNFSF9), and MYC Some include:

[0054] For example, a macrophage phenotype, post-treatment marker profile induced or resulting from treatment with a therapeutic agent herein, e.g., a therapeutic agent in which TLR2 / 4 / 5 activation is attenuated such that type I IFN expression is not inhibited, and a therapeutic agent encoding the IL-15 / IL-15R alpha chain complex plus eSTING (a STING as described herein with constitutive activity) in a non-integrated nucleic acid vehicle, e.g., a plasmid, is as follows:

[0055] [Table 1]

[0056] Alternatively, post-treatment markers that are upregulated in the resulting macrophages include the costimulatory molecules CD80 and CD86, the chemokine signaling molecules CCR7, CXCL10, and CXCL11, the pattern recognition receptors (PRRs) CD206 and CD209, which are upregulated compared to M1 macrophages and downregulated compared to M2 macrophages, and the scavenger receptors CD68 and CD163, which are upregulated.

[0057] In the methods, therapeutics and uses provided herein, proliferative macrophages, such as proliferative M2 macrophages, can be identified by the presence of biopsy surface markers: CD68+KI67 and / or PCNA, MERTK, and / or by gene expression of a G2M module in which half or more than half (≥ or > 14 genes of the set) are expressed, and optionally STMN1 is expressed. Proliferative macrophages can also be identified by a G2M module that includes at least half of the genes of the STMN1+ set, e.g., ≥ or > 14 genes, by a G2M module that includes at least half of the genes, or by the markers CD68, MERTK and KI67 and / or PCNA. For example, the M1 / M2 hybrid phenotype is characterized by or identified by hybrid markers (lower than M2, higher than M1): SPP1, CD209, CD206, e.g., the phenotype includes inducible markers: MERTK, C1QC, IFN-α2a, IFNβ, CXCL10, 4-1BBL and / or MYC, etc.

[0058] In some embodiments, the hybrid M1 / M2 macrophage phenotype is characterized by the following markers: hybrid markers (lower than M2, higher than M1): SPP1, CD209, CD206 and / or inducible markers: MERTK, C1QC, IFN-α2a, IFNβ, CXCL10, 4-1BBL, MYC. In others, the resulting macrophages are CIQChiSPP1low. In all embodiments, the macrophages containing the therapeutic agent or delivery vehicle may be proliferative macrophages, e.g., proliferative M2 macrophages, which convert to M1 / M2 hybrid phenotype macrophages upon expression of the payload encoded in the therapeutic agent.

[0059] As described above and herein, therapeutic agents that result in an M1 / M2 hybrid phenotype in macrophages generally comprise nucleic acids that are in a non-integrating form, thereby not integrating into the host genome. The nucleic acids encode immune stimulatory proteins, including but not limited to those described in this disclosure, such as cytokines and constitutive STING, and combinations thereof.

[0060] Provided is a method of treating a tumor, comprising identifying a subject whose tumor contains proliferating macrophages, and administering a therapeutic agent that delivers a non-integrated genetic payload into the proliferating macrophages, such that the encoded payload is transcribed.

[0061] Also provided is a method of increasing the therapeutic effect of immunostimulatory bacteria such as those provided herein, comprising administering a pro-apoptotic agent to a subject prior to administration of the immunostimulatory bacteria. Exemplary agents that promote tumor apoptosis include chemotherapeutic agents such as docetaxel (DTX), paclitaxel (PTX), doxorubicin (DOX), 5-fluorouracil (5-FU), carboplatin (CARB), cyclophosphamide (CTX), and other such chemotherapeutic agents.

[0062] Also provided is a method of pretreating a subject with anti-PD-1 treatment to suppress PD-1 expression in macrophages, thereby promoting phagocytosis of the immunostimulatory bacteria prior to or along with the immunostimulatory bacteria, and treating with anti-PD-L1 after administering the immunostimulatory bacteria and after a sufficient time such that nucleic acids encoding the payload(s) are delivered to macrophages, such that PD-L1 is then induced in macrophages, thereby increasing the therapeutic effect of the immunostimulatory bacteria in a subject.

[0063] Methods for treating cancer and uses of therapeutic agents for treating cancer are provided. Provided are methods for treating cancer, comprising administering immunostimulatory bacteria to a subject that has been pretreated with a pro-apoptotic agent prior to administration of the immunostimulatory bacteria to the subject. Provided are methods for treating cancer in a subject, comprising first treating the subject with a pro-apoptotic agent and then administering the immunostimulatory bacteria. These include methods and uses in which administration of a therapeutic agent, such as immunostimulatory bacteria, converts macrophages to an M1 / M2 hybrid phenotype. Exemplary immunostimulatory bacteria include those that have TLR2, TLR4, and / or TLR5 activity, thereby not inhibiting type I IFN production by macrophages containing the therapeutic agent, and that contain nucleic acids encoding at least two different immunostimulatory proteins, one of which induces type I IFN when introduced into macrophages, and another of which stimulates an antiviral or anti-cancer immune response.

[0064] Also provided are methods and uses for enhancing the therapeutic efficacy of immunostimulatory bacteria in a subject, comprising pretreating the subject with an anti-PD-1 antibody or other PD-1 antagonist to suppress PD-1 expression in macrophages in the subject's tumor, thereby promoting their phagocytic ability, administering immunostimulatory bacteria, the bacteria encoding one or more immunostimulatory proteins, and then treating with an anti-PD-L1 agent after a sufficient time for nucleic acids encoding the payload to be delivered to the macrophages. Also provided are methods and uses for enhancing the therapeutic efficacy of immunostimulatory bacteria in a subject, comprising pretreating the subject with an anti-PD-1 antibody or other antagonist to suppress PD-1 expression in macrophages, thereby promoting their phagocytic ability before or together with the immunostimulatory bacteria, and then administering the immunostimulatory bacteria. In these embodiments, the anti-PD-1 treatment may be administered at least about 3, 6, 12, 24, 36, 48 hours or more, e.g., about 2 days, before administration of the immunostimulatory bacteria. Anti-PD-1 agents include antagonists and antibodies, including antibodies and single chain or other forms thereof, that bind to or inhibit PD-1.

[0065] In accordance with the methods, therapeutic agents, and uses provided herein, subjects selected for treatment with a therapeutic agent encoding a non-integrated nucleic acid are identified by obtaining a tumor biopsy or body fluid from the subject and selecting the subject for treatment if the phagocytes in the tumor biopsy or body fluid are proliferative. Body fluids include, but are not limited to, urine, blood, plasma, sweat, CSF, and other such samples. Proliferative macrophages can be identified by detecting the following markers: tumor gene expression of the G2M module (>14 genes in the set) alone or in combination with stathmin 1 (STMN1), and / or biopsy surface markers: CD68+KI67 and / or PCNA, MERTK, and / or SPP1 in lung or gastric tumors, and / or C1QC in colon and breast cancers. In embodiments of the methods, therapeutic agents, and uses, the macrophages are M2 macrophages.

[0066] Therapeutic agents herein that convert macrophages to M1 / M2 hybrid phenotype macrophages can also be used to treat fibrotic diseases. This phenotype is effective for the treatment of fibrotic diseases, and therefore any of the therapeutic agents provided herein that convert the phenotype can be used for such treatment.

[0067] In the methods, therapeutics, and uses, the therapeutic agent comprises a nucleic acid encoding an immunostimulatory protein, and the encoded protein may comprise a cytokine and a cytoplasmic DNA / RNA sensor that induces expression of type I IFN, and the cytoplasmic DNA / RNA sensor is modified to have increased activity or constitutive activity in inducing type I IFN. The cytoplasmic DNA / RNA sensor is modified to have constitutive activity, whereby type I IFN can be induced in the absence of a ligand and / or cytoplasmic DNA / RNA. Exemplary cytoplasmic DNA / RNA sensors and modified forms thereof are described and exemplified below.

[0068] Therapeutic agents, methods and uses include those in which the therapeutic agent comprises a delivery vehicle and a nucleic acid, e.g., DNA, where the delivery vehicle encodes cytokines and STING pathway proteins that constitutively induce type I IFN, resulting in a hybrid M1 / M2 proliferative and phagocytic macrophage phenotype, in which TLR2, particularly TLR / 2 / 4 / 5, induction is attenuated or eliminated.

[0069] In other embodiments, the therapeutic agents for use in the methods and uses, and identified by the screening methods, include those wherein the nucleic acid in the therapeutic agent encodes an immunostimulatory protein selected from among STING, MDA5, IRF-3, IRF-7 and RIG-I, and the immunostimulatory protein comprises a modification that is a gain-of-function (GOF) mutation(s) that renders STING, MDA5, IRF-3, IRF-7 or RIG-I constitutively active, thereby causing expression of type I IFN to be constitutive.

[0070] Therapeutic agents for use herein and in the methods and uses described and claimed include any of those described herein or any that comprise a nucleic acid provided in a delivery vehicle that generally is non-integrated, e.g., in a non-integrated (into the genome) plasmid, encoding an immunostimulatory protein, particularly one that does not inhibit TLR2 or TLR2, TLR4 and / or TLR5. The nucleic acid can encode any of the payloads described herein and combinations thereof, and therapeutic agents include the immunostimulatory bacteria provided herein or those known in the art that have the required properties.

[0071] Due to the similarities in immune responses between antitumor and antiviral responses, the immunostimulatory bacteria provided herein can also be used to treat infectious diseases. The immunostimulatory bacteria can encode antiviral or antibacterial therapeutics, such as inhibitors of viral or bacterial products, inhibitors of the expression of viral or bacterial products, or viral or bacterial antigens. Combinations of immune responses derived from immunostimulatory bacteria with therapeutic antipathogen products, and combinations of immunostimulatory proteins with other such therapeutics, also provide therapeutic immunostimulatory bacteria for vaccinating against and / or treating infectious diseases, particularly diseases associated with viral infections, such as chronic and latent viral infections. Of interest are chronic viral infections, such as those caused by hepatitis virus, herpes virus, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), respiratory syncytial virus (RSV), measles virus, and other such viruses that chronically infect a subject. Immunostimulatory bacteria can also be used to treat acute infectious diseases such as chronic influenza, oral bacteria (P. gingivalis), and early infections with coronaviruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, which causes COVID-19). Targeted pathogenic bacteria include, for example, species of Escherichia, Staphylococcus, Pseudomonas, and Porphyromonas.

[0072] Also provided herein are immunostimulatory bacteria that can be used and / or formulated as vaccines for administration into tissues, such as by intramuscular injection, inhalation, and other such direct routes. These bacteria are designed to be non-replicative in vivo and therefore contain auxotrophy, such as thyA, so that they do not express active thymidylate synthase, and they encode a payload under the control of a promoter recognized in bacteria. If they are intended to deliver a protein payload to a vaccinated host, the encoded payload contains or is designed to be translated in the bacterial host. If they are intended to deliver RNA, the encoded nucleic acid is designed so that bacterial ribosomes cannot translate them, but eukaryotic ribosomes can. This can be done, for example, by including an IRES in the encoded nucleic acid. The vaccine payload includes a nucleic acid encoding an immunizing antigen or protein, such as an antigen derived from a viral or bacterial pathogen. The payload may also include products such as STING, particularly modified STING, which is part of a cytoplasmic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), and optionally immune stimulatory proteins such as cytokines, e.g., IL-15, the IL-15 / IL-15R alpha chain complex, etc. Vaccines are formulated for suitable routes of administration, including aerosols as well as emulsions, tablets, and powders.

[0073] The immunostimulatory bacteria provided herein can encode an antigen(s) derived from a pathogen, such as a viral antigen, and are used as a vaccine to prevent infection or treat an existing infection. Antigens include, but are not limited to, any known to those skilled in the art to induce a protective immune response or ameliorate disease caused by a pathogen. These immunostimulatory bacteria can prime T cell responses against pathogens, such as viruses, by their ability to accumulate in immune cells, such as antigen-presenting cells. For example, as described in detail herein, some of the immunostimulatory bacteria provided herein are deficient in asparaginase II, an enzyme that suppresses T cell function. Any of the immunostimulatory bacteria described and provided herein can be used. It has been described and shown herein that eliminating asparaginase II activity, for example, by modifying the bacterial genome to eliminate expression of the active enzyme, can be used to encode an antigen or combination of antigens. The resulting bacteria promote anti-pathogen, e.g., anti-viral, T cell responses. The expression of antigens, such as those derived from pathogens, bacteria, viruses, or other organisms, combined with their ability to accumulate in immune cells, such as antigen-presenting cells, provides protection from infection by the pathogen. For example, immunostimulatory bacteria can encode viral antigens, such as antigens from essential viral core proteins shared between or across viral families. For example, in the case of coronaviruses such as SARS-COV2, antigens derived from the nucleocapsid and / or nonstructural M proteins can enhance CD8+ T cell responses to the highly conserved and less mutated core protein, thereby providing broad pan-coronavirus protection and providing effective vaccines and treatments. Proteins and antigens derived from this coronavirus and coronavirus family that can be used for immunization and / or treatment are known, and exemplary ones are described herein and known to those skilled in the art. In addition to spike proteins, portions thereof, and modified spike proteins, other proteins have been identified for this purpose.See, for example, Cohen et al., (2021) Cell Reports Medicine 2:1000354.

[0074] The immunostimulatory bacteria can encode an antiviral or antibacterial therapeutic agent. Such therapeutic agents include inhibitors of viral genes and proteins (such as proteins required for replication and / or packaging), or the immunostimulatory bacteria can encode a therapeutic agent that prevents viral binding or interaction with a viral receptor(s) that facilitates or provides for viral entry into target cells. In some embodiments, expression of the encoded therapeutic protein, such as an antigen or antigenic protein, can be under the control of a prokaryotic promoter. In other embodiments, the protein can be expressed under the control of a eukaryotic promoter. The choice of promoter depends on whether it is to be expressed in the bacteria, such as prior to administration as described herein for delivery of mRNA that is translated in the host, or whether the protein is to be expressed in host cells, such as immune cells, after delivery.

[0075] The immunostimulatory bacteria provided herein include genome modifications, such as deletions, disruptions, and other alterations that result in an inactivated encoded product, such as changing the orientation of all or part of a gene so that a functional gene product is not expressed. Some of the immunostimulatory bacteria provided are modified so that the resulting bacterium is msbB- / purI-. In some embodiments, the bacterium is msbB- and purI-, whereby the entire length of at least the coding portion of the msbB and / or purI gene is deleted. The bacterial genome may also be modified so that the bacterium lacks flagella. This occurs in bacteria that normally express flagella. In such bacteria, for example, the fliC and fljB genes in Salmonella, or genes equivalent to fliC and fljB in other species, may be deleted or otherwise modified so that functional gene products are not expressed. The bacterium may also be modified to be an adenosine auxotroph and / or to be msbB- / pagP-. Also provided are immunostimulatory bacteria and pharmaceutical compositions containing the same, wherein the bacteria do not express L-asparaginase II, thereby rendering the bacteria ansB-. Elimination of the encoded asparaginase activity improves or preserves T cell viability / activity. Therapeutic bacteria, such as inactivated or attenuated bacteria used as vaccines, can be improved by modifying the bacterial genome to remove asparaginase activity. Exemplary such vaccines include the BCG (Bacillus Calmette-Guerin) vaccine and related vaccines used to immunize against tuberculosis. The BCG vaccine is known to have variable efficacy, and removing asparaginase can improve the efficacy of such vaccines because endogenous bacterial asparaginase inhibits or reduces T cell activity.

[0076] Provided herein is an immunostimulatory bacterium containing a plasmid encoding a therapeutic product or combination of therapeutic products under the control of a eukaryotic promoter. The genome of the bacterium contains modifications, for example, one, two, or more modifications selected from the following: a) deletion or disruption of all or a sufficient portion of the gene, whereby the bacterium is modified to produce pentaacylated lipopolysaccharide (LPS); The genome of an immunostimulatory bacterium is modified by deletion or disruption of all or a sufficient portion of a gene, thereby modifying the bacterium to produce pentaacylated lipopolysaccharide (LPS); and deletion or disruption of hexaacylated lipopolysaccharide, such that it is substantially reduced to 10-fold or more or absent compared to wild-type bacteria; b) deletion or disruption of all or a sufficient portion of the gene, whereby the bacterium is attenuated for recognition by Toll-like receptor (TLR) 2, TLR4, and / or TLR5; c) deletion or disruption of all or a sufficient portion of the gene so that the bacterium does not activate the synthesis of curli and / or cellulose; d) deletion or disruption of all or a sufficient portion of the gene so that the bacterium does not activate the synthesis of secreted asparaginase;

[0077] e) deletion or disruption of all or a sufficient portion of a gene or genes, which renders the bacterium auxotrophic for purines, adenosine, and / or ATP; f) deletion or disruption of all or a sufficient portion of the gene, whereby the bacterium loses, deletes or disrupts the flagellum; g) deletion or disruption of all or a sufficient portion of the gene, whereby the bacterium is modified to specifically infect tumor-resident myeloid cells; h) deletion or disruption of all or a sufficient portion of the gene, whereby the bacterium is modified to specifically infect tumor-resident myeloid cells and is unable to replicate within the tumor-resident myeloid cells; and i) Deletion or disruption of either or both of lppA and lppB, which reduces or eliminates lipoprotein expression at the membrane, thereby increasing expression of the encoded therapeutic protein within the tumor microenvironment and / or tumor-resident immune cells.

[0078] For example, the immunostimulatory bacteria contain modifications, including deletions, insertions, and substitutions, of a), d), and f), or modifications c) and d), or modifications a), c), d), e), and f), or modifications a), c), d), e), f), and i), or modifications a), d), f), and i), or modifications c), d), and i), or modifications f) and i), or modifications a) through i), or modifications a), b), d), and f), or other combinations of modifications a), b), c), and d), and modifications a) through i). Deletions or disruptions include any modification of a gene such that an active gene product is not expressed.

[0079] In particular, immunostimulatory bacteria are provided whose genomes have been modified by deletion or disruption, including insertion of all or a sufficient portion of a gene(s), thereby reducing the bacteria's recognition by TLR2, TLR4, and TLR5. Such bacteria have low virulence and accumulate / colonize the tumor microenvironment and tumor-resident myeloid cells such as macrophages. These bacteria contain plasmids encoding therapeutic products, particularly complementary products such as cytokines, in combination with modified STING polypeptides, including gain-of-function / constitutively active STING proteins, STING chimeras, and chimeric STING proteins containing gain-of-function (GOF) mutations. Cytokines include, for example, the IL-15 / IL-15R alpha chain complex (also referred to herein as IL-15Rα / IL-15sc, IL-15 / IL-15Rα, or IL-15 complex), IL-15, IL-12, or other anti-tumor immunostimulatory cytokines or chemokines. The bacterium can further encode other products, such as anti-tumor antibodies. Product combinations are described and provided herein. Product combinations that stimulate or promote anti-tumor responses and / or deliver therapeutic products are described throughout the disclosed specification and are delivered by immunostimulatory bacteria whose genomes have been modified so that the bacteria have low virulence and effectively colonize tumors, tumor microenvironments, and / or tumor-resident immune cells, such as macrophages. Exemplary such bacteria are species such as Salmonella, Listeria, and Escherichia coli, which have been modified to lack flagella and contain lipopolysaccharide (LPS) with pentaacylated lipid A, for example, by rendering the bacteria msbB- / pagP-. The bacteria may also be further modified by removal of Curli pili and / or to reduce or eliminate cellulose production and biofilm formation, such as by modifying the bacteria to be csgD-.It is shown herein that bacteria with these modifications do not have a maximum tolerated dose (MTD) and exhibit high tumor colonization.

[0080] In all embodiments, the immunostimulatory bacteria can also contain, or further contain, a deletion or disruption of a gene encoding a flagellum, such that the bacteria are flagellin- (e.g., fliC- / fljB- in Salmonella) and do not produce flagella (i.e., sometimes referred to as flagellin-deficient or flagellin-), whereas wild-type bacteria have flagella. The immunostimulatory bacteria can be auxotrophic for purines, e.g., adenosine, or adenosine, adenine, and / or ATP. The immunostimulatory bacteria can also be purI-. The immunostimulatory bacteria can also be pagP-. The immunostimulatory bacteria can also be aspartate semialdehyde dehydrogenase- (asd-), e.g., the bacteria are asd- due to disruption or deletion of all or part of the endogenous gene encoding aspartate semialdehyde dehydrogenase (asd), such that endogenous asd is not expressed. The bacterium may encode aspartate semialdehyde dehydrogenase (asd) on a plasmid under the control of a bacterial promoter. The immunostimulatory bacterium may also be msbB- or pagP- / msbB-. For example, the immunostimulatory bacterium may be asd-, purI-, msbB-, flagellin- (e.g., fliC- / fljB-), and pagP-, or asd-, csgD-, purI-, msbB-, flagellin- (e.g., fliC- / fljB-), and pagP-. In some embodiments, the immunostimulatory bacterium is ansB-, asd-, csgD-, purI-, msbB-, flagellin- (e.g., fliC- / fljB-), and pagP-.

[0081] Provided are immunostimulatory bacteria containing a plasmid encoding a therapeutic product under the control of a eukaryotic promoter, or encoding multiple products under the control of multiple eukaryotic promoters or under the control of a single promoter. The genome of the immunostimulatory bacteria has been modified by deletion of a sufficient portion of a gene(s) or disruption of a gene(s), such that the bacteria are one or more of ansB-, asd-, csgD-, purI-, msbB-, flagellin- (e.g., fliC- / fljB-), and pagP-. Also provided herein are immunostimulatory bacteria in which the genes lppA (lpp1) and / or lppB (lpp2), which encode the major outer membrane lipoproteins Lpp1 (LppA) and Lpp2 (LppB), respectively, have been deleted or disrupted to eliminate or substantially reduce expression of the encoded lipoproteins. In particular, the immunostimulatory bacteria are lppA- and lppB-. Provided are immunostimulatory bacteria that contain a plasmid encoding an anti-cancer therapeutic or an anti-pathogen therapeutic under the control of eukaryotic regulatory sequences and that are lppA- and lppB-. For example, the immunostimulatory bacteria can be ansB-, asd-, csgD-, purI-, msbB-, flagellin- (such as fliC- / fljB-), pagP-, lppA-, and / or lppB-.

[0082] In embodiments herein, the therapeutic product is an anti-cancer therapeutic or therapeutic agent used in cancer treatment. The encoded product may be operably linked to a nucleic acid encoding a secretion signal, such that upon expression, the therapeutic product is secreted, e.g., from tumor-resident immune cells.

[0083] Additionally, any of the immunostimulatory bacteria can have one or more genes or operons involved in Salmonella pathogenicity island 1 (SPI-1) invasion deleted or inactivated, such that the immunostimulatory bacteria do not invade or infect epithelial cells. For example, the one or more genes / operons are selected from avrA, hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP.

[0084] The plasmid in the immunostimulatory bacterium can be present at low or medium copy number. The plasmid can contain a medium-to-low copy number origin of replication, e.g., a low copy number origin of replication. The plasmid may be present at medium to low copy number depending on the ORI sequence, the size of the plasmid, and the culture conditions. In some embodiments, the plasmid is present at a higher copy number. Typically, a medium copy number is less than 150 or less than about 150 and more than 20 or more than about 20, or 20 or 25 to 150; a low copy number is less than 25, or less than 20, or less than about 25, or less than about 20 copies. In particular, a low to medium copy number is less than about 150 copies, or less than 150 copies; a low copy number is less than about 25 copies, or less than 25 copies.

[0085] Encoded therapeutic products include nucleic acids and proteins. A plasmid can encode two or more therapeutic products. Exemplary products include, but are not limited to, cytokines, proteins that constitutively induce type I IFN, and costimulatory receptors or ligands. Further exemplary combinations are described below. In some embodiments, the costimulatory molecule lacks all or part of the cytoplasmic domain for expression on antigen-presenting cells (APCs), such that the truncated molecule is capable of constitutive immunostimulatory signaling to T cells via costimulatory receptor binding but is unable to counterregulatory signaling to antigen-presenting cells (APCs) due to the lack of the deleted cytoplasmic domain or portion thereof.

[0086] The encoded therapeutic product can be operably linked to a nucleic acid encoding a regulatory sequence recognized by a eukaryotic host, e.g., a secretion signal that causes secretion from a cell or plasmid containing the bacterium. In embodiments in which the immunostimulatory bacterium encodes two or more products, expression of each product can be under the control of a separate promoter. Alternatively, two or more products can be expressed under the control of a single promoter, with each product separated, for example, by an internal ribosome entry site (IRES) or a nucleic acid encoding a 2A peptide, to cause separate expression of each of the therapeutic products. Exemplary 2A peptides are T2A, F2A, E2A, or P2A, which can be flanked by the nucleic acid encoding the therapeutic product to cause separate expression of the therapeutic products expressed under the control of a single promoter. The therapeutic product is expressed under the control of a eukaryotic promoter, e.g., an RNA polymerase (RNAP) II promoter or an RNA polymerase III promoter. These include viral promoters, RNA polymerase II promoters, or mammalian RNA polymerase II promoters, such as, but not limited to, the cytomegalovirus (CMV) promoter, SV40 promoter, Epstein-Barr virus (EBV) promoter, herpesvirus promoter, adenovirus promoter, elongation factor-1 alpha (EF-1α) promoter, UBC promoter, PGK promoter, CAGG promoter, adenovirus 2 or 5 late promoter, eIF4A1 promoter, CAG promoter, or CD68 promoter. The plasmid may further include other eukaryotic regulatory sequences for controlling expression of the therapeutic product, such as terminators and / or promoters selected from SV40, human growth hormone (hGH), bovine growth hormone (bGH), MND (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), chicken beta-globin, and rbGlob (rabbit globin) genes. Other regulatory sequences include a poly(A) tail, a woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE), and a hepatitis B virus posttranscriptional regulatory element (HPRE).

[0087] Immunostimulating bacteria and vaccines Provided are immunostimulatory bacteria containing a plasmid encoding a therapeutic product, wherein the bacteria contain genomic modifications, such as insertions, deletions, substitutions, or transposons, that cause the bacteria to not produce active thymidylate synthase and require supplementation for growth. The supplementation includes nutrients that can bypass the reaction catalyzed by thymidylate synthase, thereby allowing the bacteria to replicate. The supplementation includes one or more of thymine, thymine derivatives, thymidine, thymidine derivatives, thymine precursor(s), thymidine precursor(s), or thymidine monophosphate precursor(s). The bacteria may further contain additional modifications that reduce or eliminate activation of TLR2, and, optionally, TLR4 and / or TLR5, in a host, such as a human or other mammal.

[0088] Also provided is an immunostimulatory bacterium comprising a plasmid encoding a therapeutic product, wherein the bacterium comprises a genomic modification whereby the bacterium does not secrete active asparaginase, and wherein the bacterium comprises a genomic modification that reduces or eliminates activation of TLR2 and, optionally, TLR4 and / or TLR5 in a host.

[0089] These immunostimulatory bacteria can further comprise a genome modification by deletion, disruption, or modification of all or a sufficient portion of the gene ansB, which encodes L-asparaginase II, such that the bacteria are ansB- and do not express active L-asparaginase II.

[0090] Also provided are immunostimulatory bacteria that contain genomic modification(s) that reduce or eliminate activation of TLR2, such that induction of type I interferon (IFN) is not inhibited by TLR2; the immunostimulatory bacteria contain genomic modification(s) that prevent it from replicating in vivo but allow it to replicate when grown in vitro under nutrient supplementation; and the genomic modification(s) that eliminate or inactivate thymidylate synthase, such that the bacteria are thyA- and / or asd- or both.

[0091] It is described herein that activation of TLR2 can inhibit the induction of type I IFN.It is shown herein that the expression of proteins such as STING protein by bacteria or in a delivery vehicle that activates TLR2 or TLR4 / 5 and TLR2 is not an advantageous combination because activation of TLRs such as TLR2 inhibits type I interferon.Type I IFN is, for example, interferon-α and / or interferon-β.

[0092] Provided are immunostimulatory bacteria containing genomic modifications that reduce or eliminate TLR2 activation, whereby induction of type I IFN is not inhibited by TLR2, the immunostimulatory bacteria containing a plasmid encoding an interferon or encoding a modified STING protein that constitutively induces type I interferon, and encoding an antigen or protein from a pathogen or tumor. These bacteria can also contain genomic modifications that reduce or eliminate TLR4 and / or TLR5 activation / induction.

[0093] Provided are immunostimulatory bacteria containing a plasmid encoding a therapeutic product, the genome of which has been modified by deletion or disruption of all or a sufficient portion of a gene(s) such that the bacterium produces lipopolysaccharide (LPS) with pentaacylated lipid A, and lipopolysaccharide with hexaacylated lipid A is substantially reduced or absent by at least 10 times compared to wild-type bacteria; the genome of the bacterium has been modified so that the bacterium itself does not inhibit or prevent the induction of type I interferon (IFN) in infected immune cells; and the genome of the bacterium has been modified so that it is auxotrophic for essential nutrients. For example, provided are immunostimulatory bacteria having a genome modification such that the bacterium does not encode or produce active asparaginase and / or thymidylate synthase. Any of the immunostimulatory bacteria provided herein may have genome modifications, including deletions, insertions, and / or substitutions, such that the bacterium is thyA- and / or asd-, or both thyA- and asd-. Additionally, in some embodiments where the bacterium is asd- due to genome modification, the bacterium may contain a nucleic acid encoding asd on a plasmid such that it is expressed in vivo. Specific embodiments and applications of particular auxotrophies and complementation with plasmids are described in the detailed description and / or known to those skilled in the art.

[0094] Also provided is an immunostimulatory bacterium comprising a plasmid encoding a therapeutic product, wherein the genome of the immunostimulatory bacterium has been modified by deletion or disruption or rearrangement of all or a sufficient portion of a gene(s), whereby the bacterium is modified to produce lipopolysaccharide (LPS) having pentaacylated lipid A, and lipopolysaccharide having hexaacylated lipid A is substantially reduced by at least 10-fold compared to wild-type bacteria or is absent, and the genome of the bacterium has been modified so that it does not produce active thymidylate synthase, whereby the bacterium is thyA-.

[0095] Also provided are immunostimulatory bacteria comprising a plasmid encoding a therapeutic product, wherein the genome of the immunostimulatory bacteria is modified by deletion or disruption of all or a sufficient portion of a gene or genes such that the bacterium loses flagella; the unmodified immunostimulatory bacteria has flagella, and the genome of the bacterium is modified such that it does not produce active thymidylate synthase.

[0096] Any of the immunostimulatory bacteria provided herein may have a genome modification, such as a modification that renders the bacterium csgD-, thereby causing the bacterium to lose curli pili. Any of the immunostimulatory bacteria may contain a genome modification that reduces or eliminates activation of TLR4 and / or TLR5. The immunostimulatory bacteria provided herein may contain a genome modification that results in the bacterium not having flagella, such that the wild-type bacterium has flagella. These bacteria may further contain a genome modification that results in the bacterium not producing curli pili. They may also contain a genome modification that results in pentaacylated lipopolysaccharide. Thus, the bacteria provided herein may lose flagella and be msbB- / pagP-.

[0097] Provided herein are immunostimulatory bacteria that contain genomic modifications that reduce or eliminate TLR2 activation, thereby preventing the induction of type I IFN from being inhibited by TLR2, and the immunostimulatory bacteria are capable of replicating in vivo in a eukaryotic host. The immunostimulatory bacteria include a plasmid encoding a therapeutic product(s) including a tumor-associated antigen, or encoding a product that is part of a cytoplasmic DNA / RNA sensor pathway that results in the expression of a tumor antigen and a type I interferon (IFN), the encoded product being interferon-alpha or interferon-beta, or the encoded product being both IFN-alpha and IFN-beta, e.g., a type I IFN that is a STING protein, or encoding a tumor-associated antigen and interferon-alpha, or encoding a tumor-associated antigen and interferon-beta. The product that is part of the cytoplasmic DNA / RNA sensor pathway includes a modified STING protein that increases the induction of type I interferon compared to unmodified human STING protein, or a modified protein that results in increased induction of type I interferon, e.g., a STING protein. Modified STING proteins include, for example, any of those described below that result in increased or constitutive expression or induction of type I interferon, particularly compared to unmodified human STING protein. Modified STING proteins may also have reduced NF-κB signaling compared to wild-type human STING protein. Included are chimeric STING proteins as described herein (see also International PCT Publication WO2020 / 176809 and U.S. Patent Publication No. 2020 / 027061). For example, the STING protein contains substitutions corresponding to N154S, R284G, or N154S / R284G with reference to the human STING protein. Exemplary immunostimulatory bacteria include those encoding modified STING proteins that constitutively induce type I interferon and tumor antigens and / or cytokines, such as the IL-15 receptor complex.Immunostimulatory bacteria have genomic modifications that render the bacteria flagellin-deficient, such that they lack flagella and have pentaacylated LPS, such as by rendering the bacteria msbB- / pagP-. The bacteria may also lose curli pili, such as by rendering them csgD-. The bacteria may also be auxotrophic for a required nutrient, e.g., thyA- and / or adenosine-auxotrophic or other such auxotrophic bacteria. The bacteria may also be ansB-. The specific combination of genomic modifications as described herein will vary depending on the intended use and desired effect of the bacterium, as will the specific choice of the encoded therapeutic product. As described herein, promoters and other regulatory sequences controlling expression of the encoded product will also vary depending on the intended use of the bacterium. As described herein throughout this application, the therapeutic protein encoded on the plasmid may be under the control of eukaryotic regulatory sequences, such as for anti-tumor therapeutic embodiments, or may be under the control of a bacterial promoter, such as for embodiments such as certain vaccines for which the encoded protein or RNA is intended for delivery.

[0098] With regard to the encoded product, the immunostimulatory bacterium may encode a therapeutic product that is part of a cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN), or the encoded product is interferon-alpha or interferon-beta, or the encoded product is both IFN-alpha and IFN-beta. Other products include cytokines, antibodies, bispecific engager antibodies, and other antigens, such as tumor antigens or pathogen antigens for vaccination. An exemplary therapeutic product that is part of a cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN) is a STING protein, particularly one that has been modified to have increased, particularly constitutive, activity, such that sensing of cytoplasmic DNA / RNA or the presence of such DNA / RNA, or any ligand of such a pathway, is not required. The plasmid may encode an antigen, epitope(s), or protein derived from a pathogen or tumor.

[0099] Provided are immunostimulatory bacteria containing plasmids encoding a combination of heterologous products, wherein the genome of the immunostimulatory bacteria is modified by deletion or disruption of all or a sufficient portion of a gene(s) so that the bacterium has attenuated recognition by TLR2, and optionally one or both of TLR4 and TLR5, one product is part of a cytoplasmic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), and the second product is an antigen, epitope(s) therefrom, or a protein for immunization against a pathogen or tumor. In some embodiments, the genome of the bacteria is modified so that the bacterium does not have curli pili.

[0100] Also provided are immunostimulatory bacteria comprising a nucleic acid operably linked to a prokaryotic promoter, particularly where the nucleic acid is encoded on a plasmid, wherein the nucleic acid expressed in the bacterium under control of the prokaryotic promoter encodes an RNA lacking sequences necessary for prokaryotic translation, thereby producing an RNA in the bacterium. Exemplary of such embodiments are those in which the encoded RNA lacks a Shine-Dalgarno sequence and / or contains an internal ribosome entry site (IRES). The nucleic acid may also encode a translational readthrough 2A peptide, such that, when the nucleic acid encodes a polycistronic message, distinct products are produced upon expression of the nucleic acid.

[0101] Provided are immunostimulatory bacteria comprising a nucleic acid operably linked to a prokaryotic promoter, such as on a plasmid, where the nucleic acid comprises an RNA lacking sequences necessary for prokaryotic translation. As a result, the bacterium can transcribe but not translate the encoded RNA. Therefore, the bacterium can be used as an RNA delivery vehicle. As described, this can be achieved when the encoded RNA lacks a Shine-Dalgarno sequence. For polycistronic nucleic acids, the nucleic acid may include one or more of the 2A peptides, such as T2A, P2A, E2A, or F2A.

[0102] Provided are immunostimulatory bacteria in which a nucleic acid encoding a therapeutic product is operably linked to a prokaryotic promoter, the nucleic acid encoding an RNA lacking sequences necessary for prokaryotic translation, whereby the RNA is produced in the bacterium, and the RNA may lack a Shine-Dalgarno sequence, contain an internal ribosome entry site (IRES), and / or may also contain a translational read-through 2A peptide. In some embodiments, the nucleic acid encoding the therapeutic product(s) may be operably linked to a nucleic acid encoding a secretion signal, whereby the therapeutic product(s) are secreted upon expression.

[0103] Bacterial promoters for expression of the encoded therapeutic product include any that are recognized by bacterial or bacteriophage RNA polymerases, such as bacterial or bacteriophage promoters. If the promoter is one that is recognized only by bacteriophage RNA polymerases, the bacterium may encode a bacteriophage polymerase, such as T7 RNA polymerase. Exemplary promoters are SEQ ID NOS: 393-396, respectively: attatgtcttgacatgtagtgagtgggctggtataatgcagcaag, or ttatgcttgacgctgcgtaaggtttttgttataatacaccaag, or attatgtcttgacatgtagtgagtgggctggtaaatgcagcaag, or (Salmonella rpsm promoter) Any of the following may be used.

[0104] Immunostimulatory bacteria contain genomic modifications, such as elimination of flagella and / or other modifications such that the bacteria do not infect epithelial cells but still infect or accumulate in, or preferentially infect (infect to a greater extent or amount than unmodified bacteria, and infect other cell types to a lesser extent or amount than unmodified bacteria) phagocytes, e.g., tumor-resident myeloid cells in a tumor-bearing subject and tissue-resident myeloid cells, such as at or near the site of vaccination if the bacteria is a vaccine. Such bacteria include those that, for flagellated bacteria, contain genomic modifications that render the bacteria flagellin-deficient, resulting in the bacteria losing their flagella.

[0105] The immunostimulatory bacteria provided herein include those containing a plasmid encoding a therapeutic product, and infection of macrophages with the bacteria converts the macrophages to M1 or M1-like phenotype macrophages. The encoded therapeutic product may be a therapeutic product that is part of a cytoplasmic DNA / RNA sensor pathway that leads to type I interferon (IFN) expression, e.g., a therapeutic product modified so that type I IFN expression is constitutive. The therapeutic product may be a gain-of-function (GOF) mutant of a therapeutic product that is part of a cytoplasmic DNA / RNA sensor pathway, where the mutant GOF product does not require a cytoplasmic nucleic acid, nucleotide, dinucleotide, or cyclic dinucleotide to lead to type I IFN expression. An exemplary such product is a modified or mutant STING protein. Infection with such bacteria can convert human M2 macrophages to M1-like type I IFN-producing cells. An exemplary such bacterium is one that lacks flagella, whereas wild-type bacteria have flagella and are pagP- / msbB-. Thus, provided are methods for converting M2 macrophages to those with an M1 or M1-like phenotype by introducing or infecting them with immunostimulatory bacteria that lack flagella and have pentaacylated LPS, e.g., bacteria that are msbB- / pagP- and encode a therapeutic product that is part of an intracytoplasmic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), e.g., a mutant or modified STING protein as described herein, including one that is modified so that expression of type I IFN is constitutive, e.g., one that leads to constitutive type I IFN expression. Exemplary of such bacteria are species or types of strains of the genus Salmonella.

[0106] The immunostimulatory bacteria can encode therapeutic products on the plasmid, including anti-cancer therapeutics, including any products used in connection with or in conjunction with cancer treatment. Therapeutic products also include products for, in connection with, or used in conjunction with the treatment of pathogens, such as viral, bacterial, yeast, or parasitic pathogens. The products can be antiviral therapeutics and / or anti-pathogenic bacterial therapeutics. Some therapeutic products are used to treat various indications; for example, it is understood that anti-cancer products may also be effective and can be used to treat viral infections. Antiviral agents include vaccines and therapeutic products that inhibit viral enzymes or inhibit viral replication. Therapeutic agents include antiviral therapeutics, e.g., viral antigens, whose expression results in an immune protective response against viruses and antibodies that bind and / or interact with viral antigens, thereby inhibiting or blocking the virus or resulting in antiviral immunity. Other therapeutic agents include antibacterial therapeutic agents, e.g., bacterial antigens, whose expression results in an immune protective response against bacterial pathogens and / or results in antibodies that bind to and / or interact with bacterial antigens, thereby inhibiting or blocking pathogenic bacteria or resulting in anti-pathogenic bacterial immunity. Antiviral agents encoded by bacteria include antiviral therapeutic agents for treating viruses or infectious pathogens that cause persistent infections. Exemplary antiviral therapeutic agents include viral antigens or epitopes of antigens, such as, but not limited to, viral surface proteins, viral nucleocapsid proteins, viral nonstructural proteins, or viral open reading frame proteins, for example, in embodiments where the therapeutic product is a viral surface antigen or a portion thereof sufficient to produce an immune response in the host, or in embodiments where the therapeutic product interferes with viral gene expression or replication.

[0107] The virus or other infectious agent or pathogen may cause chronic and / or latent and / or delayed infection. Exemplary viral pathogens include T-cell leukemia viruses, Epstein-Barr virus, cytomegalovirus, herpes viruses, varicella-zoster virus, measles virus, papovavirus, prions, hepatitis viruses A, B, C, D, and E, adenovirus, parvovirus, human immunodeficiency virus (HIV), coronavirus, smallpox virus, poliovirus, influenza virus, rotavirus, yellow fever virus, mumps virus, rubella virus, and papillomavirus, e.g., HIV or hepatitis viruses. Other infectious agents include prions and protozoa.

[0108] Therapeutic products encoded by immunostimulatory bacteria include immunostimulatory proteins, such as the aforementioned stimulator of interferon genes (STING) protein, modified STING proteins, cytokines, chemokines, or costimulatory receptors or ligands. Immunostimulatory bacteria include those in which the bacteria have lost flagella and / or contain pagP- or msbB- / pagP genome modifications. The product includes an immunostimulatory protein that confers or contributes to anti-tumor immunity in the tumor microenvironment, such as a cytokine or chemokine.

[0109] Immunostimulatory bacteria include any of those described herein and include genome modifications such that they do not express asparaginase or do not activate the synthesis of secreted asparaginase, and / or the genome of the immunostimulatory bacteria has been modified by deletion or disruption of all or a sufficient portion of the gene ansB, which encodes L-asparaginase II, such that the bacterium is ansB- and does not express active L-asparaginase II. Such bacteria can encode any therapeutic product of interest, including any of those provided or described herein, whereby the resulting bacterium is an anti-cancer therapeutic that colonizes tumors and / or tumor microenvironments, whereby the ansB- phenotype reduces or eliminates the production of active asparaginase.

[0110] Provided herein is an immunostimulatory bacterium comprising a nucleic acid operably linked to a prokaryotic promoter, the nucleic acid comprising RNA that is missing sequences necessary for prokaryotic translation, whereby the RNA is produced in the bacterium but cannot be translated by the bacterium, the bacterium has a genomic modification whereby infection is restricted to myeloid cells, and the RNA encodes or is a therapeutic product.

[0111] Provided herein are immunostimulatory bacteria that primarily or exclusively infect myeloid cells, including an RNA delivery system comprising bacterially encoded RNA under the control of a prokaryotic promoter, where the RNA lacks regulatory sequences necessary for bacterial translation, and the RNA encodes or is a therapeutic product. As discussed, the transcribed RNA lacks a Shine-Dalgarno sequence or contains or lacks other sequences that are not translated by bacterial ribosomes but are translated by eukaryotic ribosomes in the host. The RNA may contain a Kozak consensus sequence, for example, ACCAUGG (SEQ ID NO: 397). Immunostimulatory bacteria include those that lack flagella and are msbB- / pagP-. In the RNA delivery system, the bacterium can contain a plasmid encoding the therapeutic product(s). In some embodiments, the nucleic acid encoding the therapeutic product(s) is operably linked to an inducible or constitutive prokaryotic promoter. The coding nucleic acid may include an internal ribosome entry site (IRES) or other sequence encoding a nucleic acid such that the transcribed RNA is not translated by bacterial ribosomes but is translated by eukaryotic ribosomes. As a result, the bacteria encode and produce RNA encoding any therapeutic proteins, such as antigens and other payloads, but do not translate the RNA. The RNA is translated after the bacteria are administered to a host, such as a human, for example, by invading phagocytes, where it can be translated.

[0112] Provided are immunostimulatory bacteria and RNA delivery systems in which the genome of the immunostimulatory bacteria has been modified by deletion, disruption, or other alteration of all or a sufficient portion of the ansB gene, which encodes L-asparaginase II, such that the bacterium is ansB- and does not express active L-asparaginase II. The immunostimulatory bacteria and RNA delivery systems may further contain genome modification(s), such as deletion, disruption, or other alteration of all or a sufficient portion of the csgD gene, such that the bacterium is ansB-, does not express active L-asparaginase II, and is csgD-, and does not activate the synthesis of curli pili, and / or the bacterium may further contain genome modification(s) that impair biofilm formation. These bacteria and RNA delivery systems may further include genome modifications in which the bacterium is flagellin- and does not produce flagella, and in which wild-type bacteria possess flagella. Thus, for example, the immunostimulatory bacterium or RNA delivery system may have a genomic modification in which the bacterium is csgD- / msbB- / pagP-, and the bacterium or RNA delivery system may also include a genomic modification in which the bacterium loses flagella. Other genomic modifications may be included in which the bacterium loses flagella and is lppA- / lppB-, and may be csgD-. Any of the immunostimulatory bacteria and RNA systems described herein may be auxotrophic for purines, for example, adenosine, and / or for adenosine, adenine, and / or ATP. Adenosine auxotrophy is advantageous for bacteria to accumulate in the tumor microenvironment or tumor-resident macrophages, where adenosine accumulation occurs. The bacteria provided herein may also include additional genomic modifications, including those that are purI-, pagP-, and / or asd- or thyA-, or both, by complete gene deletion.

[0113] The immunostimulatory bacteria provided herein can be aspartate semialdehyde dehydrogenase- (asd-), where the bacteria are asd- by ​​disruption or deletion or rearrangement or other modification of all or part of the endogenous gene encoding aspartate semialdehyde dehydrogenase (asd), such that endogenous asd is not expressed or a functional enzyme is not produced, or are thyA- by disruption or deletion of all or part of the endogenous gene(s) such that endogenous thymidylate synthase is not expressed or a functional enzyme is not produced. Thus, provided are immunostimulatory bacteria that are aspartate semialdehyde dehydrogenase- (asd-), where the bacterium is asd- due to disruption or deletion of all or part of the endogenous gene encoding aspartate semialdehyde dehydrogenase (asd), such that endogenous asd is not expressed or a functional enzyme is not produced, and the bacterium is thyA- due to disruption or deletion of all or part of the endogenous gene(s), such that endogenous thymidylate synthase is not expressed or a functional enzyme is not produced. The immunostimulatory bacteria can encode aspartate semialdehyde dehydrogenase (asd) on a plasmid under the control of a bacterial promoter, such that asd can be produced in vivo. Exemplary immunostimulatory bacteria provided herein are those in which the unmodified bacterium is a Salmonella bacterium.

[0114] The immunostimulatory bacteria provided herein can be msbB- due to genome modifications, including, but not limited to, complete or partial deletion of the gene locus, which results in bacteria that grow better than those that retain part of the gene.

[0115] The bacterium provided herein is an immunostimulatory bacterium that is asd-, purI-, msbB-, flagellin-, and pagP-; or any immunostimulatory bacterium described above or herein that is asd-, csgD-, purI-, msbB-, flagellin-, and pagP-; or thyA-, csgD-, purI-, msbB-, flagellin-, and pagP-; or ansB-, asd-, csgD-, purI-, msbB-, flagellin-, and pagP-; or ansB-, thyA-, csgD-, purI-, msbB-, flagellin-, and pagP-, or ansB-, thyA-, csgD-, purI-, msbB-, flagellin-, and pagP-.

[0116] The immunostimulatory bacteria encode therapeutic products, such as, but not limited to, anti-cancer therapeutics and / or therapeutics for treating diseases, disorders, and conditions caused by pathogens or other diseases, disorders, and conditions. Exemplary encoded products include, but are not limited to, combinations of products, such as modified STING and IL-15 or IL-15 / IL-15R alpha chain complexes, where STING constitutively induces type I IFN in the absence of cGAS and / or any STING ligand.

[0117] Provided are immunostimulatory bacteria as described herein that encode a therapeutic product that is an antiviral product, e.g., a viral antigen and / or an antiviral antibody, including, for example, viruses that cause chronic or latent infections, such as, but not limited to, hepatitis viruses, herpes viruses, varicella-zoster viruses, pox viruses, measles viruses, and retroviruses.

[0118] In immunostimulatory bacteria, the copy number of the plasmid ranges from low to high. In some embodiments, the copy number is low to moderate, e.g., the copy number of the plasmid is less than 150. In other embodiments, the copy number is from 150 to greater than 150. Thus, in embodiments, the copy number of the plasmid is equal to or less than 150 copies, or is 150 or less. In other embodiments, the plasmid is present at a low copy number, where a low copy number is less than 25, or less than 20, or less than about 25, or less than about 20 copies, typically less than 25.

[0119] Encoded therapeutic products include proteins and also nucleic acids, e.g., RNA products, antigens, and antibodies. A plasmid may encode two or more products. Exemplary products include any used in the treatment of cancer. Also included are products used as antiviral treatment(s). For example, a bacterium may encode two or more products selected from a cytokine, a protein that constitutively induces type I IFN, and a costimulatory receptor or molecule. The costimulatory molecule may be modified to lose its cytoplasmic domain. The products may have complementary activities, and in some embodiments, the activities are synergistic. Nucleic acids encoding one or more of the therapeutic product(s) include nucleic acids that can encode signals for secretion of the therapeutic product(s) from cells, including bacteria. The nucleic acid encoding the product of the plasmid can be operably linked to regulatory sequences recognized by a eukaryotic host. In embodiments in which the immunostimulatory bacterium encodes two or more products, expression of each product may be under the control of a separate promoter, or expression of all two or more products may be under the control of a single promoter, e.g., the nucleic acid encoding each product may be separated by a nucleic acid encoding a 2A peptide to drive separate translation of each encoded therapeutic product. Exemplary 2A peptides include T2A, F2A, E2A, or P2A peptides, which drive separate expression of therapeutic products expressed under the control of a single promoter. Eukaryotic regulatory signals can control the expression of the product(s). Eukaryotic promoters include RNA polymerase II promoters and RNA polymerase III promoters. Eukaryotic RNA polymerase II promoters include viral promoters from viruses that infect eukaryotes and mammalian RNA polymerase II promoters.Exemplary promoters include, but are not limited to, viral promoters such as cytomegalovirus (CMV) promoter, SV40 promoter, Epstein-Barr virus (EBV) promoter, herpesvirus promoter, and adenovirus promoter; elongation factor-1 (EF-1) alpha promoter, MND promoter, UBC promoter, PGK promoter, or CAG promoter, such as EF-1 alpha, adenovirus 2 or 5 late, CMV, SV40, MND, PGK, EIF4A1, CAG, or CD68 promoter. Viral promoters include those that are late promoters. Immunostimulatory bacteria include those in which the plasmid contains regulatory sequences, including terminator and / or promoter(s) selected from SV40, hGH, BGH, MND, chicken beta-globulin, and rbGlob (rabbit globulin) genes, to control the expression of the therapeutic product(s). The encoded therapeutic product(s) can be operably linked to a signal sequence for secretion from cells containing the plasmid; or, in some embodiments, can be designed or modified to be expressed on the surface of the cell in which they are produced. Plasmids encoding therapeutic product(s) can include nucleic acid constructs including an enhancer, a promoter, an open reading frame encoding the therapeutic product or heterologous protein, and a polyA tail. Exemplary plasmids in bacteria include those containing constructs including an enhancer, a promoter, an IRES, an open reading frame encoding the therapeutic product or heterologous protein, and a polyA tail, and those containing constructs including an enhancer, a promoter, an IRES, a localization sequence, an open reading frame encoding the therapeutic product or heterologous protein, and a polyA tail. The construct can include a post-transcriptional regulatory element, such as a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) or a hepatitis B virus post-transcriptional regulatory element (HPRE).

[0120] The bacteria provided herein contain a plasmid encoding a therapeutic product or a mutant therapeutic product that is part of a cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN). These products can be modified to have increased or constitutive activity for the expression of type I IFN. In their unmodified forms, these products directly or indirectly sense or interact with cytoplasmic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides to induce the expression of type I IFN. Just as the therapeutic product is a mutant that leads to constitutive expression of type I IFN when expressed in a subject, the mutant or modified protein induces the expression of type I IFN without sensing or interacting with cytoplasmic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides. Mutations in the mutant protein include those that result in a gain-of-function form in which the mutant does not require a cytoplasmic nucleic acid, nucleotide, dinucleotide, or cyclic dinucleotide, or ligand, to induce the expression of type I IFN. These products, which are part of the cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN), include, but are not limited to, STING, RIG-I, MDA-5, IRF-3, IRF-5, IRF-7, IRF-8, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, and SNRNP200, such as STING, RIG-I, IRF-3, IRF-5, IRF-8, or MDA5, particularly mutants of these proteins that result in increased activity or constitutive expression of type I interferon (IFN). Mutations include those that promote or cause interferonopathy in humans. Other mutations in these proteins include mutations and combinations of mutations that eliminate phosphorylation sites in the proteins, thereby reducing nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling.Provided are immunostimulatory bacteria wherein the therapeutic product is a variant thereof having increased or constitutive activity, wherein the therapeutic product is STING, RIG-I, IRF-3, IRF-5, IRF-8, or MDA5, for example, where the therapeutic product is a mutant of STING, RIG-I, IRF-3, IRF-5, IRF-8, or MDA5 that comprises a gain-of-function mutation that results in increased or constitutive expression of type I IFN, and, optionally, a mutation or substitution in the C-terminal tail (CTT) that results in reduced NF-κB signaling activity, for example, where the therapeutic product is a mutant of STING, RIG-I, IRF-3, IRF-5, IRF-8, or MDA5 in which one or more serine (S) or threonine (T) residues that become phosphorylated as a result of viral infection are replaced with aspartic acid (D), thereby making the resulting mutant a phosphomimetic that constitutively induces type I IFN.

[0121] The therapeutic product can be IRF-3 having one or more substitutions at residues at positions 396, 398, 402, 404, and 405 with reference to SEQ ID NO: 312, where the residues are substituted with aspartic acid residues, e.g., IRF-3 comprising a S396D substitution with reference to SEQ ID NO: 312, such as where IRF-3 comprises S396D / S398D / S402D / T404D / S405D substitutions with reference to SEQ ID NO: 312. Other examples include therapeutic products that sense cytoplasmic DNA / RNA and are mutant STING, MDA5, RIG-I, or IRF-3, where unmodified STING has the sequence set forth in any of SEQ ID NOs: 305-309, unmodified MDA5 has the sequence set forth in SEQ ID NO: 310, unmodified RIG-I has the sequence set forth in SEQ ID NO: 311, and unmodified IRF-3 has the sequence set forth in SEQ ID NO: 312. In other examples, the therapeutic product is selected from among STING, MDA5, IRF-3 and RIG-I and contains a gain-of-function mutation(s) that renders STING, MDA5, IRF-3, IRF-5, IRF-8 or RIG-I constitutively active, whereby expression of type I IFN is constitutive. a) With reference to SEQ ID NOS: 305 to 309 in STING, S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R one or more selected from 310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A, N154S / R284G and S324A / S326A; b) in MDA5, one or more of T331I, T331R, A489T, R822Q, G821S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F and A452T with reference to SEQ ID NO: 310; c) in RIG-I, one or both of E373A and C268F with reference to SEQ ID NO: 311, and d) S396D with reference to SEQ ID NO: 312 in IRF-3, and e) any of the above conservative substitutions For example, a therapeutic product may be selected from the group consisting of S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316A, E31 and a mutant STING protein containing one or more amino acid substitutions selected from 6N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A, N154S / R284G, and S324A / S326A, and conservative substitutions thereof.

[0122] The encoded therapeutic product includes, for example, any form of antibody known to those of skill in the art, and also includes multispecific, e.g., bispecific, antibodies, e.g., bispecific antibodies such as bispecific T cell engagers. For example, a plasmid in a bacterium encodes a bispecific T cell engager antibody that binds to DLL3 and CD3, e.g., a bispecific T cell engager antibody comprising the heavy and light chains of an anti-DLL3 antibody and an anti-CD3 antibody, such as those encoded in constructs designated SC16.15, SC16.34, and SC16.56, which encode the heavy and light variable chains of antibodies that bind to DLL3 and CD3, respectively, the sequences of which are set forth in SEQ ID NOS: 485-491, or humanized variants thereof and variants with at least 95% or 98% sequence identity thereto. For example, the encoded bispecific T cell engager antibody may comprise any of a) to f): a) a light chain comprising amino acid residues 154 to 260 of SEQ ID NO: 487, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and b) a heavy chain comprising the sequence of amino acid residues set forth as amino acid residues 22 to 138 of SEQ ID NO: 487, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and c) a light chain comprising the sequence of amino acid residues set forth as amino acid residues 155 to 261 of SEQ ID NO: 489, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and d) a heavy chain comprising the sequence of amino acid residues set forth as amino acid residues 22 to 139 of SEQ ID NO: 489, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and e) heavy and light chains, the light chain comprises the sequence of amino acid residues set forth as amino acid residues 155 to 261 of SEQ ID NO: 485, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and A heavy chain and a light chain, wherein the heavy chain comprises the sequence of amino acid residues set forth as amino acid residues 22 to 139 of SEQ ID NO: 485, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and f) the heavy and light chains of an anti-CD3 antibody, the light chain of the anti-CD3 antibody comprises the sequence of amino acid residues set forth as amino acid residues 398 to 504 of SEQ ID NO: 485, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and The bispecific antibody may comprise a combination of anti-CD3 antibody heavy and light chains, wherein the heavy chain of the anti-CD3 antibody comprises the sequence of amino acid residues set forth as amino acid residues 267-382 of SEQ ID NO: 485, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto, such that the resulting construct is capable of binding to each of DLL3 and CD3. The encoded bispecific T cell engager antibody construct may comprise a leader sequence, such as an IgGK leader sequence. The bispecific antibody may also comprise a Gly-Ser linker linking one or more light and heavy chains, and may also comprise a linker, e.g., a Gly-Ser linker linking moieties that bind to different targets, e.g., a Gly-Ser linker linking the anti-DLL3 and anti-CD3 moieties of an exemplary bispecific T cell engager antibody. An exemplary linker comprises the sequence of amino acids set forth as residues 383-397 of SEQ ID NO: 485, and variants thereof. The bispecific T cell engager antibody can comprise a flag tag, for example, a flag tag comprising the sequence of amino acids set forth as residues 505-512 of SEQ ID NO: 485. Exemplary constructs encoding bispecific T cell engager antibodies include nucleic acid constructs encoding a leader sequence, the heavy and light chains of an anti-DLL antibody and the heavy and light chains of an anti-CD3 antibody, and optionally one or more peptide linkers and, optionally, a flag tag, such as where the encoded bispecific T cell engager antibody construct comprises the sequence of amino acid residues set forth in any of SEQ ID NOs: 485-491 or a humanized variant thereof or a variant having at least 95% sequence identity thereto, and a sequence having at least 95% or 98% sequence identity thereto and retaining bispecific binding.

[0123] Another example of a therapeutic product that can be encoded in the plasmid of the provided immunostimulatory bacteria is a tumor-associated antigen(s). These plasmids can encode other therapeutic products, such as a protein that is part of a cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN), such as a modified STING protein that constitutively induces type I interferon. Exemplary of such STING proteins are modified STING proteins containing substitutions corresponding to N154S or R284G or N154S / R284G. These include human STING, non-human STING with reduced NF-κB signaling activity compared to human STING, and chimeric STING proteins containing a CTT derived from non-human STING with reduced NF-κB activity compared to human STING. Plasmids can also encode, for example, cytokines with antitumor or antiviral activity, such as IL-15 or the IL-15 / IL-15R alpha chain complex. The plasmid can encode a modified STING, IL-15 / IL-15R alpha chain complex or a combination of modified STING, IL-15 and a tumor-associated antigen and / or a bispecific T cell engager antibody.Other encoded therapeutic products include: IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-15, IL-2 with reduced binding to IL-2Ra, IL-15 / IL-15R alpha chain complex (IL-15Rα-IL-15sc), IL-18, IL-21, IL-23, IL-36γ, IL-2 modified to not bind IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, interferon-γ, CCL3, CCL4, CCL5, and those involved in or associated with T cell recruitment and / or persistence. and immunostimulatory protein(s) that confer or contribute to an anti-tumor immune response in the tumor microenvironment selected from one or more of: proteins that cause or enhance an anti-tumor immune response, CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), a member of the B7-CD28 family, a CD47 antagonist, an anti-IL-6 antibody or IL-6 binding decoy receptor, a TGF beta polypeptide antagonist, and a member of the tumor necrosis factor receptor (TNFR) superfamily. Other immunostimulatory proteins that can be encoded include costimulatory molecules selected from among CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, and 4-1BB ligand (4-1BBL), appropriately truncated and lacking their cytoplasmic domains for expression on antigen-presenting cells (APCs), such that the truncated gene product can constitutively transmit immunostimulatory signals to T cells via costimulatory receptor engagement and, due to the deleted cytoplasmic domain, cannot transmit counterregulatory signals to antigen-presenting cells (APCs). Other immunostimulatory proteins that confer or contribute to anti-tumor immune responses in the tumor microenvironment include cytokines, chemokines, and / or costimulatory molecules, such as cytoplasmic domain-deleted versions thereof, e.g., one or more of 4-1BBL, CD80, CD86, CD27L, CD24L, B7RP1, and OX40L. Other therapeutic products include, for example, TGF-beta polypeptide antagonists.Other therapeutic products include antibodies or antigen-binding fragments or forms thereof, such as, but not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, nanobodies, diabody fragments, and single-chain antibodies. Antibodies or antigen-binding fragments thereof can be humanized or human. Exemplary antibodies and antigen-binding fragments include antagonists of PD-1, PD-L1, CTLA-4, VEGF, VEGFR2, CD24, or IL-6.

[0124] The immunostimulatory bacterium can contain a plasmid encoding two or more therapeutic products selected from: a) an immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment; b) a protein that is part of a cytoplasmic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), or a mutant thereof with increased activity that increases type I IFN expression, or a mutant thereof that leads to constitutive expression of type I IFN; and c) an anti-cancer antibody or antigen-binding portion thereof. In some embodiments, the immunostimulatory protein is a costimulatory molecule that is missing a cytoplasmic domain or a portion thereof sufficient for expression on antigen-presenting cells (APCs), such that the truncated costimulatory molecule can constitutively transmit immunostimulatory signaling to T cells via costimulatory receptor engagement and cannot transmit counter-regulatory signaling to antigen-presenting cells (APCs). In some embodiments, the immunostimulatory bacterium comprises a plasmid encoding two or more therapeutic products under the control of a single promoter, wherein the therapeutic products are selected from one or more of: a) an immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment; b) a protein that is part of a cytoplasmic DNA / RNA sensor pathway that leads to expression of type I interferon (IFN), or a variant thereof that has increased activity that increases expression of type I IFN, or a variant thereof that leads to constitutive expression of type I IFN; and c) an anti-cancer antibody or antigen-binding portion thereof, wherein the encoding nucleic acids are separated by an IRES sequence or a 2A peptide, and each nucleic acid encoding each product may be operably linked to a nucleic acid encoding a signal sequence, whereby, upon translation of the encoded mRNA, each product is individually expressed and secreted from the bacterium and / or cell containing the plasmid. If the immunostimulatory protein is a costimulatory molecule, it may be missing its cytoplasmic domain or a portion thereof sufficient for expression on antigen-presenting cells (APCs), such that the truncated costimulatory molecule is capable of constitutively transmitting immunostimulatory signaling to T cells via costimulatory receptor engagement and is unable to transmit counterregulatory signaling to antigen-presenting cells (APCs).For example, the immunostimulatory bacteria provided herein may contain a plasmid encoding at least two therapeutic products selected from a cytokine, a protein constitutively inducing type I IFN, a costimulatory molecule, and an anti-cancer antibody or its antigen-binding portion. The immunostimulatory bacteria may contain a plasmid encoding at least two therapeutic products selected from two or more or all of a cytokine, a protein constitutively inducing type I IFN, a costimulatory molecule, and an anti-cancer antibody or its antigen-binding portion, as well as an antigen or antigenic protein, e.g., one that provides an immune response against tumors and / or pathogens. The antigen or antigenic protein may be, for example, a tumor-associated antigen. Examples of tumor-associated antigens and proteins include, but are not limited to, carcinoembryonic antigens, cancer viral antigens, and overexpressed / accumulated antigens, cancer testis antigens, lineage-restricted antigens, mutated antigens, post-translationally altered antigens, or idiotypic antigens. Exemplary of such antigens and proteins include:

[0125] [Table 2-1] [Table 2-2]

[0126] In some embodiments, the encoded payload, e.g., a therapeutic protein, is expressed under the control of a eukaryotic promoter. In other embodiments described herein, the encoded payload is expressed under the control of a prokaryotic promoter that is recognized by the bacterium, e.g., when an immunostimulatory bacterium is the RNA delivery vehicle.

[0127] In all embodiments, the immunostimulatory bacteria can include genomic modifications such that the bacteria are flagellin-, asd-, msbB-, pagP-, and csgD-; or ansB-, asd-, csgD-, purI-, msbB-, flagellin-, and pagP-; or thyA-, asd-, csgD-, purI-, msbB-, flagellin-, and pagP-; or thyA-, csgD-, purI-, msbB-, flagellin-, and pagP-; or other combinations of modifications as described herein. Included are genomic modifications of the immunostimulatory bacteria that, upon administration, render them less inflammatory than wild-type bacteria. Combinations of modifications include those that render the bacteria msbB- / pagP- and lack flagella, whereas wild-type bacteria have flagella.

[0128] The immunostimulatory bacteria provided herein can be anti-cancer therapeutics. The immunostimulatory bacteria can also be vaccines for treating or preventing cancer or infectious diseases caused by pathogens, or for reducing the risk thereof. In such embodiments, the encoded payload is expressed under the control of a prokaryotic promoter, and the nucleic acid encoding the payload may contain a translational regulatory signal that is recognized by eukaryotic ribosomes but not by bacterial ribosomes. As a result, the encoded product and construct are transcribed by the bacteria but not translated by the bacteria; they are translated when administered to a host and in host cells, such as phagocytes. The immunostimulatory bacteria can encode a pathogen-derived antigen or protein or epitope(s) thereof. Exemplary pathogens include, but are not limited to, viruses or other pathogens that cause chronic viral infections or chronically infect a subject, such as infections with hepatitis virus, herpes virus, varicella-zoster virus (VZV), Epstein-Barr virus, human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), respiratory syncytial virus (RSV), and measles virus, as well as pathogens that cause acute infections, such as chronic influenza and early infections with coronaviruses, e.g., severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, which causes COVID-19).

[0129] In some embodiments, the plasmid encodes an antigen from a pathogen or an epitope thereof or a combination of epitopes thereof, e.g., an antigen from an essential viral protein, e.g., in the case of coronavirus, an antigen from the nucleocapsid, M and / or S protein, that can result in the enhancement of neutralizing antibodies and long-lived circulating, tissue-resident CD8+ T cells. Provided are immunostimulatory bacteria in which a nucleic acid encoding the antigen, epitope, or antigenic protein is operably linked to a prokaryotic promoter recognized by the bacterium, and the coding sequence contains regulatory sequences for translation recognized by eukaryotic ribosomes, thereby preventing the bacterium from translating the encoded RNA, or the coding sequence does not contain a Shine-Dalgarno sequence recognized by bacterial ribosomes, thereby preventing the encoded mRNA from being translated, and the mRNA is delivered to a eukaryotic host cell to which the bacterium is delivered.

[0130] Any of the immunostimulatory bacteria provided herein may comprise a plasmid encoding two or more therapeutic products under the control of a single promoter, wherein expression of nucleic acids encoding at least two or all of the products is under the control of a single promoter, and the nucleic acid encoding each product is separated by a nucleic acid that provides a separate translation product, e.g., a nucleic acid encoding a 2A polypeptide, such that, upon translation, each product is expressed separately.

[0131] In all of the immunostimulatory bacteria provided herein, the nucleic acid encoding one or more of the therapeutic products is operably linked to a nucleic acid encoding a sequence that directs secretion of the expressed product(s).

[0132] Therapeutic products include costimulatory molecules, particularly those with a cytoplasmic domain deletion for expression on antigen-presenting cells (APCs), whereby the resulting truncated gene product is capable of constitutively transmitting immunostimulatory signaling to T cells via costimulatory receptor engagement and is incapable of counterregulatory signaling to APCs due to the cytoplasmic domain deletion. Exemplary of such costimulatory molecules are one or more of 4-1BBL, CD80, CD86, CD27L, B7RP1, CD24L, or OX40L, among others, with a cytoplasmic domain deletion.

[0133] The immunostimulatory bacteria can encode multiple products. In some embodiments, at least one product is selected from a) and at least one product is selected from b), wherein a) is selected from IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-23, IL-36gamma, IL-2 with reduced binding to IL-2Ra, IL-15 / IL-15R alpha chain complex (IL-15Rα-IL-15sc), IL-18, IL-2 modified to not bind IL-2Ra, CXCL9, CXCL10, CXCL11, interferon alpha, interferon beta, CCL3, CCL4, CCL5, or a product that is involved in, causes, or enhances T cell recruitment and / or persistence. a) is a protein that is a member of the B7-CD28 family, a TGF-beta polypeptide antagonist, or a member of the tumor necrosis factor receptor (TNFR) superfamily, and b) is STING, RIG-I, MDA-5, IRF-3, IRF-5, IRF-7, IRF-8, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, or SNRNP200.

[0134] Additional therapeutic products include, for example, one or more of a TGF-beta inhibitory antibody, a TGF-beta binding decoy receptor, an anti-IL-6 antibody, and an IL-6 binding decoy receptor. The immune stimulatory bacteria may be a combination of the following therapeutic products: IL-2 and IL-12p70; IL-2 and IL-21; IL-2, IL-12p70 and STING GOF mutants; IL-2, IL-21 and STING GOF mutants; IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (4-1BBLΔcyt), in which Δcyt is a deletion of the cytoplasmic domain; IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα and STING GOF mutants; IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα and IL-12p70; IL-15 / IL-15Rα and IL-21; IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; IL-15 / IL-15Rα, IL-21, and STING GOF mutants; IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-21; IL-12p70, IL-21, and STING GOF mutants; IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and STING GOF mutants; IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-18; IL-12p70, IL-18, and STING GOF mutants; IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-2, and IL-12p70; TGF-β decoy receptor, IL-2, and IL-21; TGF-β decoy receptor, IL-2, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-2, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, and IL-12p70; TGF-β decoy receptor, IL-15 / IL-15Rα, and IL-21; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-12p70, and IL-21; TGF-β decoy receptor, IL-12p70, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor and IL-12p70; TGF-β decoy receptor, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-12p70, and IL-18; TGF-β decoy receptor, IL-12p70, IL-18, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor and STING GOF mutant; anti-CTLA-4 antibody, IL-2, and IL-12p70; anti-CTLA-4 antibody, IL-2, and IL-21; anti-CTLA-4 antibody, IL-2, IL-12p70, and STING GOF mutants; anti-CTLA-4 antibody, IL-2, IL-21, and STING GOF mutants; anti-CTLA-4 antibody, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, and STING GOF mutant; anti-CTLA-4 antibody, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, and IL-12p70; anti-CTLA-4 antibody, IL-15 / IL-15Rα, and IL-21; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-12p70, and IL-21; anti-CTLA-4 antibody, IL-12p70, IL-21, and STING GOF mutants; anti-CTLA-4 antibody, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody and IL-12p70; anti-CTLA-4 antibody, IL-12p70, and STING GOF mutant; anti-CTLA-4 antibody, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-12p70, and IL-18; anti-CTLA-4 antibody, IL-12p70, IL-18, and STING GOF mutants; anti-CTLA-4 antibody, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies and STING GOF mutants; CD40 agonist, IL-2, and IL-12p70; CD40 agonist, IL-2, and IL-21; CD40 agonist, IL-2, IL-12p70, and STING GOF mutants; CD40 agonist, IL-2, IL-21, and STING GOF mutants; CD40 agonist, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, and IL-12p70; CD40 agonist, IL-15 / IL-15Rα, and IL-21; CD40 agonist, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-12p70, and IL-21; CD40 agonist, IL-12p70, IL-21, and STING GOF mutants; CD40 agonist, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist and IL-12p70;CD40 agonist, IL-12p70, and STING GOF mutant; CD40 agonist, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-12p70, and IL-18; CD40 agonist, IL-12p70, IL-18, and STING GOF mutants; CD40 agonist, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); tumor-associated antigens; CD40 agonists and STING GOF mutants STING GOF variants include chimeric STING and non-human STING, including those described or exemplified herein, including those detailed above and below.

[0135] In all embodiments, the immunostimulatory bacteria provided herein can also encode tumor-associated antigens. These bacteria are of interest for use as vaccines and as therapeutic agents. Other exemplary combinations of encoded products include: IL-2 and IL-12p70; IL-2 and IL-21; IL-2, IL-12p70, and STING GOF mutants; IL-2, IL-21, and STING GOF mutants; IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (4-1BBLΔcyt), in which Δcyt is a deletion of the cytoplasmic domain; IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα, and STING GOF mutants; IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα and IL-12p70; IL-15 / IL-15Rα and IL-21; IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; IL-15 / IL-15Rα, IL-21, and STING GOF mutants; IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-21; IL-12p70, IL-21, and STING GOF mutants; IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and STING GOF mutants; IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-18; IL-12p70, IL-18, and STING GOF mutants; IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-2, and IL-12p70; TGF-β decoy receptor, IL-2, and IL-21; TGF-β decoy receptor, IL-2, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-2, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, and IL-12p70; TGF-β decoy receptor, IL-15 / IL-15Rα, and IL-21; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-12p70, and IL-21; TGF-β decoy receptor, IL-12p70, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor and IL-12p70; TGF-β decoy receptor, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-12p70, and IL-18; TGF-β decoy receptor, IL-12p70, IL-18, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor and STING GOF mutant; anti-CTLA-4 antibody, IL-2, and IL-12p70; anti-CTLA-4 antibody, IL-2, and IL-21; anti-CTLA-4 antibody, IL-2, IL-12p70, and STING GOF mutants; anti-CTLA-4 antibody, IL-2, IL-21, and STING GOF mutants; anti-CTLA-4 antibody, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, and STING GOF mutant; anti-CTLA-4 antibody, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, and IL-12p70; anti-CTLA-4 antibody, IL-15 / IL-15Rα, and IL-21; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-12p70, and IL-21; anti-CTLA-4 antibody, IL-12p70, IL-21, and STING GOF mutants; anti-CTLA-4 antibody, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody and IL-12p70; anti-CTLA-4 antibody, IL-12p70, and STING GOF mutant; anti-CTLA-4 antibody, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-12p70, and IL-18; anti-CTLA-4 antibody, IL-12p70, IL-18, and STING GOF mutants; anti-CTLA-4 antibody, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies and STING GOF mutants; CD40 agonist, IL-2, and IL-12p70; CD40 agonist, IL-2, and IL-21; CD40 agonist, IL-2, IL-12p70, and STING GOF mutants; CD40 agonist, IL-2, IL-21, and STING GOF mutants; CD40 agonist, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, and IL-12p70; CD40 agonist, IL-15 / IL-15Rα, and IL-21; CD40 agonist, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-12p70, and IL-21; CD40 agonist, IL-12p70, IL-21, and STING GOF mutants; CD40 agonist, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist and IL-12p70;** CD40 agonist, IL-12p70, and STING GOF mutants; CD40 agonist, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-12p70, and IL-18; CD40 agonist, IL-12p70, IL-18, and STING GOF mutants; CD40 agonist, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist and STING GOF mutants; Bispecific T cell engagers (BiTe) targeting DLL3, EGFR, Her2, CEA, mesothelin, PSMA, EpCAM, CD74, folate receptor, nectin-4, EphA2, CA-IX, B7H3, Siglec-15, Muc1, or Lewis Y antigens; BiTe+STING protein, BiTe+IL-15, BiTe+IL-15+STING protein; tumor antigen(s) + STING gain-of-function mutant; Therapeutic compositions of tumor antigen(s) and IL-15; A therapeutic composition comprising tumor antigen(s) + IL-15 + STING gain-of-function mutant; one or more antigens and IFN; one or more antigens and IFNα; one or more antigens, and IFNα2 or IFNα1-16; one or more antigens and any of IFNα1-16; one or more antigens and IFN-β; one or more antigens, IFNα2, and IFN-β; IRF3 GOF mutant with one or more antigens and mutation S396D; one or more antigens, IFNα2 or IFNα1-16, and an IRF3 GOF mutant with mutation S396D; IFN alpha 2+IRF3-S396D; IFNα1–16+IRF3-S396D; IFN alpha 2 + IFN-beta; IFNα1-16 + IFN-beta FLT-3L, sialidase, or IL-12p35, or azurin, or membrane-anchored IL-2, IL-12, IL-12p35, IL-21, IL-15, FLT-3L, alone or in combination with other immune-stimulating proteins; and A TLR8 agonist, either alone or in combination with any of the immunostimulatory proteins, wherein the agonist is polyU or polyU / G, microRNA, or miR-21. In all embodiments, the immunostimulatory bacterium can encode a tumor-associated antigen, such as any of those listed in the table above and described herein.

[0136] Other encoded therapeutic products include, for example, bispecific T cell engagers, such as those that bind delta-like ligand 3 (DLL3) and CD3. In all embodiments, the immunostimulatory bacteria can encode a cytokine, e.g., a cytokine and a modified or mutant STING protein. The immunostimulatory bacteria encode an immunostimulatory protein(s) that confer or contribute to anti-tumor immunity in the tumor microenvironment, e.g., a cytokine or chemokine that confer or contribute to anti-tumor immunity in the tumor microenvironment. Exemplary cytokines include IL-15, IL-2, and IL-12, e.g., the IL-15 / IL-15R alpha chain complex. Exemplary cytokines include IL-15, IL-2, and IL-12, e.g., the IL-15 / IL-15R alpha chain complex. Exemplary STING proteins include any of those described herein, particularly those that contain a gain-of-function mutation such that the STING protein constitutively induces type I IFN. STING proteins also have lower NF-κB signaling activity than human STING, and can be engineered or selected to result in low NF-κB signaling and constitutive type I IFN induction.

[0137] Exemplary STING proteins include chimeric STING proteins comprising a human STING protein with a CTT from Tasmanian devil, or chimeric STING proteins that are chimeric STINGs comprising a human STING protein with a CTT from Tasmanian devil and one or more gain-of-function mutations, for example, one or both of N154S and R284G, or any of the mutations described herein or known in the art that achieve constitutive activity. Exemplary modified STING gain-of-function mutants include any of those described herein.

[0138] The encoded therapeutic product can include a multimerization domain, e.g., an Fc domain. Other encoded therapeutic products include any of those described herein, such as a B7 protein transmembrane domain and / or a product that is a bispecific T cell engager antibody. The encoded therapeutic product may be GPI-anchored or may include a moiety that increases the serum half-life of the encoded product, e.g., a polypeptide, e.g., human serum albumin (HSA) or a portion thereof. The therapeutic product may be a fusion with another fusion protein, e.g., collagen.

[0139] The immunostimulatory bacteria can be derived from any suitable bacterial species, including, but not limited to, species such as Salmonella, Listeria, and Escherichia coli, and any of those listed or described herein.The immunostimulatory bacteria contain genome modifications that prevent the bacteria from infecting epithelial cells or reduce their ability to infect epithelial cells, and contain modified LPS to attenuate the bacteria and / or increase the uptake or infection of phagocytes, such as tumor-resident macrophages, and increase tumor colonization.Various genome modifications that achieve these characteristics are described herein.Strains include those that lack flagella and have pentaacylated LPS. Exemplary strains include those designated YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI / ΔthyA and other strains containing genomic modifications that render the bacterium auxotrophic for adenosine, lose flagella, and have pentaacylated LPS, such as by rendering the bacterium msbB- / pagP- and, optionally, losing or reducing curli pili.

[0140] The bacteria provided herein are useful as therapeutic agents for diseases, disorders, and conditions, such as cancer. They are also useful as vaccines for preventing (reducing the risk or severity of) or treating diseases, disorders, and conditions, such as those caused by pathogens or cancer. They can be designed to deliver RNA as described herein. Provided are genomically modified bacteria containing genomic modifications that result in reduced responses by toll-like receptors (TLR) 2, 4, and 5 compared to bacteria that do not contain the genomic modifications, the bacterium contains further genome modifications such that it is auxotrophic for a required nutrient or factor, such that it is unable to replicate in a eukaryotic host, but can replicate in vitro when the nutrient or factor is supplied; the bacterium comprises a plasmid containing nucleic acid encoding the product, or comprises RNA encoding the product; the product encoded by the nucleic acid or RNA is an antigen sequence or a sequence from a pathogen that is a pathogenic virus, bacterium, or parasite, or is a tumor antigen, whereby upon expression of the encoded antigen in a host, the host mounts an immune defense or immune response against the pathogenic virus, bacterium, parasite, or tumor antigen, or the product is a therapeutic product; expression of the antigen sequence(s) is under the control of a prokaryotic promoter such that RNA encoding the antigen(s) is produced in the bacterium; the nucleic acid encoding the antigen comprises regulatory sequences that inhibit or prevent translation of the encoded RNA by bacterial ribosomes but do not inhibit or prevent translation of the encoded RNA by eukaryotic host ribosomes, thereby uncoupling translation from transcription in the bacterium; The resulting bacteria, when administered to a eukaryotic subject, are selective for infecting phagocytes and deliver the nucleic acid to the phagocyte, where the RNA is translated.

[0141] These immunostimulatory bacteria can encode multiple products, which can be encoded as a polycistronic message, under the control of separate promoters, or in any suitable configuration. The nucleic acid encoding the product or at least the antigen sequence may contain a sequence that prevents or inhibits translation by a prokaryotic host, e.g., a bacterium, and / or a sequence that inhibits or prevents translation in the bacterium while promoting translation in a eukaryotic host. An exemplary sequence is an internal ribosome entry site (IRES), which promotes or enhances host cell translation and inhibits or prevents bacterial translation. As a result, products encoded in the bacterium are transcribed into RNA but are not translated until they are in the eukaryotic host. The bacterium thus serves as an RNA delivery vehicle. An exemplary IRES may be the vascular endothelial growth factor and type 1 collagen-induced protein (VCIP) IRES. Exemplary bacteria are provided in which the nucleic acid encoding the antigen(s) contains a VCIP or other IRES that inhibits or reduces translation in the bacterium and allows, and optionally promotes or enhances, translation in the eukaryotic host. A translational regulatory sequence, such as an IRES or VCIP IRES, can be included in the plasmid at a position 3' of the promoter and 5' of the antigen(s) coding sequence. An exemplary VCIP IRES is set forth in SEQ ID NO: 434, or a sequence having at least 98% sequence identity thereto, and having IRES activity.

[0142] The pathogen from which the encoded antigen is derived or against which the encoded antigen is directed can be derived from or include any pathogen, such as a bacterium or a virus. The encoded antigen also includes a tumor antigen. The resulting bacterium can be a vaccine for preventing or treating a viral or bacterial infection or for preventing or treating cancer. The pathogen can be selected from viruses that cause chronic viral infections. Exemplary infectious diseases include those caused by hepatitis virus, herpes virus, varicella-zoster virus (VZV), Epstein-Barr virus, human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), respiratory syncytial virus (RSV), measles virus, and other viruses that chronically infect subjects. The infection can be an acute infection, such as an infection caused by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or a severe acute respiratory syndrome species of Escherichia, Staphylococcus, Pseudomonas, Actinobacteria, Archaeobacteria, Mycobacteria, or Porphyromonas. Other pathogens include P. gingivalis, SARS-CoV2, or Escherichia coli or Haemophilus influenzae.

[0143] The plasmid in the bacterium can encode the antigen and can further encode an immunostimulatory protein or other adjuvant, as well as a combination of immunostimulatory proteins or other therapeutic proteins, such as a STING protein, particularly a modified STING containing a gain-of-function mutation and / or a chimeric STING protein, such as any of those described herein. Thus, any of the bacteria described herein can be provided such that they deliver mRNA encoding any of the products and combinations described herein. The product can be encoded in a plasmid as part of a polycistronic sequence expressing the antigen under the control of a prokaryotic promoter recognized by the bacterium, or the immunostimulatory protein(s) and / or other therapeutic protein can be encoded on a plasmid under the control of a eukaryotic promoter recognized by the eukaryotic host. The resulting bacterium can contain mRNA encoding the antigen(s) and any other proteins expressed under the control of a prokaryotic promoter, where the mRNA is produced by culturing the bacterium in vitro. Bacteria include those containing genome modifications that cause the bacterium to lose its flagellum and produce LPS with pentaacylated lipid A. The bacterium may be asd- or thyA- or both and / or adenosine auxotroph and csgD-, or may be ansB-. The bacterium may comprise or further comprise a nucleic acid encoding a TLR8 agonist, such as polyU, polyU / G, microRNA, or miR-21. Exemplary bacteria include those that are msbB- / pagP-, have lost flagella, and are asd- or thyA-, or both asd- and thyA-. Exemplary bacterial species include, but are not limited to, species or strains of Escherichia, Listeria, Mycobacterium, or Salmonella. The bacterium may be a Salmonella strain, such as Salmonella typhimurium. The unmodified Salmonella may be a wild-type strain, or the unmodified Salmonella strain may be an attenuated strain.Exemplary starting bacteria are those derived from strains VNP20009 or YS1646, or those derived from strain ATCC14028 or strains having all of the identifying characteristics of strain ATCC14028.

[0144] Genome modification includes nucleic acid sequence and generally any modification that causes phenotypic changes.Genome modification includes one or more of deletion, insertion, disruption, transposition and other modifications in genes, so that the product coded by the gene is not produced or is inactive when produced.

[0145] The promoter controlling expression of the product encoded in the plasmid may be a prokaryotic promoter, particularly in embodiments in which bacteria are provided as vaccines and / or RNA delivery vehicles. These include embodiments in which the product is expressed in bacteria in vitro prior to administration to a subject, e.g., a human or animal. Prokaryotic promoters include bacterial promoters and bacterial phage promoters. Any promoter recognized by bacterial RNA polymerase or by the encoded phage polymerase.

[0146] Provided are vaccines comprising any of the bacteria provided herein, particularly those encoding antigens for therapeutic or prophylactic immunization, in a vehicle in an amount for administration to a subject to induce an adaptive immune response in the subject. Vaccines and other compositions containing the bacteria provided herein can be formulated for any route of administration, for example, as an aerosol, powder, tablet, or suppository. They can be formulated for oral, nasal, inhalation, rectal, vaginal, ocular, intracranial, intradermal, or intramuscular administration.

[0147] Provided herein is a vaccine containing a nucleic acid encoding an antigen from a protein derived from a viral pathogen, such as a respiratory virus, e.g., a coronavirus, e.g., SARS-COV2, formulated for nasal or pulmonary inhalation. The vaccine is designed and formulated to not sufficiently activate TLR2, thereby inducing type I IFN. Activation of TLR2 inhibits or reduces type I IFN activation, and thus, vaccines such as those provided herein, such as immunostimulatory bacteria and vaccines, do not activate TLR2 or activate TLR2 at a sufficiently low level that type I IFN is activated.

[0148] Vaccines can also be formed / formulated so as not to activate TLR4 and / or TLR5, or to have sufficiently low activation of TLR4 and / or TLR5 responses sufficient to reduce or inhibit type I IFN, resulting in type I IFN expression. As described herein, many vaccines and delivery vectors designed to stimulate type I IFN expression also have the property of activating TLR2, 4, and / or 5, with activation at a level sufficient to inhibit or reduce type I IFN expression. The immunostimulatory bacteria and vaccines provided herein are designed so that they do not sufficiently activate TLR2, and in particular TLR4 and / or TLR5, so that type I IFN expression is not reduced or inhibited by the bacteria or vaccine.

[0149] Provided are vaccines containing nucleic acids encoding antigens, proteins, or epitopes from a pathogen or tumor, which induce an immune response against the pathogen or tumor, where the pathogen is a respiratory pathogen that infects the respiratory system, including the lungs and / or nasopharynx, and the tumor is a lung or airway tumor. The vaccine is formulated for inhalation through the nasal cavity or lungs, and delivers the nucleic acid to phagocytic macrophages to convert them into immunostimulatory phagocytic macrophages capable of in situ antigen cross-presentation to CD8+ T cells and migration to lymph nodes to prime CD4+ and CD8+ T cells. The vaccine is designed, configured, and / or formulated so as not to activate TLR4 and / or TLR5 responses sufficiently to reduce or inhibit type I IFN. Vaccines are provided that do not activate TLR2 / 4 / 5 sufficiently to reduce or inhibit type I IFN. A vaccine can encode a product that induces an immune response against a pathogen, e.g., a virus. Viral pathogens include mRNA viruses, e.g., coronaviruses or influenza viruses. Coronaviruses include SARS viruses, e.g., SARS-COV2 viruses. A vaccine encodes an antigen, protein, or epitope from a pathogen, e.g., a viral antigen, protein, or epitope. Exemplary proteins include capsid or nucleoprotein. For example, if the virus is SARS-COV2, the protein or epitope may be or be derived from proteins designated or encoded by S1, S2, envelope (E), membrane (M), nucleocapsid (N), ORF3a, ORF6, ORF7a, ORF7b, and ORF8, e.g., a protein or epitope derived from or consisting of spike protein. A vaccine includes the bacteria provided herein that deliver mRNA, e.g., mRNA encoding a protein or antigen. mRNA includes mRNA that has been modified to increase the stability of the mRNA and / or the stability of the encoded protein or antigen or epitope.The encoded protein can be modified, such as by altering the protein's structure to alter its interaction with host cell proteins. For example, mRNAs and proteins have been designed to improve, increase, or stabilize the interaction of the encoded protein or epitope with cell surface receptors. Modified mRNAs have been designed to encode the spike protein from the SARS-COV2 virus, and those skilled in the art can similarly design other modified proteins / mRNAs from other viruses to enhance or improve their effectiveness in preventing, reducing, or ameliorating diseases caused by the virus. Vaccines can be delivery vehicles, such as oncolytic viruses and immunostimulatory bacteria, and contain nucleic acids encoding antigens or proteins from pathogens or encoding tumor antigens.

[0150] Immunostimulatory bacteria contain pentaacylated lipopolysaccharide (LPS) and lack flagella, whereas wild-type bacteria contain flagella. As a result, the bacteria do not induce an inflammatory response or induce a reduced inflammatory response, for example, compared to a bacterium designated VNP20009. Furthermore, bacteria may contain genomic modifications that result in the absence of curli pili production. In addition to encoding immunostimulatory proteins, such as cytoplasmic DNA / RNA sensor pathway proteins, e.g., eSTING and cytokines, such as the IL-15 / IL-15R alpha chain complex or IL-15, they can also encode tumor-associated antigens (TAAs). Products that are part of the cytoplasmic DNA / RNA sensor pathway that result in type I interferon (IFN) expression include, for example, STING, IRF3, IRF5, IRF7, IRF8, MDA5, RIG-I, and particularly modified forms thereof containing gain-of-function mutations that result in constitutive type I interferon expression. The immunostimulatory bacteria can be an attenuated bacterium or a gram-negative bacterium, or is a gram-positive bacterium.

[0151] Exemplary bacteria from which the immune stimulatory bacteria may be derived include, but are not limited to, Salmonella, Shigella, Escherichia coli, Bifidobacteriae, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, and the like. Examples of suitable bacterial strains include strains of Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus, or Erysipelothrix, or archaea, or attenuated or modified strains of any of the bacterial strains listed above. The bacterium can be, for example, a strain of Shigella, Escherichia coli, Listeria, or Salmonella.

[0152] For example, bacteria such as Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium ovis, pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Mycobacterium tuberculosis, Staphylococcus aureus, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintanaquintana or Agrobacterium tumefaciens bacteria. Included are strains of Salmonella typhimurium, for example, where the unmodified Salmonella is a wild-type strain, for example, where the unmodified Salmonella strain is attenuated, or where, for example, the immunostimulatory bacterium is derived from strain VNP20009 or YS1646 or strain ATCC14028 or a strain having all of the identifying characteristics of strain ATCC14028.

[0153] The immunostimulatory bacteria can be ansB-, asd-, csgD-, purI-, msbB-, flagellin- and pagP-, or are ansB-, thyA-, csgD-, purI-, msbB-, flagellin- and pagP-.

[0154] Bacteria can be engineered to encode and express a gene that confer resistance to complement killing (rck), for example, a Salmonella rck gene, such as a strain of E. coli that has been engineered to express rck, e.g., Nissle.

[0155] Also provided are pharmaceutical compositions containing the therapeutic agents provided herein, including any of the immunostimulatory bacteria in a pharmaceutically acceptable vehicle. They can be formulated for systemic administration, e.g., parenteral, or intravenous, or intramuscular, or intratumoral, or intraperitoneal, or oral, or rectal, or vaginal, or intraocular, or intradermal, or intracranial, or mucosal administration, or by oral or oral or nasal inhalation, or by rectal, or by aerosol to the lungs and / or nose, or mucosally, or intracranial, or intradermal, or intratumoral administration.

[0156] Methods for treating cancer and the use of therapeutic agents containing immune-stimulating bacteria are provided. The cancer may include solid tumors, hematological malignancies, or any other malignancies. The methods include administering a composition. Subjects can be selected by biopsy to identify subjects whose tumors contain proliferative macrophages, e.g., proliferative M2 macrophages, for example, by identifying macrophages with markers of proliferation, e.g., biopsy surface markers: CD68+KI67 and / or PCNA, MERTK. Proliferative macrophages can exhibit all or a subset of the above markers. For example, gene expression of the G2M module, in which more than half (>14 genes in the set) are expressed, can be used to further confirm proliferation. Alternatively, tumor macrophages can be biopsied and assessed for expression of at least two of CD68, MERTK, and KI67 and / or PCNA.

[0157] Also provided are combination therapies, such as regimens in which a subject is first treated with a chemotherapeutic agent that induces apoptosis in tumors, or an agent such as a checkpoint inhibitor, e.g., an anti-PD-1 or anti-PD-L1 antibody, prior to administration of a therapeutic agent provided herein. A second anti-cancer agent or treatment is administered before, simultaneously with, after, or intermittently with the immunostimulatory bacteria or pharmaceutical composition. The second anti-cancer agent or treatment can be immunotherapy, chemotherapy, surgery, radiation, or a combination thereof.

[0158] Cancers to be treated include, but are not limited to, leukemia, lymphoma, gastric cancer, and cancers selected from among cancers of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, colorectal, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicle, cervix, and liver. The cancer may be metastatic.

[0159] The second agent includes, but is not limited to, an agent selected from among anti-PD-1, anti-PD-L1 or anti-CTLA-4 antibodies, anti-IL-6, anti-Siglec-15, anti-VEGF, anti-CD73, and anti-CD38 antibodies. Other exemplary second agents may be selected from among poly(ADP-ribose) polymerase (PARP) inhibitors, histone deacetylase (HDAC) inhibitors, chemotherapeutic agents, anti-EGFR antibodies, CAR-T cells, anti-Her2 antibodies, anti-mesothelin antibodies, and anti-B-cell maturation antigen (BCMA) antibodies.

[0160] Pharmaceutical Composition Provided are pharmaceutical compositions comprising any of the bacteria and / or vaccines provided herein. The bacteria or other delivery vehicle are formulated in a pharmaceutically acceptable medium. The formulation may be a liquid, powder, such as a lyophilized powder or tablet, or other suitable formulation. Vaccines can be administered locally, particularly, but not limited to, by inhalation, intramuscular, and transdermal administration, resulting in a local immune response and preventing or reducing the likelihood or severity of infection. To treat tumors, bacteria and vaccines can be administered systemically, for example, intravenously, or intratumorally or by other routes, such as intrahepatically, intraperitoneally, and other modes. Immunostimulatory bacteria accumulate and colonize phagocytes, particularly those at the site of administration and / or in the tumor microenvironment and tumor.

[0161] Methods and Uses The vaccines, bacteria, and pharmaceutical compositions provided herein are intended for use in the treatment of diseases, disorders, and conditions, including cancer and infectious diseases, and for the prevention, treatment, or reduction of symptoms thereof. Provided herein are bacteria referred to herein as immunostimulatory bacteria due to their ability to accumulate in phagocytes, including tumor-resident macrophages, and due to their properties and composition, as well as their ability to stimulate an immune response through the encoded payload and the combination of the properties and structure of the bacteria and the payload. The bacteria can be used as therapeutic agents for the treatment of diseases, disorders, and conditions, and can produce proteins encoded by components of nucleic acid constructs in plasmids in the bacteria, and can also be used to deliver mRNA.

[0162] Methods for treating cancer and / or viral or other pathogen infections are provided. The uses and methods include administering or using the RNA delivery systems and vaccines and immunostimulatory bacteria provided herein to treat or prevent (reduce the risk or severity of) cancer and / or viral infections. The immunostimulatory bacteria and vaccines encode tumor-associated antigens or viral or other pathogen antigens, proteins, or epitopes.

[0163] Thus, provided are methods of use and treatment using the immunostimulatory bacteria, vaccines, delivery vehicles and compositions provided herein for use in treating or preventing (reducing the risk of occurrence of) a disease or condition or infection or cancer.

[0164] Also provided are methods for converting M2 macrophages to an M1 or M1-like phenotype by administering immunostimulatory bacteria modified as described herein and encoding an immunostimulatory protein, e.g., STING, particularly modified STING, and a combination of modified STING and a cytokine, e.g., IL-15, e.g., the IL-15 / IL-15R alpha chain complex. These immunostimulatory bacteria are used to treat or administered to a subject having a condition, disease, or disorder that can be treated by enhancing an antiviral or antitumor immune response. Use of the immunostimulatory bacteria to convert M2 macrophages to M1 or M1-like phenotype macrophages in a subject having a condition, disease, or disorder that can be treated by enhancing an antiviral or antitumor immune response. The subject may have a disease, disorder, or condition that is cancer and / or a viral or other pathogen infection.

[0165] Provided are methods for delivering RNA encoding a therapeutic product, comprising administering immunostimulatory bacteria designed as described above and below to deliver RNA for the treatment of a disease, condition, or disorder. Uses of such bacteria for treatment are also provided. Diseases, disorders, and conditions include cancer and / or viral or other pathogen infections. As described herein, immunostimulatory bacteria transcribe the encoded product in vitro but do not translate it, so that when administered to a subject, they are used to deliver RNA, e.g., mRNA. Provided are bacteria for use in delivering RNA to a subject, comprising a plasmid encoding a heterologous product, wherein the nucleic acid encoding the heterologous product is linked to a promoter recognized by the bacterium, and the nucleic acid encoding the product contains a eukaryotic sequence for translation not recognized by the bacterium, thereby allowing the bacterium to produce but not translate the RNA. The bacterium delivers RNA encoding a therapeutic product and / or an antigen or protein from a pathogen or tumor to elicit an immune response against the antigen or protein.

[0166] Encoded therapeutic products include anything described herein in the original claims, such as nucleic acids encoding proteins, or variants thereof, that are part of an intracellular DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN). Type I IFNs include interferon-α and interferon-β. Variants include those that, when expressed in a subject, lead to constitutive expression of type I IFN. These include gain-of-function (GOF) variants that do not require an intracellular nucleic acid, nucleotide, dinucleotide, or cyclic dinucleotide (CDN) to result in the expression of type I IFN. Examples of these proteins include proteins selected from STING, RIG-I, MDA-5, IRF-3, IRF-5, IRF-7, IRF-8, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, and SNRNP200, and mutants thereof, that have increased activity or result in constitutive expression of type I interferon (IFN). Mutants include mutants of STING, RIG-I, IRF-3, or MDA5 in which one or more serine (S) or threonine (T) residues that are phosphorylated as a result of viral infection are replaced with aspartic acid (D), whereby the resulting mutant is a phosphomimetic that constitutively induces type I IFN, and any of those known to those of skill in the art and / or described herein. Examples of variants include those in which the mutations are selected as follows: a) in STING, with reference to SEQ ID NOS: 305-309: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R 284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R31 0A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A,b) in MDA5, with reference to SEQ ID NO: 310: T331I, T331R, A489T, R822Q, G821S, A946T, R337G, D39 c) in RIG-I, one or both of E373A and C268F, with reference to SEQ ID NO: 311; and d) in IRF-3, S396D, with reference to SEQ ID NO: 312, e.g., S102P, V147L, V147M, N154S, V155M, with reference to SEQ ID NOs: 305-309. G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R 293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E29 and mutant STINGs containing one or more amino acid substitutions selected from: N154S / R284G, N154S / R284G, N154S / R284G, and S324A / S326A.

[0167] The immunostimulatory bacteria may also encode immunostimulatory proteins that confer or contribute to an anti-tumor immune response in the tumor microenvironment, including, but not limited to, cytokines, chemokines, or costimulatory molecules. Examples of these include 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-2 modified to not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, interferon-γ, CCL3, CCL4, CCL5, and proteins involved in T cell recruitment and / or persistence. or proteins that cause or enhance these, CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), members of the B7-CD28 family, CD47 antagonists, anti-IL-6 antibodies or IL-6 that bind to decoy receptors, TGF-beta polypeptide antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily. A costimulatory molecule selected from CD40, CD40 ligand, CD28, OX40, OX40 ligand, 4-1BB, and 4-1BB ligand can be truncated so that the molecule lacks the cytoplasmic domain for expression on antigen-presenting cells (APCs), or a portion thereof; due to the deleted or partially deleted or truncated cytoplasmic domain that eliminates immunosuppressive counter-signaling, the truncated gene product can transmit constitutive immunostimulatory signaling to T cells through costimulatory receptor binding, but cannot transmit counter-regulatory signaling to antigen-presenting cells (APCs). Other such proteins are TGF-beta polypeptide antagonists, such as anti-TGF-beta antibodies or fragments thereof, anti-TGF-beta receptor antibodies or fragments thereof, soluble TGF-beta antagonist polypeptides, or TGF-beta binding decoy receptors.

[0168] The plasmid may encode a therapeutic antibody or antigen-binding fragment thereof, such as a Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, nanobody, diabody fragment, or single-chain antibody. Examples include, but are not limited to, antagonists of PD-1, PD-L1, CTLA-4, VEGF, VEGFR2, or IL-6.

[0169] The plasmids can encode complementary products whose expression results in enhanced anti-tumor or other activity. For example, the combination of a modified, e.g., constitutively active and / or chimeric STING protein described herein with a cytokine such as the IL-15 / IL-15R alpha chain complex (IL-15Rα-IL-15sc) has synergistic activity.

[0170] The immunostimulatory bacteria provided herein can be used to treat benign nervous system tumors. Examples of tumors include those in which the subject has or has been diagnosed with a benign tumor or tumor-related condition selected from among neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), schwannomatosis, meningioma, schwannoma, vestibular schwannoma, sporadic schwannoma, neurofibroma, neurofibromatosis (NF), and combinations thereof. Provided are methods of treatment and uses of the immunostimulatory bacteria provided herein for treating a subject having or at risk of having a benign nervous system tumor by administering to the subject a therapeutically effective amount of a composition comprising the immunostimulatory bacteria described herein, for example, one comprising the phenotype YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD. The bacteria may be combined with immune checkpoint inhibitors, such as anti-PD-1 antibodies or antagonists, or other checkpoint and / or angiogenesis inhibitors. Nervous system tumors include schwannomas, using attenuated Salmonella typhimurium and, optionally, one or more checkpoint inhibitors. Uses and methods using VNP20009 to treat such tumors are known (see, e.g., U.S. Patent Application Publication No. 2022 / 0125906 and Ahmed et al. (2022) Proc. Natl. Acad. Sci. USA 119:e2202719119, which describe treatments using VNP20009). The properties of the immune-stimulating bacteria provided herein are superior to VNP20009, as described herein and throughout this application, and therefore provide improved treatments for such conditions.

[0171] Bacteria provided herein, for example, those containing the phenotype YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD or YS1646Δasd / ΔFLG / ΔpagP / ΔcsgD, exhibit a broadly reduced systemic inflammatory signature, e.g., reduced levels of IL-2, TNF-alpha, IFN-gamma, IL-2, and IL-10, when administered compared to, for example, the VNP20009 strain. The bacteria demonstrated safety, including low inflammatory cytokines and no antibacterial antibodies, up to the highest dose tested in primate studies (3e9). These bacteria are abundant in tumors and immune-privileged tissues, are taken up by phagocytes such as macrophages, and do not infect epithelial or endothelial cells. The bacteria can deliver complementary payload combinations and are internalized by macrophages. When DNA is delivered, it is expressed by proliferating macrophages. The bacteria exhibit significant T cell infiltration in T cell-deficient tumors. Treated tumors show an expansion of activated CD8+ T cells and a decrease in exhausted T cells and Treg cells. The data in the Examples demonstrate cure in rodent models, including protection from metastatic disease and tumor rechallenge.

[0172] Modified STING proteins and encoding nucleic acids A delivery vehicle containing immunostimulatory bacteria can deliver a nucleic acid encoding a part of a cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN) or a protein that is a part of the cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN). These include STING, MDA5, IRF-3, IRF-7, IRF-5, IRF8, and RIG-I, and mutants thereof, which have increased or constitutive activity in inducing type I interferon (IFN) upon infection of cells, such as macrophages. Also contemplated is the delivery of agonists of one or more of STING, MDA5, IRF-3, IRF-5, IRF-7, IRF-8, and / or RIG-I. Consistent with the methods and uses herein, the delivery vehicle can deliver DNA for transcription and translation in eukaryotic host cells, as well as RNA and proteins, for example, as produced in bacteria as described herein. Achieving a phenotypic shift to a hybrid M1 / M2 phenotype can be achieved by delivering bacteria, proteins, and RNA into macrophages. Due to the expression of proteins, the macrophages are proliferative.

[0173] An exemplary product included for delivery is a modified STING protein that has increased or constitutive activity, or that results in increased or constitutive type I IFN expression. Generally, the STING protein contains a mutation that results in constitutive type I IFN expression when introduced into eukaryotic cells, such as humans. The STING protein may also have lower NF-κB signaling activity than human STING. STING proteins are provided in delivery vehicles, such as the bacteria provided herein, and other delivery vehicles, such as oncolytic vectors and nanoparticles, that encode the modified STING protein for expression in a subject to which the delivery vehicle is administered.

[0174] Provided are modified STING proteins and encoding nucleic acids, including plasmids and constructs for their expression. Reference to a modified STING protein includes reference to the encoding nucleic acid, plasmid, and construct. Provided are modified stimulator of interferon genes (STING) proteins from non-human species, where the non-human STING has reduced NF-κB signaling activity compared to human STING and, optionally, increased type I interferon (IFN) pathway signaling activity compared to human STING, where the non-human STING protein is modified to contain mutation(s) such that it has increased activity or acts constitutively in the absence of cytoplasmic nucleic acid, where the mutations are amino acid insertions, deletions, and / or substitutions, and the STING protein may have a deletion or disruption of a TRAF6 binding site.

[0175] Also provided are modified stimulator of interferon genes (STING) proteins from non-human species or chimeric human STING proteins and modified versions thereof, which contain one or more mutations associated with a gain-of-function (GOF) that result in constitutive activation of the encoded STING protein and / or enhanced sensitivity or increased affinity or binding to an endogenous ligand, whereby the STING protein is modified by one or more of an insertion, deletion, and substitution of amino acid(s), and the STING protein has IFN-beta signaling activity and attenuated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling activity compared to human STING, the mutation(s) resulting in increased or constitutive STING activity in inducing IFN-beta production. The human STING protein comprises a sequence set forth in any of SEQ ID NOs: 305-309, or is a human allelic variant thereof having at least 98% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 305-309.

[0176] The modified STING protein is a chimera in which the STING protein comprises a substitution of the C-terminal tail (CTT) region of a STING protein from a first species with the CTT of a STING protein from a second species, the second species having NF-κB signaling activity lower than that of human STING, and optionally a deleted TRAF6-binding site in the CTT. Provided are proteins that contain a TRAF6-binding site and proteins that do not contain a TRAF6-binding site. Mutations corresponding to those that result in constitutive type I IFN expression include any mutation(s) that correspond to those associated with STING-associated vasculitis (SAVI), an autoinflammatory disease in humans.

[0177] Provided are modified stimulator of interferon genes (STING) proteins that are chimeric, comprising a substitution of the CTT (C-terminal tail) region of a STING protein from a first species with the CTT of a STING protein from a second species, where the STING protein of the second species has NF-κB signaling activity lower than that of human STING, and optionally a deletion of the TRAF6-binding site in the CTT. The chimera can be a human STING protein with a substituted CTT and, optionally, a TRAF6-binding site. An exemplary human STING protein comprises a sequence set forth in any of SEQ ID NOS: 305-309, or a human allelic variant thereof having at least 98% sequence identity to the amino acid sequence set forth in any of SEQ ID NOS: 305-309. When referring to human STING protein NF-κB signaling activity for comparative purposes, the human STING protein has a sequence set forth in any of SEQ ID NOS: 305-309, and when a specific allele needs to be specified, reference is made to the protein of SEQ ID NOS: 305. Exemplary chimeric STING proteins include those in which the first species is human and the second species is selected from among Tasmanian devil, marmoset, cow, cat, ostrich, wild boar, bat, manatee, ibis, coelacanth, and chimaera shark. The chimeric STING protein may contain one or more mutations that render its activity for inducing type I IFN expression constitutive.

[0178] In selecting a non-human STING protein for use in a chimera or for modification, the type I IFN signaling activity is at least about 30%, 50%, 70%, 80% or more of that of wild-type human STING protein, and generally close to or higher than that of human STING. The NF-κB signaling activity is less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of that of wild-type human STING NF-κB signaling activity. Exemplary non-human or second species are selected from Tasmanian devils, marmosets, cattle, cats, ostriches, wild boars, bats, manatees, Japanese crested ibises, coelacanths, and chimaera sharks.

[0179] Modifications of STING are referenced by SEQ ID NOs: 305-309, e.g., by alignment with human STING of SEQ ID NO: 305. Mutations that render STING constitutive include, for example, mutation(s) corresponding to mutations that occur in interferonopathies, by reference to and alignment with human STING, the sequence of human STING to which alignment is achieved is set forth in any of SEQ ID NOs: 305-309. Exemplary of such mutations are N154S, R284G, and N154S / R284G, and others listed herein or known in the art. Exemplary modified STING proteins include those that include a replacement of the C-terminal tail (CTT) with a CTT derived from a STING protein that has reduced NF-κB signaling activity compared to that of human STING, for example, the replaced CTT may be derived from a Tasmanian devil, marmoset, cow, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, or chimaera STING protein. Exemplary replaced CTTs include any selected from the following species: Tasmanian devil, marmoset, cow, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, or chimaera STING protein, which replaces the human STING CTT. Exemplary CTT sequences include those from the following species and the following sequences: Tasmanian devil RQEEFAIGPKRAMTVTTSSTLSQEPQLLISGMEQPLSLRTDGF SEQ ID NO: 371, Marmoset EEEEVTVGSLKTSEVPSTSTMSQEPELLISGMEKPLPLRSDLF SEQ ID NO: 372, Bovine EREVTMGSTETSVMPGSSVLSQEPELLISGLEKPLPLRSDVF SEQ ID NO: 373; Cat EREVTVGSVGTSMVRNPSVLSQEPNLLISGMEQPLPLRTDVF SEQ ID NO: 374, Ostrich RQEEYTVCDGTLCSTDLSLQISESDLPQPLRSDCL SEQ ID NO: 375, Wild boar EREVTMGSAETSVVPTSSTLSQEPELLISGMEQPLPLRSDIF SEQ ID NO: 376; Bat EKEEVTVGTVGTYEAPGSSTLHQEPELLISGMDQPLPLRTDIF SEQ ID NO: 377, Manatee EREEVTVGSVGTSVVPSPSSPSTSSLSQEPKLLISGMEQPLPLRTDVF SEQ ID NO: 378, Toki CHEEYTVYEGNQPHNPSTTLHSTELNLQISESDLPQPLRSDCF SEQ ID NO: 379 coelacanth (Mutant 1) QKEEYFMSEQTQPNSSSTSCLSTEPQLMISDTDAPHTLKRQVC SEQ ID NO: 380, coelacanth (Mutant 2) QKEEYFMSEQTQPNSSSTSCLSTEPQLMISDTDAPHTLKSGF SEQ ID NO: 381, and Chimaera LTEYPVAEPSNANETDCMSSEPHLMISDDPKPLRSYCP SEQ ID NO: 383 and allelic variants or variants of each of these sequences having at least 98% sequence identity thereto.

[0180] The human CTT that can be replaced is, for example, an allelic or other variant that includes the sequence EKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (SEQ ID NO: 370) or has at least 98% sequence identity thereto. An exemplary chimeric STING is one in which the human STING CTT is replaced with a CTT from Tasmanian devil STING. The chimera may contain a mutation(s) that renders type I IFN expression constitutive, such that the STING protein is active in the absence of an inducible ligand and / or an inducible cytoplasmic nucleic acid. The replacing CTT from Tasmanian devil STING is an allelic or other variant that includes the sequence RQEEFAIGPKRAMTVTTSSTLSQEPQLLISGMEQPLSLRTDGF (SEQ ID NO: 371) or has at least 98% sequence identity thereto. The modified STING protein may contain a deleted or disrupted TRAF6 binding site, for example, a TRAF6 binding site containing amino acid residues DFS at the C-terminus, as in human STING.

[0181] Modifications that render the activity constitutive include, for example, S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / and one or more amino acid substitutions corresponding to one or more of D205A, S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A, and S324A / S326A. Exemplary of these are substitutions corresponding to C206Y or R284G or N154S, and combinations thereof, with reference to the sequence of human STING as set forth in any of SEQ ID NOs: 305-309.

[0182] The immunostimulatory bacteria provided herein can be used as vaccines (and as cancer therapeutics) by encoding an antigen against which an immune response or immunization or immune protection is desired. The immunostimulatory bacteria provided herein can be used to deliver RNA, such as mRNA or other forms, for use as a vaccine or for delivery of therapeutic drugs. As described herein, the bacteria contain a plasmid encoding a product of interest, e.g., a therapeutic product such as an antigen derived from a pathogen, under the control of a bacterial or other prokaryotic promoter recognized by the bacteria. The encoded nucleic acid cassette contains regulatory or other sequences that block, inhibit, or prevent translation by bacterial ribosomes but allow, provide, or enhance translation by eukaryotic ribosomes, such as those present in human cells. The bacteria are modified so that they cannot grow or replicate in eukaryotes, such as by rendering them asd-, which requires DAP for growth in vitro, or by altering them to thyA-, which requires a thymidine monophosphate precursor for growth but can be cultured in vitro to produce the encoded RNA. Those skilled in the art can inactivate a gene or product by modifying the endogenous gene, such as by deletion, insertion, substitution, or transposition, or by any such modification such that an active enzyme is not produced. See SEQ ID NO: 464 for an exemplary thyA gene from Salmonella, and SEQ ID NO: 465 for the encoded protein. RNA encoding a protein and / or antigen for immunization is encoded in a plasmid, but the encoded nucleic acid contains a translation signal / sequence that prevents the bacterium from translating the RNA. The resulting bacterium delivers the encoded RNA into host phagocytes, where it is translated by host cell ribosomes. ThyA- immunostimulatory bacteria have genome modifications, such as insertions, deletions, substitutions, or other changes, that result in the inactivation or elimination of the production of thymidylate synthase, which catalyzes the reductive methylation of dUMP to dTMP (a DNA biosynthetic precursor (precursor to dTTP)).

[0183] ΔthyA auxotrophy for other nutrients and essential products can be introduced instead of or in addition to asd inactivation / deletion. Other deletions or inactivations of genes or gene products required for growth, such as genes that produce nutrients, can be used instead of or in addition to asd, such as thyA (see, e.g., Loessner et al. (2006) FEBS Lett 265:81-88). Elimination of expression or production or other attenuating mutations in the bacterial genome for the production of such products results in the release of the encoded macromolecules upon bacterial cell death in vivo after administration. Asd is an essential enzyme for the synthesis of the bacterial cell wall, and thyA is an enzyme required for DNA synthesis. Mutations in the respective genes render the strain auxotrophic for diaminopimelic acid (DAP) or the thymidine monophosphate precursor. Upon depletion of the complementary substrate, such bacteria die by DAP starvation or thymine starvation, resulting in the release of bacterial proteins and plasmids. Inactivation or removal of Asd results in the release of macromolecules, and removal or inactivation of thyA expression / activity (to generate ΔThyA bacteria) does not result in the release of macromolecules, including proteins and plasmids, upon thymidine starvation (Leossner et al. (2006) FEBS Lett 265:81-88). ΔThyA is therefore advantageous for in vivo delivery of plasmids into host cells because the bacteria do not prematurely release their contents. The bacteria provided herein infect or accumulate in phagocytes, e.g., myeloid cells such as macrophages, dendritic cells, monocytes, and neutrophils, which ingest the bacteria, and intact ΔthyA bacteria release plasmids encoding therapeutic products inside the targeted cells.

[0184] Bacteria such as those herein have been genomically modified so that they are attenuated, have a reduced response by toll-like receptors (TLR) 2, 4, and 5 compared to bacteria that do not contain such genomic modifications, optionally encode rck (resistance to complement killing) to reduce inactivation by complement, and optionally contain modifications so that they infect primarily or exclusively phagocytes such as tissue-resident macrophages. It has been shown herein that genomic modifications, such as a combination of modifications that reduce responses by TLR2, 4, and 5, are required for the production of type I IFN by human antigen-presenting cells.

[0185] Provided are bacteria having genomic modifications that result in reduced responses by toll-like receptors (TLRs) 2, 4, and 5 compared to bacteria that do not contain such genomic modifications. Such modifications include those that result in the removal of pentaacylated LPS and flagella, e.g., pagP / msbB bacteria that lack flagella, and those that are deficient in or do not produce or express asparaginase II, e.g., ΔansB. The bacteria can also contain additional genomic modifications, such as one or more modifications that render the bacteria auxotrophic for a required nutrient or factor, such that the bacteria cannot replicate in a eukaryotic host but can replicate in vitro when nutrients or factors are provided, such as auxotrophy for thymidine (ΔthyA) due to a genomic modification that renders them unable to produce or express thymidylate synthase (ΔthyA), or auxotrophy for Asd.

[0186] The bacteria provided herein that combine some or all of these traits can be used to express therapeutic products, including anti-cancer products and antigens, depending on their intended use. For administration to subjects with cancer, bacteria that accumulate within tumor-resident myeloid cells can encode anti-cancer therapeutics, such as products that stimulate an immune response and / or products that suppress the immune system, or products that treat tumors and encode combinations of products that can act synergistically to treat cancer. The bacteria provided herein that accumulate within or infect phagocytes can also be used in subjects without cancer, such as as vaccines by delivering or encoding antigens or delivering RNA. Various embodiments and combinations of properties and products, as well as uses, are described throughout this disclosure.

[0187] In some embodiments, the bacterium comprises a plasmid containing a nucleic acid encoding a product, or comprises RNA encoding a product, wherein the product encoded by the nucleic acid or RNA is an antigen sequence or a sequence from a pathogen that is a pathogenic virus, bacterium, or parasite, or is a tumor antigen, whereby upon expression of the encoded antigen in the host, the host mounts an immunodefense or immune response against the pathogenic virus, bacterium, parasite, or tumor antigen, or the encoded product is a therapeutic product; expression of the antigen sequence(s) is under the control of a prokaryotic promoter such that RNA encoding the antigen(s) is produced in the bacterium; the nucleic acid encoding the antigen comprises regulatory sequences that inhibit or prevent translation of the encoded RNA by bacterial ribosomes but not by eukaryotic host ribosomes, whereby translation is uncoupled from transcription in the bacterium, and the resulting bacterium, when administered to a eukaryotic subject, is selective for infection of phagocytes and delivers the nucleic acid to phagocytes where the RNA is translated.

[0188] Upon administration, bacteria cultured in vitro to produce RNA infect phagocytes and deliver their contents, but they fail to survive and / or replicate, thereby providing RNA, such as mRNA, to host cells, which translate the RNA to produce the encoded product, such as an immunogenic protein or antigen. The RNA is typically mRNA, but may also be other forms of RNA, such as RNAi or eRNA (circular RNA), or other therapeutic forms. Immunostimulatory bacteria used for this purpose can contain a plasmid encoding the RNA at a high or higher (typically 150 or more) copy number to increase the amount of RNA delivered. Various embodiments are described, claimed, and exemplified herein. The mRNA can encode pathogen proteins, pathogen antigens, tumor antigens, therapeutic products for the treatment of tumors or infectious diseases, and combinations thereof. The mRNA may be synthetic, e.g., designed for immunization (see, e.g., U.S. Patent Publication No. 20190351040 and others describing mRNA for immunization or treatment). The resulting bacterium is a vaccine for therapy or immunization. The payload may be an adjuvant, an immunostimulatory protein, or may include products that induce type I interferon (IFN) and activate T cells in cooperation with the immunizing antigen / protein.

[0189] In some embodiments, the immunostimulatory bacteria provided herein contain a plasmid encoding two or more therapeutic proteins selected from: a) an immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment; b) one or more proteins that are part of a cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN), or a mutant thereof with increased activity that increases the expression of type I IFN, or a mutant thereof that leads to constitutive expression of type I IFN; and c) an anti-cancer antibody or antigen-binding portion thereof. For example, the immunostimulatory protein may be a costimulatory molecule that lacks a cytoplasmic domain or portion thereof sufficient for expression on antigen-presenting cells (APCs), such that the truncated costimulatory molecule can transmit constitutive immunostimulatory signaling to T cells through costimulatory receptor binding but cannot transmit counter-regulatory signaling to antigen-presenting cells (APCs). In some embodiments, the immunostimulatory bacteria encode at least two therapeutic products selected from a cytokine, a protein that constitutively induces type I IFN, a costimulatory molecule, and an anti-cancer antibody or antigen-binding portion thereof, which may be under the control of a single promoter. For example, expression of nucleic acids encoding at least two or all of the products is under the control of a single promoter, and the nucleic acid encoding each product is separated by a nucleic acid encoding a 2A peptide, which, upon translation, results in separate expression of each product. The nucleic acid encoding each product can be operably linked to a nucleic acid encoding a sequence that directs secretion of the expressed product from the cell.

[0190] Provided are immunostimulatory bacteria encoding two or more therapeutic products, at least one product selected from a) and at least one product selected from b), wherein a) is selected from IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-23, IL-36 gamma, IL-2 with reduced binding to IL-2Ra, IL-15 / IL-15R alpha chain complex (referred to herein as IL-15 / IL-15Rα, IL-15 complex, or other variations), IL-18, IL-2 modified to not bind IL-2Ra, CXCL9, CXCL10, CXCL11, interferon alpha, interferon beta, CCL3, CCL4, CCL5, T cell recruitment and / or persistence. a) is a protein involved in, causing, or enhancing inflammatory bowel disease, CD40, CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), a member of the B7-CD28 family, a TGF beta polypeptide antagonist, or a member of the tumor necrosis factor receptor (TNFR) superfamily, and b) is STING, RIG-I, MDA-5, IRF-3, IRF-5, IRF-7, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, or SNRNP200. They may also encode one or more of a TGFbeta inhibitory antibody, a TGFbeta binding decoy receptor, an anti-IL6 antibody, or an IL-6 binding decoy receptor.

[0191] Examples of combinations of encoded therapeutic products include the following combinations of therapeutic products: IL-2 and IL-12p70; IL-2 and IL-21; IL-2, IL-12p70, and STING GOF mutant; IL-2, IL-21, and STING GOF mutant; IL-2, IL-12p70, STING GOF mutant, and 4-1BBL (including 4-1BBLΔcyt) (Δcyt is a deletion of the cytoplasmic domain); IL-2, IL-21, STING GOF mutant, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα and STING GOF mutant; IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα and IL-12p70; IL-15 / IL-15Rα and IL-21; IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; IL-15 / IL-15Rα, IL-21, and STING GOF mutants; IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-21; IL-12p70, IL-21, and STING GOF mutants; IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and STING GOF mutants; IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-18; IL-12p70, IL-18, and STING GOF mutants; IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-2, and IL-12p70; TGF-β decoy receptor, IL-2, and IL-21; TGF-β decoy receptor, IL-2, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-2, IL-21, and STING GOF mutants;TGF-β decoy receptor, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, and IL-12p70; TGF-β decoy receptor, IL-15 / IL-15Rα, and IL-21; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-12p70, and IL-21; TGF-β decoy receptor, IL-12p70, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor and IL-12p70; TGF-β decoy receptor, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-12p70, and IL-18; TGF-β decoy receptor, IL-12p70, IL-18, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor and STING GOF mutants; anti-CTLA-4 antibody, IL-2, and IL-12p70;Anti-CTLA-4 antibodies, IL-2, and IL-21; anti-CTLA-4 antibodies, IL-2, IL-12p70, and STING GOF mutants; anti-CTLA-4 antibodies, IL-2, IL-21, and STING GOF mutants; anti-CTLA-4 antibodies, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-15 / IL-15Rα, and STING GOF mutants; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, and STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-15 / IL-15Rα, and IL-12p70; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, and IL-21; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-12p70, and IL-21; anti-CTLA-4 antibodies, IL-12p70, IL-21, and STING GOF mutants; anti-CTLA-4 antibodies, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies and IL-12p70; anti-CTLA-4 antibodies, IL-12p70, and STING GOF mutants; anti-CTLA-4 antibodies, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-12p70, and IL-18; anti-CTLA-4 antibodies, IL-12p70, IL-18, and STING GOF mutants;Anti-CTLA-4 antibodies, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies and STING GOF mutants; CD40 agonists, IL-2, and IL-12p70; CD40 agonists, IL-2, and IL-21; CD40 agonists, IL-2, IL-12p70, and STING GOF mutants; CD40 agonists, IL-2, IL-21, and STING GOF mutants; CD40 agonists, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt), CD40 agonists, IL-2, IL-21, and STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, and IL-12p70; CD40 agonist, IL-15 / IL-15Rα, and IL-21; CD40 agonist, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-12p70, and IL-21; CD40 agonist, IL-12p70, IL-21, and STING GOF mutants; CD40 agonist, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist and IL-12p70; CD40 agonist, IL-12p70, and STING GOF mutants; CD40 agonist, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt);CD40 agonist, IL-12p70, and IL-18; CD40 agonist, IL-12p70, IL-18, and STING GOF mutant; CD40 agonist, IL-12p70, IL-18, STING GOF mutant, and 4-1BBL (including 4-1BBLΔcyt); and CD40 agonist and STING GOF mutant.

[0192] Other combinations of products include, for example, combinations of IL-15 with STING gain-of-function mutants, including STING chimeras with gain-of-function mutation(s), or combinations of IL-15Rα-IL-15sc with STING gain-of-function mutants, including STING chimeras with gain-of-function mutation(s), as provided herein. Other products or combinations thereof include bispecific T cell engagers (BiTe®); Bite® and a STING protein, e.g., a modified GOF STING protein or STING chimera, as described herein; Bite® and IL-15; BiTe® and IL-15Rα-IL-15sc; BiTe®, IL-15 and a STING protein, e.g., a modified GOF STING protein or STING chimeric protein; and BiTe®, IL-15Rα-IL-15sc and a STING protein, e.g., a modified GOF STING protein or STING chimeric protein, where BiTe® targets, for example, DLL3, EGFR, Her2, CEA, mesothelin, PSMA, EpCAM, CD74, folate receptor, Nectin 4, EphA2, CA-IX, B7H3, Siglec-15, Muc1, Lewis Y antigen, and other such tumor antigens / tumor targets.

[0193] Also provided are therapeutic compositions containing tumor antigen(s) and a STING gain-of-function mutant or STING chimera; therapeutic compositions of tumor antigen(s) and IL-15; therapeutic compositions of tumor antigen(s) and IL-15Ra-IL-15sc; therapeutic compositions of tumor antigen(s), IL-15, and a STING gain-of-function mutant or STING chimera; and therapeutic compositions of tumor antigen(s), IL-15Ra-IL-15sc, and a STING gain-of-function mutant or STING chimera. These products can be encoded in immunostimulatory bacteria. The tumor antigen can be any of those listed or described herein (e.g., in Example 35) or known in the art.

[0194] The combination of products also includes the combination of antigens and immunostimulatory proteins.Antigens can be tumor antigens, or they can be immunization antigens, such as pathogen antigens, and pathogens include, for example, bacteria, protozoa, viruses, and prions and other prion-like particles that cause diseases and disorders.Antigens include any of those described or listed herein or known in the art.Combinations include, for example, the combination of one or more antigens with IFNα2; the combination of one or more antigens with IFN-β; the combination of one or more antigens with IFNα2 and IFN-β; the combination of one or more antigens with IRF3 GOF mutants having mutation S396D; and the combination of one or more antigens with IFNα2 and IRF3 GOF mutants having mutation S396D.

[0195] Other products and product combinations encoded in the immunostimulatory bacteria provided herein include, but are not limited to, a combination of IFNα2 and an IRF3 GOF mutant having the mutation S396D; a combination of IFNα2 and IFN-β; FLT-3L (FMS-like tyrosine kinase 3 ligand; see, e.g., SEQ ID NO: 436); a sialidase (see, e.g., SEQ ID NO: 435); the IL-12p35 subunit of IL-12p70 alone; azurin; a membrane-anchored / bound cytokine or molecule such as IL-2, IL-12, IL-12p35, IL-21, IL-15, IL-15Rα-IL-15sc, or FLT-3L; or FLT-3L; or a TLR8 agonist (e.g., the TLR8 agonist is polyU or polyU / G, a microRNA, or miR-21).

[0196] Also provided are modified non-human stimulator of interferon genes (STING) proteins and STING protein chimeras, as well as delivery vehicles, pharmaceutical compositions, cells encoding or containing these STING proteins, and uses thereof, and methods for treating cancer, all of which are described herein. In particular, the immunostimulatory bacteria provided herein encode the modified non-human STING proteins, non-human STING proteins, and STING chimeras described herein. These STING proteins encoded by the immunostimulatory bacteria are provided and described throughout the present specification.

[0197] Provided herein are modified non-human STING proteins that have lower NF-κB activation than human STING proteins and, optionally, higher type I interferon activation activity compared to wild-type (WT) human STING proteins. These non-human STING proteins are modified to contain mutations that enhance activity or act constitutively in the absence of intracellular nucleic acid signaling. The mutations are typically amino acid mutations that occur in human interferonopathies, such as those previously described for human STING. Corresponding mutations are introduced into the non-human species STING protein, and the corresponding amino acid residues are identified by alignment. In some embodiments, the TRAF6 binding site within the C-terminal tail (CTT) of the STING protein is deleted to reduce NF-κB signaling activity.

[0198] Provided are modified STING proteins, particularly chimeric human STING proteins, in which the CTT (C-terminal tail) region in a STING protein from one species, such as human, is replaced with a CTT from a STING protein from another species that has lower NF-κB signaling activity and / or higher type I IFN signaling activity than human STING, and in these chimeras, the TRAF6 binding site is optionally deleted.

[0199] Modified STING proteins also include mutations as described throughout this disclosure.

[0200] Also provided are delivery vehicles, such as immunostimulatory bacteria, any of those provided herein or known to those of skill in the art, including, for example, exosomes, nanoparticles, minicells, cells, liposomes, lysosomes, oncolytic viruses, and other viral vectors, encoding any of the modified STING proteins of 1 to 3.

[0201] Also provided are delivery vehicles, e.g., immunostimulatory bacteria, any of those provided herein or known to those of skill in the art, including, for example, exosomes, nanoparticles, minicells, cells, liposomes, lysosomes, oncolytic viruses, and other viral vectors, encoding unmodified STING from a non-human species, wherein the STING protein has reduced NF-κB signaling activity compared to human STING, and optionally, increased type I interferon stimulating / signaling activity compared to human STING.

[0202] Also provided are cells (if human, non-zygotic), e.g., cells used in cell therapy, e.g., T cells and stem cells, and cells used to produce STING proteins as described herein. Also provided are pharmaceutical compositions containing STING proteins, or delivery vehicles, or cells, or combinations thereof.

[0203] Uses and methods are provided for treating cancer and vaccinating against pathogens or cancer by administering any of the immune stimulatory bacteria as described herein.

[0204] Assays and methods for assessing NF-κB activity (signaling activity) and type I interferon-stimulating activity or interferon-β-stimulating activity of STING are described herein and are known to those skilled in the art. Exemplary methods include those described in de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175, which describes, inter alia, the interferon-β and NF-κB signaling activity of STING proteins from various species, including humans, thereby identifying STING proteins from various species that have lower NF-κB activity than human STING, as well as those with interferon-β activity comparable to or higher than human STING. de Oliveira Mann et al. (2019) provide a species alignment to identify domains of STING in each species, including the CTT domain [see also Supplemental Information for de Oliveira Mann et al. (2019)].

[0205] The non-human STING protein can be a STING protein from, but is not limited to, the following species: Tasmanian devil (Sarcophilus harrisii; SEQ ID NO: 349), marmoset (Callithrix jacchus; SEQ ID NO: 359), cow (Bos taurus; SEQ ID NO: 360), cat (Felis catus; SEQ ID NO: 356), ostrich (Struthio camelus australis; SEQ ID NO: 361), Japanese crested ibis (Nipponia nippon; SEQ ID NO: 362), coelacanth (Latimeria chalumnae; SEQ ID NOs: 363-364), wild boar (Sus scrofa; SEQ ID NO: 365), bat (Rousettus aegyptiacus; SEQ ID NO: 366), manatee (Trichechus manatus latirostris; SEQ ID NO: 367), chimaera (Callorhinchus milii; SEQ ID NO: 368), and mouse (Mus musculus; SEQ ID NO: 369). These vertebrate STING proteins readily activate immune signaling in human cells, indicating that the molecular mechanisms of STING signaling are shared among vertebrates [see de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175].

[0206] The immunostimulatory bacteria provided herein are shown to be capable of infecting myeloid cells, such as tumor-resident and tissue-resident macrophages, retaining viability for at least a limited time, and / or converting macrophages having an M2 phenotype into M1 or M1-like macrophages with reduced or eliminated immunosuppressive properties and enhanced or added immunostimulatory, antitumor, or antiviral properties through their ability to deliver a plasmid encoding a therapeutic product that results in the expression of type I IFN and / or other immunostimulatory products, e.g., a gain-of-function (GOF) mutant that does not require an intracytoplasmic nucleic acid, nucleotide, dinucleotide, or cyclic dinucleotide (CDN) to result in the expression of type I IFN. Provided are immunostimulatory bacteria containing a plasmid encoding a therapeutic product, wherein infection of macrophages, including human macrophages, with the bacteria converts M2 macrophages into M1 or M1-like phenotype macrophages. Provided are immunostimulatory bacteria containing a plasmid encoding a therapeutic product whose expression in macrophages results in or induces the conversion of M2 macrophages, e.g., human M2 macrophages, to an M1 or M1-like phenotype. Immunostimulatory bacteria with such properties include any of the bacteria provided herein that contain genomic modifications that result in infection of tumor-resident (in subjects with cancer) and tissue-resident myeloid cells. These genomic modifications include those that result in the bacteria lacking flagella (wild-type bacteria have flagella), and others, such as those that result in bacteria that are pagP- / msbB-. Other modifications include those that result in the elimination of asparaginase activity, e.g., in bacteria that infect myeloid cells, resulting in bacteria that are ansB-, thereby enhancing T cell activity, and other modifications that alter lipopolysaccharide (LPS). These immunostimulatory bacteria provided herein convert immunosuppressive phagocytic macrophages into immunostimulatory phagocytic macrophages capable of in situ antigen cross-presentation to CD8+ T cells and migration to lymph nodes to prime CD4+ and CD8+ T cells.

[0207] Included are immunostimulatory bacteria that encode therapeutic products within macrophages that facilitate or result in the conversion of M2 macrophages to an M1 or M1-like phenotype, or that convert M2 macrophages to an M1 or M1-like phenotype (having a profile of some or all of the characteristics of M1 macrophages). Exemplary therapeutic products are those that are part of an intracellular DNA / RNA sensor pathway that leads to type I interferon (IFN) expression, particularly constitutive expression. This includes gain-of-function (GOF) mutants of therapeutic products that are part of a cytoplasmic DNA / RNA sensor pathway and do not require cytoplasmic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides (CDNs) to result in type I IFN expression, such as mutant and non-human STING proteins, STING chimeras, and STING chimeras with gain-of-function mutations, as described and provided herein. Bacteria include any that can be modified as described herein, including the species described herein, such as Salmonella species and strains.

[0208] Also provided are immunostimulatory bacteria comprising a nucleic acid operably linked to a prokaryotic promoter, wherein the nucleic acid comprises RNA lacking a sequence necessary for translation by a prokaryotic cell, such that the RNA is produced in the bacterium but is not translated into protein. For example, the RNA lacks a Shine-Dalgarno sequence and contains an internal ribosome entry site (IRES) and / or a translational read-through 2A peptide. The IRES sequence prevents translation by prokaryotic ribosomes but provides translation by eukaryotic ribosomes. Bacteria include immunostimulatory bacteria in which the 2A peptide is one or more of T2A, P2A, E2A, or F2A, for producing individual products from a polycistronic construct.

[0209] Also provided are immunostimulatory bacteria as described herein that can be used as delivery vehicles for delivering RNA to eukaryotic cells, such as myeloid cells. These bacteria comprise a nucleic acid operably linked to a prokaryotic promoter, which is generally encoded on a plasmid, but in some embodiments is encoded within the bacterial genome, and the nucleic acid comprises an RNA lacking sequences necessary for translation by bacterial ribosomes, whereby the RNA produced within the bacterium lacks a Shine-Dalgarno sequence and includes an internal ribosome entry sequence (IRES) or translation read-through 2A peptide. The prokaryotic promoter, when operably linked to a nucleic acid encoding a therapeutic protein (or a non-bacterial protein), can be a bacterial promoter or a phage promoter, e.g., a bacteriophage promoter. An RNA polymerase that recognizes the phage promoter can be encoded within the bacterial genome or on a plasmid for expression in bacteria. Exemplary prokaryotic promoters include any known to those skilled in the art, including, but not limited to, those whose sequences are set forth in SEQ ID NOS: 393-396, respectively: attatgtcttgacatgtagtgagtgggctggtataatgcagcaag (SEQ ID NO: 393) ttatgcttgacgctgcgtaaggtttttgttataatacaccaag (SEQ ID NO: 394), or attatgtcttgacatgtagtgagtgggctggtaaatgcagcaag (SEQ ID NO: 395), or (Salmonella rpsM promoter; SEQ ID NO: 396), , pp. 131-134, 1997. (sufficient to initiate transcription of an operably linked nucleic acid). These immunostimulatory bacteria contain genome modifications as described herein, allowing the bacteria to infect tissue-resident myeloid cells and / or tumor-resident myeloid cells, or infect phagocytes such as macrophages in non-tumor-bearing subjects.The bacteria infect cells and deliver RNA, which is translated in eukaryotic host cells.Exemplary of such bacteria are those that are modified to lack flagella, such as by deleting or disrupting the genes involved in flagella production.Without genome modifications, bacteria are species and strains that have flagella.

[0210] Also provided are immunostimulatory bacteria in which a therapeutic product, such as an encoded protein, is linked to a moiety that confers improved pharmacological properties, such as pharmacokinetic or pharmacodynamic properties, e.g., extended serum half-life. Thus, provided are immunostimulatory bacteria in which the encoded therapeutic product comprises an Fc domain or a half-life extending moiety, e.g., human serum albumin or a portion thereof. Examples of half-life extension forms or methods include PEGylation, glycosylation modifications, sialylation, PASylation (modification with PAS amino acid polymers approximately 100 to 200 residues in length), ELPylation [see, e.g., Floss et al. (2010) Trends Biotechnol. 28(1): 37-45], HAPylation (modification with glycine homopolymers), fusion to human serum albumin, fusion to GLK, fusion to CTP, fusion to GLP, fusion to the constant fragment (Fc) domain of human immunoglobulin (IgG), fusion to transferrin, and non-structural polypeptides such as XTEN [also called rPEG, which is a genetic fusion of non-exact repeating peptide sequences containing A, E, G, P, S, and T; see, e.g., Schellenberger et al. (2009) Nat. Biotechnol. 27(12): 37-45]. 1186-1190], as well as other modifications and fusions that increase size, increase hydrodynamic radius, alter charge, or target receptors for recycling rather than clearance, and combinations of such modifications and fusions.

[0211] Also provided are immunostimulatory bacteria in which the encoded therapeutic product comprises a B7 protein transmembrane domain, or the therapeutic product is GPI-anchored by an endogenous or additional GPI anchor. The encoded therapeutic product may comprise a fusion to collagen.

[0212] The immunostimulatory bacteria in any and all embodiments may be of any suitable species. When referring to specific genes and genetic modifications, the genes and modifications correspond to those with reference to the genus Salmonella as an exemplary species. Examples of species and strains include Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Includes strains of the genera Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix.For example, Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium ovis pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, and Agrobacterium tumefaciens.

[0213] Provided herein is a genomically modified bacterium that comprises a genomic modification whereby TLR2, TLR4, and TLR5 signaling is reduced compared to a bacterium that does not comprise the genomic modification; the bacterium may comprise further genome modifications whereby the bacterium is unable to replicate in a eukaryotic host but is auxotrophic for a required nutrient or factor such that it can replicate in vitro when the nutrient or factor is provided; the bacterium comprises a plasmid containing nucleic acid or encodes an antigen sequence(s) derived from a pathogenic virus, bacterium, or parasite, or comprises RNA encoding a tumor antigen, whereby upon expression of the encoded antigen in the host, the host mounts an immune defense response against the pathogenic virus, bacterium, or parasite; expression of the antigen sequence(s) is under the control of a prokaryotic promoter such that RNA encoding the antigen(s) is produced in the bacterium; the nucleic acid encoding the antigen comprises regulatory sequences that inhibit or prevent translation of the encoded RNA by bacterial ribosomes but not by eukaryotic host ribosomes, thereby uncoupling translation from transcription in the bacterium; The resulting bacteria, when administered to a eukaryotic subject, infect and deliver the nucleic acid to phagocytes, where the RNA is translated.

[0214] The nucleic acid encoding the antigen sequence(s) contains an internal ribosome entry site (IRES) sequence, which facilitates or enhances host cell translation and inhibits or prevents bacterial translation. The IRES can be vascular endothelial growth factor and type 1 collagen-induced protein (VCIP; see, for example, SEQ ID NO: 434), and the nucleic acid encoding the antigen(s) can contain the VCIP IRES or other IRES that inhibits bacterial translation. The IRES or VCIP IRES can be included in the plasmid at a position 3' of the promoter and 5' of the antigen(s) coding sequence.

[0215] The pathogen may be a bacterium or a virus, or the encoded antigen may be a tumor antigen. The immunostimulatory bacteria provided herein may be a vaccine for preventing or treating viral or bacterial infections, including chronic and acute viral infections. The infection may result from infection with hepatitis virus, herpes virus, varicella-zoster virus (VZV), Epstein-Barr virus, human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), respiratory syncytial virus (RSV), measles virus, or other viruses that chronically infect a subject. The infectious agent may be severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, which causes COVID-19).

[0216] The pathogen can be E. coli, Staphylococcus, Pseudomonas, or Porphyromonas species, or the pathogen can be Porphyromonas gingivalis (P. gingivalis), SARS-CoV, or E. coli.

[0217] The plasmids in these immunostimulatory bacteria may further encode immunostimulatory proteins or other adjuvants, or may encode a combination of immunostimulatory proteins or other therapeutic proteins. The immunostimulatory protein may be a STING protein, for example, one containing a gain-of-function mutation, or a chimeric STING protein. The bacteria may contain a plasmid encoding a combination of therapeutic products. The immunostimulatory protein(s) and / or other therapeutic proteins may be encoded on a plasmid as part of a polycistronic sequence with an antigen under the control of a prokaryotic promoter recognized by the bacteria, or the immunostimulatory protein(s) and / or other therapeutic proteins may be encoded on a plasmid under the control of a eukaryotic promoter recognized by a eukaryotic host. The prokaryotic promoter may be a bacterial promoter or a bacteriophage promoter, and the prokaryotic host may be human.

[0218] The immunostimulatory bacteria may comprise mRNA encoding the antigen(s) and any other proteins expressed under the control of a prokaryotic promoter produced by culturing the bacteria in vitro. The immunostimulatory bacteria may comprise genomic modifications such that the bacteria have lost flagella, produce LPS with pentaacylation, and / or the bacteria may be asd- and / or adenosine auxotrophic, and / or csgD- and / or ansB-.

[0219] The bacterium may comprise a nucleic acid encoding a TLR8 agonist.

[0220] The bacteria may be msbB- / pagP-, and / or may have lost flagella, and / or may be asd-.

[0221] The bacterium can be a species or strain of Escherichia coli, Listeria, or Salmonella. For example, the bacterium can be a Salmonella typhimurium strain, where the unmodified Salmonella is a wild-type strain or an attenuated strain. The immunostimulatory bacterium can be derived from the AST-100 strain (VNP20009 or YS1646), or from the ATCC14028 strain, or from a strain having all of the identifying characteristics of the ATCC14028 strain.

[0222] As described herein, the immunostimulatory bacteria can contain one or more genomic modifications that are one or more of deletions, insertions, disruptions, and other modifications in a gene, such that the product encoded by the gene is not produced or, if produced, is inactive.

[0223] Also provided herein is a pharmaceutical composition comprising any of the immunostimulatory bacteria described or provided herein in a pharmaceutically acceptable medium. The pharmaceutical composition can be formulated as a vaccine, for example, as a liquid, powder, or tablet. Also provided are methods and uses of the bacteria or pharmaceutical compositions for treating or preventing (reducing the risk of developing) a disease or condition or an infectious disease or cancer, as well as the use of the bacteria to deliver RNA, such as mRNA, and a method of delivering RNA to a subject, comprising administering the bacteria herein.

[0224] Also provided is a bacterium containing a plasmid encoding a product(s), wherein the product(s) are therapeutic product(s), and wherein the plasmid in the bacterium encodes the product(s) to produce mRNA that is not translated by the bacterium.

[0225] Bacteria can be attenuated, or made less virulent or non-virulent by the modifications described herein. Exemplary bacteria include Salmonella species, such as Salmonella Typhimurium strains. The immunostimulatory bacteria provided herein include those that endogenously encode and express, or have been modified to encode and express, a gene encoding complement killing resistance (rck), such as a Salmonella rck gene. For example, therapeutic E. coli have been modified to encode rck and can be administered systemically.

[0226] Therapeutic protocols, methods and uses provided herein Provided are immune-stimulating bacteria that, when administered, convert immune-desert or immune-eliminated tumors into hot tumors, the bacterium comprises a genomic modification whereby the bacterium has attenuated TLR2 or attenuated TLR2 and / or TLR4 and / or TLR5 activity, whereby the bacterium, when administered to a subject, does not result in an inflammatory response in the subject or results in a reduced inflammatory response compared to a bacterium that does not comprise the genomic modification, thereby having low toxicity and high tumor colonization; The bacterium contains a plasmid encoding an immunostimulatory protein that is a type I interferon (IFN), or a plasmid encoding two or more type I interferons (IFNs), or a plasmid encoding one or more type I interferons (IFNs) and another immunostimulatory protein and / or a tumor-associated antigen; and Hot tumors are responsive to immunotherapy or more responsive than before treatment with immune stimulating bacteria.

[0227] Exemplary of such immunostimulatory bacteria are strains designated YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI, or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI / ΔthyA, or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / ΔpurI, or other strains that are pagP- / msbB-, have lost the curli pili, and are adenosine auxotrophs.

[0228] The plasmid in these bacteria encodes an immunostimulatory bacterium, for example, a type I interferon (IFN), such as IFN-a and / or IFN-b. The nucleic acid encoding the IFN(s) is operably linked to a eukaryotic regulatory sequence. Exemplary promoters and regulatory sequences are well known to those skilled in the art, for example, when the regulatory sequence includes an RNA polymerase type II promoter, such as an inducible or constitutive promoter, and optionally an enhancer; for example, when the promoter and enhancer are of viral origin, they may be a cytomegalovirus promoter and / or enhancer. The immunostimulatory bacterium can encode at least two immunostimulatory proteins. They may be encoded to yield at least two proteins when transcribed as a polycistronic message and translated, such as by including a 2A protein or other such regulatory protein or a sequence that yields at least two proteins when transcribed by a eukaryotic ribosome. When the immunostimulatory bacterium contains a plasmid encoding a type I interferon (IFN) or multiple types thereof. Exemplary type I interferons include those having the amino acid sequence set forth in SEQ ID NOs: 550, 552, 549, and 551, and allelic or other variants thereof having at least 95% or 98% sequence identity and having interferon activity. Exemplary sequences are set forth in SEQ ID NOs: 549 and 551, which set forth the nucleotide sequences of human IFNa2 and human IFN-b, respectively. SEQ ID NOs: 550 and 552 set forth the amino acid sequences of human IFNa2 and human IFN-b, respectively.

[0229] The methods, therapeutics, protocols and uses and immune stimulatory bacteria described herein, including those described above, may include plasmids comprising the sequences of nucleotides set forth as SEQ ID NOs:502-545 and degenerate sequences thereof, or portions thereof, or sequences having at least 95% sequence identity to the coding portions and regulatory regions of SEQ ID NOs:502-545, including nucleic acids encoding immune stimulatory protein(s), eukaryotic transcriptional and / or translational regulatory sequences.

[0230] Provided are methods for assessing whether a treatment using a delivery vehicle that is targeted to or phagocytosed by macrophages and encodes a therapeutic product(s) is effective for treating a tumor in a subject. These methods include identifying proliferating macrophages in a tumor biopsy or tumor sample. The delivery vehicle encodes a nucleic acid that is transcribed in the nucleus of the macrophage and is not integrated into the chromosome of the genome (non-integrated). The delivery vehicle comprises an immunostimulatory bacterium, any of those provided herein, that infects or is phagocytosed by the macrophage. It has been shown herein that expression of the encoded payload occurs in proliferating macrophages. Proliferating macrophages can be identified by any method known in the art, including the methods described herein, in which proliferating macrophages are identified in a biopsy by any of the following markers: G2M module (>14 genes in the set), oncogene expression of stathmin 1 (STMN1); Biopsy surface markers: CD68+KI67 and / or PCNA, MERTK, Some tumor types: SPP1 in lung, stomach, and / or Some tumor types: C1QC in colon and breast.

[0231] For example, proliferative macrophages have a hybrid SPP1+ and C1QC+ (expression of both SPP1 and C1QC) macrophage phenotype and exhibit enhanced phagocytic and proliferation properties.

[0232] Also provided is a method of rendering tumors responsive to immunotherapy, comprising administering a therapeutic agent that converts macrophages to an M1 / M2 hybrid phenotype, where the macrophages are identified as proliferative macrophages. Delivery vehicles and therapeutic agents that achieve such conversion are described herein throughout this disclosure. The resulting macrophages with a hybrid M1 / M2 phenotype have a hybrid SPP1+ and C1QC+ (expression of both SPP1 and C1QC) macrophage phenotype, thereby providing the macrophages with enhanced phagocytic and proliferative properties.

[0233] Provided are plasmids comprising the sequence of nucleotides set forth in SEQ ID NO:501 or degenerate codons thereof in the protein coding region, or a sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO:501, wherein the plasmid encodes a protein that is an IL-15 / IL-15R alpha chain complex or a protein with at least 95% sequence identity thereto, encodes a chimeric STING that constitutively induces type 1 interferon activity and has reduced NF-κB signaling activity compared to human STING, and encodes a protein that is a protein with activity of the IL-15 receptor complex and a STING protein with constitutive activity and reduced NF-κB signaling activity compared to human STING. Provided are immunostimulatory bacteria containing a plasmid containing one that has the phenotype YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD, where YS1646 is ΔmsbB / ΔpurI and F-ΔpurI represents a strain in which purI is deleted. Bacteria further include those that are thyA- as described herein. Genomic modifications, in all embodiments, include those that render a product or locus inactive, such as by insertion, deletion, rearrangement, and / or any other alteration such that the encoded active product is not produced.

[0234] Provided are immunostimulatory bacteria comprising a plasmid encoding IL-15, e.g., the IL-15 / IL-15R alpha chain complex, and encoding constitutive STING, wherein the plasmid encoding the IL-15 / IL-15R alpha chain complex and constitutive STING comprises the sequence set forth in SEQ ID NO: 501, or a portion thereof encoding IL-15 and eSTING, or a degenerate or variant thereof having at least 95% sequence identity to a portion of SEQ ID NO: 501 or to a degenerate thereof. The immunostimulatory bacteria can be a Salmonella strain designated YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI or a related strain with genomic modifications that render the strain msbB− / pagP−, aflagellated, csgD−, and adenosine auxotrophic, and any other genomic modifications described herein throughout this disclosure.

[0235] Provided is a protocol or regimen for treating cancer in which PD-1 expression on macrophages in a subject is first suppressed, and then a therapeutic agent, such as an immunostimulatory bacterium provided herein, converts immune desert or T cell-negative tumors or other tumors that are not responsive to immunotherapy into hot tumors that are responsive to immunotherapy. After treatment with the therapeutic agent, the subject may then be treated with immunotherapy, for example, anti-PD-L1 therapy. An exemplary protocol is as follows: A subject to be treated with the delivery vehicle is pretreated with an agent that suppresses PD-1 expression in macrophages, thereby promoting the phagocytic ability of macrophages, and the delivery vehicle contains a nucleic acid encoding an anti-cancer product, targets phagocytic macrophages, and can be phagocytosed by phagocytic macrophages; and Then, after a predetermined time sufficient to suppress or reduce PD-1 expression, a delivery vehicle is administered. The predetermined time can be at least 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours, or 1 to 2 days, or up to 72 hours, generally between 4 and 48 hours, e.g., between 8 and 12 hours, or between 4 and 24 hours, or between 12 and 48 hours. Regarding the predetermined time period as used throughout this disclosure, the determination of the period is within the skill of one in the art and may vary depending on other parameters and factors, including the particular agent and delivery vehicle administered and the subject. An additional immunotherapy, e.g., an anti-PD-L1 therapy, can also be administered.

[0236] Provided is a cancer treatment protocol or regimen that includes pretreating a subject to be treated with a delivery vehicle with an anti-PD-1 agent, thereby suppressing PD-1 expression in macrophages and thereby promoting macrophage phagocytosis, the delivery vehicle comprising a nucleic acid encoding an anti-cancer product and targeting or being phagocytosed by phagocytic macrophages, and then administering the delivery vehicle. The delivery vehicle is administered after a time sufficient to suppress or reduce PD-1 expression, e.g., at least 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day to 2 days, up to 72 hours, or, for example, between 8 hours and 48 hours, 4 hours and 12 hours, or 8 hours and 24 hours. Generally, the delivery vehicle, e.g., an immunostimulatory bacterium, is administered within 24 to 48 hours after administration of the anti-PD-1 therapy. The protocol may include a further step of treating with an immunotherapeutic agent, e.g., an immune checkpoint inhibitor. The immunotherapy is generally administered after a predetermined time following the delivery agent, sufficient to allow the tumor to be sensitive to the immunotherapy. For example, the protocol may include administering an anti-PD-L1 agent after the anti-cancer product encoded in the delivery vehicle has been expressed, thereby causing PD-L1 to be expressed in macrophages. The predetermined time for immunotherapy may be at least 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day to 2 days, or up to 72 hours, or for example, 8 hours to 48 hours, or 4 hours to 12 hours, or 8 hours to 24 hours. In these protocols or regimens, the anti-PD-L1 agent may be an anti-PD-L1 antibody, e.g., an anti-PD-L1 antibody or other antagonist.

[0237] The delivery agent in the protocols and regimens can be one that converts macrophages that phagocytose the delivery vehicle into macrophages with an M1 / M2 hybrid phenotype as described herein, for example, any of the immunostimulatory bacteria or other therapeutic agents described herein throughout this disclosure as possessing this characteristic. Exemplary of such delivery agents are the bacteria designated "Test Strain 4" in the table in Example 39 and related strains. The protocol or regimen can include administering a PD-1 antibody, followed by administering bacteria, and then administering an immunotherapy, e.g., an anti-PD-L1 antibody.

[0238] 1. A method of treating a subject having an immune desert or T cell-negative tumor, comprising: First, a drug that suppresses PD-1 expression in macrophages is administered, thereby increasing the phagocytic ability of macrophages compared to before treatment; and Then, after a predetermined time, a delivery vehicle containing a nucleic acid encoding an anti-cancer product is administered, where the delivery vehicle targets or accumulates in phagocytic macrophages, and then the delivery vehicle is administered. The predetermined time can be at least 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1-2 days, up to 72 hours, or between, for example, 8 hours and 48 hours, or 4 hours and 12 hours, or 8 hours and 24 hours. The predetermined time period is sufficient for suppression of PD-1 expression on macrophages, e.g., at least 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 to 2 days, up to 72 hours, or for example, between 8 and 48 hours, or 4 and 12 hours, or 8 and 24 hours, or 12 and 48 hours, or other such time period as determined by one of skill in the art. The method further includes administering to the subject an immunotherapy, e.g., a checkpoint inhibitor, e.g., an anti-PD-L1 antibody or other inhibitor, after a second predetermined time period, e.g., a time period recited above.

[0239] Consistent with the above protocols, regimens, methods, and uses, the delivery vehicle can be an immunostimulatory bacterium encoding an immunostimulatory protein, such as any of those described herein, for example, a Salmonella species with genomic modifications that cause the bacterium to lose flagella, be an adenosine auxotroph, have pentaacylated LPS, lose curli pili, and / or be asd-negative, and optionally, other genomic modifications that reduce toxicity / inflammatory responses to the bacterium and / or promote accumulation / targeting in phagocytic macrophages. The immunostimulatory bacterium can encode one or more immunostimulatory proteins and, optionally, tumor-associated antigens under the control of eukaryotic regulatory signals. Combinations of immunostimulatory proteins include those described above and throughout this disclosure. Exemplary immunostimulatory proteins include cytoplasmic DNA / RNA sensors, cytokines, and / or tumor-associated antigens, such as one or more of the combinations described above and below. Exemplary of immune stimulatory proteins are intracytoplasmic DNA / RNA sensors, for example, modified STING protein (referred to herein in this disclosure as eSTING) that constitutively induces type I interferon (IFN) in macrophages, cytokines that are IL-15 or the IL-15 / IL-15R alpha chain complex and / or type I interferon (IFN), or other cytokines with properties similar to IL-15 and the IL-15 / IL-15R alpha chain complex. An exemplary bacterium is YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI containing a plasmid encoding an IL-15 / IL-15R alpha chain complex and a chimeric STING containing CTT and substitutions N154S / R284G from Tasmanian devil, e.g., the strain designated CRST-2000 (see Example 39) or its derivatives and related strains with additional genomic modifications, are exemplary of immune stimulatory bacteria for use in the methods, uses, protocols and other embodiments provided herein.

[0240] Provided are immunostimulatory bacteria, e.g., bacteria having genomic modifications as described herein, for use in or as a method for converting immune desert or T cell-negative tumors into hot tumors. Consistent with these uses and methods, a subject to be treated is identified as having an immune desert or T cell-negative tumor. The bacteria are administered, and after treatment, the tumor is sensitive to treatment with an immune checkpoint inhibitor, or other immunotherapies have not been effective in treating the tumor prior to treatment with the immunostimulatory bacteria. The immunostimulatory bacteria can be any of those described herein, e.g., those that result in an M1 / M2 hybrid phenotype, e.g., those that encode eSTING, which results in constitutive expression of the IL-15 / IL-15R alpha chain complex and type I interferon (IFN), and also immunostimulatory bacteria encoding one or more of IFNa and / or IFNb, e.g., bacteria containing a plasmid (or portion thereof) described in Example 57, e.g., bacteria containing all or a portion of a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NOs: 502-545, or containing degenerate codons thereof. The plasmid and portions thereof encode IFNa and / or IFNb or variants thereof having IFNa or IFNb activity. The immunostimulatory proteins are encoded and expressed under the control of a eukaryotic promoter and, optionally, other regulatory sequences.

[0241] Isolated macrophages for cell therapy Provided are isolated macrophages containing a therapeutic agent that, when introduced into the macrophages, results in an M1 / M2 hybrid macrophage phenotype. The therapeutic agent is introduced into the macrophages in vitro or ex vivo. After culturing, treating, or formulating the macrophages containing the therapeutic agent, the resulting composition containing the macrophages can be administered to a subject in need of treatment with the macrophages, for example, a subject with cancer, such as a T-cell-negative tumor or a tumor known as a desert tumor or cold tumor, thereby converting the tumor into a warm tumor susceptible to immunotherapy. The macrophages can be allogeneic or autologous to the subject to be treated. The therapeutic agent can induce a hybrid M1 / M2 phenotype, thereby enabling macrophages to phagocytose apoptotic tumor cells, and induce constitutive type I IFN to recruit and prime tumor antigen-specific CD8+ T cells, thereby inducing durable antitumor immunity, such as the immunostimulatory bacteria described herein. Macrophages can be isolated from a subject or previously obtained, cultured, and optionally genetically modified, and a therapeutic agent is typically introduced into the cultured macrophages. Macrophages can be formulated for storage or administration prior to use. Therapeutic agents include any of those described herein or identified as converting macrophages to the M1 / M2 phenotype. Therapeutic agents include immunostimulatory bacteria encoding payloads for cancer treatment, RNA delivery, as vaccines, or other applications such as the treatment of diseases, disorders, and conditions. Included are immunostimulatory bacteria encoding two or more complementary immunostimulatory proteins. Exemplary immunostimulatory bacteria include those comprising the phenotype YS1646 / ΔFLG / ΔpagP / ΔcsgD or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD or YS1646Δasd / ΔFLG / ΔpagP / ΔcsgD, or other such phenotypes that result in a reduced or eliminated TLR2 / 4 / 5 response, such that the bacteria have reduced inflammatory properties compared to VNP20009 and infect primarily or exclusively phagocytic cells, e.g., macrophages.Exemplary therapeutic agents are delivery vehicles comprising a nucleic acid molecule of SEQ ID NO:501 or a sequence having at least 90%, or 95%, or 97%, or 98%, or 99% or more sequence identity to SEQ ID NO:501, or a nucleic acid molecule that includes one or more degenerate codons to either SEQ ID NO:501 or a sequence having at least 90%, or 95%, or 97%, or 98%, or 99% or more sequence identity to SEQ ID NO:501.

[0242] Thus, provided is the use of a therapeutic agent and macrophages for the treatment of cancer, wherein the macrophages are introduced into a subject with cancer after introduction of the therapeutic agent, or used to treat cancer in subjects with T cell elimination (or immune desert or cold tumors) or who have failed or are unresponsive to immunotherapy, e.g., anti-PD1 immunotherapy. Provided is a method for converting an immune desert tumor or a T cell elimination tumor or a cold tumor into a hot tumor, comprising administering isolated macrophages containing a therapeutic agent.

[0243] Also provided, described, and claimed herein are delivery vehicles, cells, pharmaceutical compositions, methods, uses, and treatments of cancer, particularly in humans. Also provided are companion diagnostics and methods for selecting subjects for treatment, and methods for monitoring treatment, which are described below and in the claims, and which are incorporated into this section in their entirety. [Brief explanation of the drawings]

[0244] [Figure 1A] Figures 1A-1C show the inserts in plasmids pATI-1.75 and pATI-1.76 (Figures 1A and 1B, respectively). Figure 1C shows that the Shine-Dalgarno sequence has been replaced with a Kozak sequence for translation in eukaryotic cells, e.g., myeloid cells. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 2] Figure 2 shows an alignment of wild-type human STING (SEQ ID NO: 306) and Tasmanian devil STING (SEQ ID NO: 349) proteins. [Figure 3] FIG. 3 shows an alignment of wild-type human STING (SEQ ID NO: 306) and marmoset STING (SEQ ID NO: 359) proteins. [Figure 4] Figure 4 shows an alignment of wild-type human STING (SEQ ID NO: 306) and bovine STING (SEQ ID NO: 360) proteins. [Figure 5] Figure 5 shows an alignment of wild-type human STING (SEQ ID NO: 306) and feline STING (SEQ ID NO: 356) proteins. [Figure 6] Figure 6 shows the alignment of wild-type human STING (SEQ ID NO: 306) and ostrich STING (SEQ ID NO: 361) proteins. [Figure 7] Figure 7 shows an alignment of wild-type human STING (sequence number 306) and Japanese ibis STING (sequence number 362) proteins. [Figure 8] Figure 8 shows an alignment of wild-type human STING (SEQ ID NO: 306) and coelacanth STING (SEQ ID NO: 363) proteins. [Figure 9] Figure 9 shows an alignment of wild-type human STING (SEQ ID NO: 306) and zebrafish STING (SEQ ID NO: 348) proteins. [Figure 10] Figure 10 shows an alignment of wild-type human STING (SEQ ID NO: 305) and boar STING (SEQ ID NO: 365) proteins. [Figure 11] Figure 11 shows an alignment of wild-type human (SEQ ID NO: 305) and bat STING (SEQ ID NO: 366) proteins. [Figure 12] Figure 12 shows an alignment of wild-type human (SEQ ID NO: 305) and manatee STING (SEQ ID NO: 367) proteins. [Figure 13] Figure 13 shows an alignment of wild-type human (SEQ ID NO: 305) and chimaera STING (SEQ ID NO: 368) proteins. [Figure 14] Figure 14 shows an alignment of wild-type human (SEQ ID NO: 305) and mouse STING (SEQ ID NO: 369) proteins. [Figure 15] Figure 15 is adapted from Roeszer et al. ((2018) Cells 7(8):103), who present a scheme summarizing the signals that prevent or promote cell cycle entry and progression in macrophages. [Figure 16] Figure 16 shows priming and activation of tumor-associated antigen (TAA)-specific CD8+ T cells and induction of anti-tumor immunity by an exemplary immunostimulatory bacterium designated STACT, which encodes the IL-15 / IL-15R alpha chain complex plus eSTING, which constitutively induces type I IFNs such as eSTING. [Figure 17A]Figure 17A displays a schematic from Anfray et al. Cells (2019) listing molecular pathways previously associated with the tumor-associated macrophage (TAM) phenotype. Figure 17B shows a pictorial representation of M1 macrophages (top) and M2 macrophages (bottom), as well as macrophages with the resulting M2 hybrid phenotype described herein. Figure 17C describes macrophage markers, including the M1 / M2 hybrid phenotype described herein, and the consequences upon infection of tumor-resident macrophages with the immunostimulatory bacteria described herein and expression of the encoded payload. Figure 17D, adapted from Roeszer, T. ((2015) Mediators Inflamm 2015:816460), depicts an example of M2 activation and markers associated with distinct M1 and M2 activation phenotypes, but not hybrid M1 / M2. Figure 17E illustrates the mechanism of action of STACT. Figure 17F is adapted from a diagram showing the cancer-immunity cycle in Chen and Mellman ((2013) Immunity 39(1):1-10) and illustrates STACT IL-15plex+eSTING as a comprehensive immunotherapy. [Figure 17B] Same as above. [Figure 17C-D] Same as above. [Figure 17E] Same as above. [Figure 17F] Same as above. [Figure 18] Figure 18 shows SPP1 expression in tumor tissue compared to normal tissue in various tumor types and tissues. The color of the p-value letter and the shape surrounding the p-value indicate direction; gray p-values ​​surrounded by an oval = lower in tumors [negative (Neg)]; light gray p-values ​​surrounded by a rectangle = higher in tumors [positive (Pos)]; and very light gray p-values ​​without a p-value surrounding them indicate no significant difference (NS). Cancer types are ordered by the median expression value of tumor samples (from left to right, corresponding to high to low). [Figure 19]Figure 19 shows C1QC expression in tumor tissue compared to normal tissue in various tumor types and tissues. The color of the p-value letter and the shape surrounding the p-value indicate direction; gray p-values ​​surrounded by an oval = lower in tumors [negative (Neg)]; light gray p-values ​​surrounded by a rectangle = higher in tumors [positive (Pos)]; and very light gray p-values ​​without a p-value surrounding them indicate no significant difference (NS). Cancer types are ordered by the median expression value of tumor samples (from left to right, corresponding to high to low). [Figure 20] FIG. 20 shows a Kaplan-Meier (KM) plot of a bioinformatics analysis of groups with high or low C1QC expression in consensus molecular subtype 1 (CMS1) colorectal cancer. [Figure 21] Figure 21 shows a forest plot showing the results for gene expression of 23 tumor types from The Cancer Genome Atlas (abbreviated as TCGA). A Cox proportional hazards regression model was calculated to test the association between prognosis and expression of SPP1 as a continuous variable. The dot shape represents the p-value; triangle represents p<0.05. The dots are the hazard ratios for each comparison, and the black lines are the 95% confidence intervals. [Figure 22] Figure 22 shows a forest plot showing results for gene expression of 23 tumor types from The Cancer Genome Atlas (TCGA). A Cox proportional hazards regression model was calculated to test the association between prognosis and expression of C1QC as a continuous variable. The dot shape represents the p-value; triangles represent p<0.05. The dots are the hazard ratios for each comparison, and the black lines are the 95% confidence intervals. [Figure 23A]Figures 23A-B show the abundance of proliferative macrophages across tissues based on analyses performed across tumor types using publicly available scRNA-seq datasets. The graphs show the proportions as percentages (0.05 = 5%), calculated using the proportion of CD68+ cells among total CD45+ cells (Figure 23A) and the proportion of G2 / M score+ among total CD68+ macrophages (Figure 23B) from tumor tissue samples from all patients in each dataset. Boxes represent the median ± interquartile range, and whiskers represent ±1.5 × interquartile range. Outliers are represented by black dots. [Figure 23B] Same as above. [Figure 24] FIG. 24 shows box plots generated from apoptosis module expression data from The Cancer Genome Atlas (abbreviated TCGA; 161 genes related to apoptosis) comparing cancer types and ordered by median expression. [Figure 25] Figures 25A-F show box plots of G2M scores (Figures 25A-C) and STMN1 expression (Figures 25D-F) in non-proliferating and proliferating macrophages in lung cancer (A, D), breast cancer (B, E), and colon cancer (not stratified by CMS subtype) (C, F). [Figure 26] Figure 26 shows a correlation plot showing the association of SPP1 expression with changes in major cancer pathways, where the y-axis represents -log10(p-value), with higher dots corresponding to lower p-values. Different dots represent tumor types. [Figure 27] Figure 27 shows an association plot showing the association of C1QC with alterations in key cancer pathways, where the y-axis represents -log10(p-value), with higher dots corresponding to lower p-values. Different dots represent tumor types. [Figure 28] Figure 28 shows box plots of CD68 expression in breast cancer patients at baseline or after either two cycles of epirubicin and docetaxol (C2, chemo only), or four cycles of chemotherapy plus surgery and bevacizumab (C4, sur+chemo+bev). [Figure 29]Figure 29 shows box plots of CD68 expression in breast cancer patients at baseline or after either two cycles of epirubicin and docetaxel (C2, chemo only), or four cycles of chemotherapy plus surgery and bevacizumab (C4, sur+chemo+bev). [Figure 30] Figure 30 shows pHRodo® reagent and CellTrace® labeling of dyes that are fluorescent only in the acidic cellular compartment of YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD cells containing a plasmid encoding the IL-15 receptor complex and chimeric STING, where the plasmid has the sequence set forth in SEQ ID NO: 501, which is internalized by THP-1 cells differentiated into M0 macrophages over time. pHRodo® and CellTrace® reagent labeling is shown at 90 minutes (top panel) and 48 hours (bottom panel) after bactofection at MOIs of 1, 5, 20, and 40. [Figure 31] Figures 31A-F show uptake of YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD containing a plasmid encoding an IL-15 receptor complex and chimeric STING, where the plasmid has the sequence set forth in SEQ ID NO: 501, which is internalized by M0, M1, and M2 macrophages after bactofection at an MOI of 1, 5, 20, and 40. Uptake is shown in M0 macrophages as measured by pHRodo® reagent (Figure 31A) and CellTrace® reagent (Figure 31D), in M1 macrophages as measured by pHRodo® reagent (Figure 31B) and CellTrace® reagent (Figure 31E), and in M2 macrophages as measured by pHRodo® (Figure 31C) and CellTrace® reagent (Figure 31F). [Figure 32A]Figures 32A and 32B show STING reporter luciferase activity in wild-type M0 THP-1-derived macrophages; THP-1 cells that were not bactofected; and THP-1 cells to which cGAMP was added. Figure 32A shows STING reporter luciferase activity in wild-type M0 THP-1-derived macrophages at MOIs of 1, 5, 20, and 40, measured at 90 minutes, 3 hours, 24 hours, and 48 hours after bactofection. Figure 32B shows STING reporter luciferase activity in wild-type M0 THP-1-derived macrophages at MOIs of 1, 5, 20, and 40, measured at 48 hours after bactofection. [Figure 32B] Same as above. [Figure 33] Figure 33 shows the cell cycle-dependent internalization of STACT (YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD containing a plasmid encoding an IL-15 receptor complex and chimeric STING, as referred to in Example 58, where the plasmid has the sequence set forth in SEQ ID NO: 501). The top left panel shows the quadrants of the graph corresponding to the cell cycle phase. The bottom left panel shows the cell cycle phase of unstained cells. The top right panel shows the cell cycle phase for non-bactofected M0 cells (left), M0 cells bactofected at an MOI of 20 and 40 (center), and cells bactofected with bacteria containing asd but no plasmid as a control (right). The middle right panel shows the cell cycle phase for non-bactofected M1 cells (left), M1 cells bactofected at an MOI of 20 and 40 (center), and cells containing ASD (right). The bottom right panel shows the cell cycle phases for non-bactofected M2 cells (left), M2 cells bactofected at MOIs of 20 and 40 (center), and cells containing ASD (right). [Figure 34] FIG. 34 is a schematic diagram showing high levels of adenosine and immune exclusion caused by hypoxia in tumors. [Figure 35A]Figures 35A and 35B show tumors with high CD73 expression and high CD39 expression. Figure 35A shows a boxplot depicting the median expression of CD73 for each tumor indication from the atlas (abbreviated TCGA). Figure 35B is a scatterplot displaying the expression of the ENTPD-1 gene (CD39) versus the expression of the NT5E gene (CD73) for tumor indications in TCGA; triangles represent the top indications, and tumor indications are graded according to Kummel's myeloid cell signature. [Figure 35B] Same as above. [Figure 36A] Figures 36A and 36B show Krummel's T cell, myeloid cell, and stromal cell signatures across solid tumor indications in TCGA. Triangles represent the top indication, and tumor indications are graded according to NT5E (CD73) expression. Figure 36A is a scatter plot displaying Krummel's T cell vs. myeloid cell signature. Figure 36B is a scatter plot displaying Krummel's stromal cell vs. myeloid cell signature. [Figure 36B] Same as above. [Figure 37A] Figures 37A-F show myeloid cell markers from TCGA across the C1-C6 immune subtypes defined in Thorsson et al. (2018) Immunity 48(4):812-830 [wound-healing (C1), IFN-γ dominant (C2), inflammatory (C3), lymphocyte-depleted (C4), immunologically quiet (C5), and TGF-β dominant (C6)]. Key characteristics of each immune subtype are shown in Figure 37A. Overall survival in years for each immune subtype is shown in Figure 37B. In Figures 37C-F, boxplots show Kreummel's T cell signature for each subtype from TCGA (Figure 37C); Kreummel's myeloid cell signature for each subtype from TCGA (Figure 37D); MRC1 expression for each subtype from TCGA (Figure 37E); and CD163 expression for each subtype from TCGA (Figure 37F). [Figure 37B] Same as above. [Figure 37C]Same as above. [Figure 37D] Same as above. [Figure 37E] Same as above. [Figure 37F] Same as above. [Figure 38] Figure 38 shows tumor indications with high myeloid signature, T cell signature, hypoxia markers, adenosine markers, TGF beta and antigen presentation in TCGA. [Figure 39] Figure 39 shows metastatic cancer indications with high myeloid cell signature, T cell signature, hypoxia markers, adenosine markers, TGF beta and antigen presentation in the MET500 dataset. [Figure 40A] Figures 40A to 40C show an allograft mouse model with high T cell, myeloid, and stromal infiltration and high adenosine from the TISMO database. Figure 40A is a plot displaying rank-based scoring of gene sets within the fibroblast signature versus the myeloid cell signature for the allograft mouse model. The model is shown with a gradient according to the rank-based scoring of gene sets within the T cell signature. Figure 40B is a plot displaying rank-based scoring of gene sets within the T cell signature versus the myeloid cell signature for the allograft mouse model. The model is shown with a gradient according to Cd8a expression. Figure 40C is a plot displaying expression of NT5E (CD37) versus CD68, a marker for anti-myeloid content, for the allograft mouse model. The model is shown with a gradient according to Sting1 expression. [Figure 40B] Same as above. [Figure 40C] Same as above. [Figure 41] Figure 41 shows a box plot generated from SPP1 expression across TCGA and comparing it with cancer types ordered by median expression. COAD and READ colorectal cancers were separated into their CMS subtypes and plotted. [Figure 42]Figure 42 shows a box plot generated from C1QC expression across TCGA and comparing it with cancer types ordered by median expression. COAD and READ colorectal cancers were separated into their CMS subtypes and plotted. DETAILED DESCRIPTION OF THE INVENTION

[0245] Overview A.Definition B. Overview of Immunostimulatory Bacteria for Cancer Treatment 1. Bacterial cancer immunotherapy 2. Upfront treatment targeting the tumor microenvironment a. Limitations of autologous T cell therapy b. Viral vaccine platform c. Bacterial cancer treatment i. Listeria spp. ii. Salmonella spp. iii.VNP20009(YS1456) iv. Wild-type strain 3. Limitations of existing bacterial cancer immunotherapy 4. Therapeutic agents that induce a hybrid M1 / M2 antitumor phenotype in tumor-resident macrophages C. Modification and Enhancement of Immunostimulatory Bacteria to Increase the Therapeutic Index and Increase Accumulation in Tumor-Resident Myeloid Cells 1. Deletion of genes in the LPS biosynthetic pathway a. msbB deletion b. pagP deletion or inactivation 2. Nutritional requirements a.purI deletion / disruption B adenosine auxotrophy C thymidine auxotrophy 3. Plasmid Maintenance and Delivery a.asd deletion b.endA deletion / disruption 4. Flagellin Knockout Strain 5. Bacterial engineering to promote adaptive immunity and enhance T cell function L-asparaginase II (ansB) deletion / disruption 6. Deletion / disruption of Salmonella genes required for the production of curli fimbriae csgD deletion 7.Improved resistance to complement Rck expression 8. Deletion of genes required for lipoprotein expression in Salmonella and other Gram-negative bacteria 9. Robust immune-stimulating bacteria with genomes optimized for antitumor therapy and encoding multiple therapeutic products 10. Vaccines and bacteria that deliver RNA, including mRNA and other forms of RNA, for expression in eukaryotic hosts 11. Bacterial vaccines against specific antigens, including those derived from pathogens and tumors, for use as anti-pathogen treatments and vaccines, and for anti-cancer treatment and / or prevention. 12. Conversion of M2 phenotype macrophages to M1 and M1-like phenotype macrophages D. Immunostimulatory bacteria with enhanced therapeutic index that encode genetic payloads that stimulate immune responses in the tumor microenvironment 1. Immunostimulatory proteins a. Cytokines and chemokines b. Co-stimulatory molecules 2. Constitutively active proteins that stimulate immune responses and / or type I IFN, non-human STING proteins, STING chimeras, and modified forms a. Constitutive STING expression and gain-of-function mutations b. Constitutive IRF3 expression and gain-of-function mutations c. Non-human STING proteins and mutants thereof with increased or constitutive activity, and STING chimeras and mutants thereof with increased or constitutive activity. d. Other gene products that act as intracellular DNA / RNA sensors and their constitutive variants i.RIG-I ii.MDA5 / IFIH1 iii.IRF7 e. Other type I IFN-regulatory proteins 3. Antibodies and antibody fragments TGF-β b. Bispecific scFv and T cell engagers c. Anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies i. Anti-PD-1 / anti-PD-L1 antibody ii. Anti-CTLA-4 antibody d. Additional Exemplary Checkpoint Targets 4. Combinations of immunomodulatory proteins may have synergistic and / or complementary effects 5. Molecules that activate prodrugs 6. Immunostimulatory bacteria resulting in combination therapy E. Immunostimulating Bacteria as Antiviral Therapeutics and Against Other Infectious Pathogens F. Construction of Exemplary Plasmids Encoding Therapeutic Products for Delivery in Bacteria 1. Constitutive promoters for heterologous expression of proteins 2. Multiple Therapeutic Product Expression Cassettes a. Single promoter construct b. Dual / multiple promoter constructs 3. Regulatory Elements a post-transcriptional regulatory element b. polyadenylation signal sequence and terminator C enhancer d. secretion signal e. Improving bacterial fitness 4. Origin of Replication and Plasmid Copy Number 5. CpG motifs and CpG islands 6. Plasmid Maintenance / Selection Components 7. DNA nuclear targeting sequence G. Exemplary Bacterial Strains and Mechanisms of Action for Use as Vaccines and Therapeutics 1. Exemplary Immunostimulatory Bacteria - Mechanism of Action (MOA) for In Situ Cancer Vaccination 2. Exemplary Immunostimulatory Bacteria and Mechanisms of Action for Peripheral Cancer Vaccination 3. Exemplary Immunostimulatory Bacteria and MOAs for Pathogen Vaccination 4. Exemplary Immunostimulatory Bacteria for the Treatment of Cancer H. Pharmaceutical Production, Compositions and Formulations 1.Manufacturing a. Cell bank production b. Manufacturing of active ingredients c. Manufacturing of drug products 2. Composition 3. Preparation a. Liquids, injections, emulsions b. Dry heat-stable formulation 4. Compositions for other routes of administration 5. Dosage and Administration 6. Packaging and Manufactured Goods I. Methods of Treatment and Use 1. Diagnostic methods for selecting patients for treatment and monitoring treatment a. Patient selection b. Diagnostic methods for assessing or detecting the activity of immune-stimulating bacteria indicate the effectiveness of treatment 2. Tumor 3. Administration 4. Monitoring J. Example

[0246] A.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications, as well as publications, GenBank sequences, databases, websites, and other published materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise specified. In the event of multiple definitions for terms herein, those in that section prevail. When referring to a URL or other such identifier or address, of course, such identifiers may change and specific information on the Internet may change, but equivalent information may be found by searching the Internet. Reference thereto evidences the availability and public dissemination of such information.

[0247] As used herein, STACT refers to the S. Typhimurium-Attenuated Cancer Therapy strain. STACT refers to an exemplary strain commonly designated YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD. YS1646, as described in the detailed description, is msbB- and purI- due to genomic modifications that disrupt expression of the gene products. STACT contains these modifications and may also include complete gene deletions of either or both msbB and purI. In the examples, the STACT strain contains a complete deletion of purI. STACT strain modifications include genomic modifications that result in the loss of flagella, rendering the bacterium csgD- in addition to msbB and purI-. Modifications that render the bacterium asd- and / or ansB- are optional and are selected by the user for a particular application or protocol. STACT strains are exemplary of the immunostimulatory bacteria described and provided herein. These immunostimulatory bacteria, including exemplary bacteria designated STACT, contain genomic modifications that eliminate flagella and biofilm (csgD-) and result in pentaacylated LPS. These include strains with the phenotype Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD, in which purI and msbB- modifications (either by insertion, deletion, transposition, or complete deletion of each gene) result in advantageous properties discussed and demonstrated throughout this disclosure. These properties include, but are not limited to, (1) enhanced tolerability after IV administration compared to the unmodified parent strain YS1467 (also referred to as VNP20009), (2) tumor-specific enrichment, (3) phagocytosis by tumor-resident antigen-presenting cells (APCs) that lack epithelial cell infectivity, (4) provision of multigene cargo delivery, and (5) attenuation of bacterial pathways that impair CD8+ T cell function. STACT bacteria can encode payloads, e.g., therapeutic proteins, e.g., immunostimulatory proteins, e.g., cytokines, costimulatory molecules, cyclic DNA / RNA sensors, particularly those engineered to constitutively induce type I interferons (IFNs), tumor-associated or other antigens, antibodies and other such proteins.

[0248] As used herein, a "therapeutic bacterium" is a bacterium that, when administered to a subject, e.g., a human, achieves a treatment, e.g., an anti-cancer treatment or an anti-tumor treatment.

[0249] As used herein, a therapeutic agent provided herein includes a delivery vehicle and a nucleic acid, e.g., DNA, that is delivered to cells or tissues, e.g., tumor-resident immune cells, tumor microenvironment, and tumors, where it is taken up by cells, such as tumor-resident immune cells, and the nucleic acid is expressed in the cells. Generally, the nucleic acid encodes one or more immunostimulatory proteins, including proteins that induce type I IFN expression, and the delivery vehicle is designed, configured, or formulated so as not to induce TLR2 or one or more combinations of TLR2, TLR4, and TLR5 activity sufficient to inhibit that induced by the encoded immunostimulatory protein, such as type I IFN, e.g., STING protein.

[0250] As used herein, specific tissue or cell, for example, tumor-targeted therapeutic agent refers to active targeting, in which therapeutic agent is directed to tissue or cell, for example, by including a protein that binds to cell surface protein, and also refers to passive targeting, in which targeted therapeutic agent accumulates to some extent in cells, such as cells that cannot be taken up by other cells and therefore end up.Tumor-targeted therapeutic agent is a therapeutic agent that ends up or accumulates in tumor microenvironment, cells in tumor microenvironment and / or tumor.Generally, tumor-targeted therapeutic agent does not end up in other cells and tissues and non-tumor sites, or ends up only minimally.

[0251] As used herein, "immunostimulatory bacteria" are therapeutic bacteria that, when introduced into a subject, accumulate in immune-privileged tissues and cells, such as tumors, tumor microenvironments, and tumor-resident immune cells, and replicate and / or express products that are immune-stimulating or result in immune stimulation. For example, immune-stimulatory bacteria are attenuated in a host by reduced virulence or pathogenicity and / or by encoded products that reduce virulence or pathogenicity. This is because immune-stimulatory bacteria are primarily unable to replicate and / or express products (or have reduced replication / product expression) except in immune-privileged environments such as the tumor microenvironment (TME). The immune-stimulatory bacteria provided herein are modified to encode a product(s) and / or exhibit a trait or characteristic that makes the product immune-stimulatory. The immune-stimulatory bacteria also include genome modifications that prevent expression of endogenous product(s). The bacteria can be said to have lost such product(s). Those skilled in the art will recognize that genes can be inactivated by deletion, disruption, including transposition or insertion of transposons, insertion, and any other change that eliminates the gene product. This can be achieved by insertion, deletion, and / or disruption, including transposition or inclusion of transposons. Examples of inactivated genes include, for example, msbB, pagP, ansB, gene(s) encoding curli pili, genes encoding flagella (inactivation of which causes the bacterium to lose its flagella), and other modifications described herein and / or known to those skilled in the art. Those skilled in the art will also understand that corresponding genes in various bacterial species may have different names.Encoded products, properties, and traits in immunostimulatory bacteria include, but are not limited to, at least one of: immunostimulatory proteins, such as cytokines, chemokines, or costimulatory molecules; cytoplasmic DNA / RNA sensors or gain-of-function or constitutively active mutants thereof (e.g., STING, IRF3, IRF7, IRF-8, MDA5, RIG-I); RNAi, e.g., siRNA (shRNA and microRNA) or CRISPR, that target, disrupt, or inhibit immune checkpoints, e.g., TREX1, PD-1, CTLA-4, and / or PD-L1; antibodies and fragments thereof, e.g., anti-immune checkpoint antibodies, anti-IL-6 antibodies, anti-VEGF antibodies, or TGF-β inhibitory antibodies; other antibody constructs, such as bispecific T cell engagers (BiTE® antibodies); soluble TGF-β receptors that act as decoys that bind TGF-β, or TGF-β antagonizing polypeptides; and IL-6 that binds to decoy receptors. The immunostimulatory bacteria may also contain modifications that render the bacteria auxotrophic for a metabolite that is immunosuppressive or is in an immunosuppressive pathway, such as adenosine.

[0252] As used herein, the strain designations VNP20009 (see, e.g., International Application PCT Publication No. WO 99 / 13053; see also U.S. Pat. No. 6,863,894), 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 S. typhimurium that contains deletions or other modifications within msbB and purI and was generated from the wild-type S. typhimurium strain ATCC #14028.

[0253] As used herein, the strain designations YS1456 and 8.7 are used interchangeably and each refer to the strain deposited with the American Type Culture Collection (ATCC) and assigned accession number 202164 (see U.S. Patent No. 6,863,894). This strain is msbB- and purI- and is strain VNP2009.

[0254] As used herein, the statement that a bacterium is "derived from" a particular strain means that such a strain can serve as a starting material and can be modified to produce the particular bacterium.

[0255] As used herein, T cell exhaustion refers to a state of T cell dysfunction that occurs during many chronic infections and cancers. It describes the T cell response to chronic antigen stimulation in these conditions. It is defined by insufficient effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from that of functional effector or memory T cells. It is characterized by a gradual, progressive loss of T cell function.

[0256] As used herein, a gene module is a set or group of genes that have similar expression profiles or that are associated with one or more genetic or cellular interactions, such as a set of co-expressed genes that are bound by the same set of transcription factors. For example, a G2M score is a set of genes that are associated with the cell cycle transition from G2 to M, and thus serve as an indicator of cell proliferation.

[0257] As used herein, proliferative macrophages are characterized by the oncogene expression of the following: G2M module (>14 genes in the set), stathmin 1 (STMN1), and / or Biopsy surface markers can be identified by some or all of the following: CD68+KI67 and / or PCNA, MERTK. Proliferative macrophages can exhibit all of the above markers or a subset thereof. For example, gene expression in the G2M module, where more than half (>14 genes in the set) are expressed. Additionally, the STMN1+G2M module can be used to confirm proliferation. Alternatively, tumor macrophages can be biopsied and assessed for expression of at least two of CD68, MERTK, and KI67 and / or PCNA.

[0258] As used herein, the M1 / M2 hybrid phenotype refers to a phenotype induced in macrophages by immunostimulatory bacteria provided herein that are attenuated by reducing or eliminating the TLR2 / 4 / 5 response to a therapeutic agent, e.g., bacteria, and encode a non-integrated immunostimulatory payload, e.g., a combination of cytokines and a STING protein that constitutively induces type I IFN. This phenotype is a proliferative and phagocytic macrophage, characterized by, for example, the following markers: Hybrid markers (lower than M2 and higher than M1): SPP1, CD209, CD206, e.g., CD209 and CD206, and / or Two or more inducible markers: MERTK, C1QC, IFN-α2a, IFN-β1, CXCL10, 4-1BBL (TNFSF9), MYC It is associated with a combination of at least two, and generally at least three, of the

[0259] The Summary above and the Tables in the Examples compare macrophage phenotypes before and after administration of an exemplary therapeutic agent, IL-15 / IL-15R alpha chain complex plus eSTING (constitutive STING), e.g., STACT, which encodes chimeric human STING with a gain-of-function mutation and Tasmanian devil CTT.

[0260] As used herein, the statement that "expression of type I IFN in macrophages is not inhibited" upon introduction of a therapeutic agent, delivery vehicle, or nucleic acid means that type I IFN is produced in macrophages at levels higher than in macrophages prior to introduction of the therapeutic agent, delivery vehicle, or nucleic acid.

[0261] As used herein, "expression cassette" refers to a nucleic acid construct that includes regulatory sequences for gene expression operably linked to a nucleic acid encoding an open reading frame (ORF) that encodes a payload, e.g., a therapeutic product or other protein.

[0262] As used herein, 2A peptides are viral oligopeptides, 18-22 amino acids (aa) long, that mediate polypeptide cleavage during translation in eukaryotic cells. The designation "2A" refers to a specific region of the viral genome, and various viral 2A peptides have typically been named after the viruses from which they originate. Examples include F2A (foot-and-mouth disease virus 2A), E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (Thosea asigna virus 2A). For coding sequences, see, e.g., Liu et al. (2017) Scientific Reports 7:2193, Figure 1. See also SEQ ID NOS: 327-330. These peptides generally share the core sequence motif of DxExNPGP and are present in numerous viral families. They aid in polyprotein fragmentation by preventing peptide binding to the ribosome. 2A peptides provide multicistronic / polycistronic vectors in which multiple proteins are expressed from a single open reading frame (ORF). For purposes herein, 2A peptides include naturally occurring ones and any modified versions thereof, e.g., any having 97%, 98%, or 99% sequence identity to any naturally occurring 2A peptide, including those disclosed herein, which result in a single polypeptide that is transcribed and translated from a transcript containing multiple (two or more) open reading frames.

[0263] The cap-independent translation enhancer (CITE) sequence used is a eukaryotic translation element that is part of the RNA molecule transcribed in bacteria, so that the RNA is not translated in bacteria but is translated in animal cells (see U.S. Pat. No. 6,500,419). CITE sequences designed for transcription of RNA that can be translated in eukaryotes but not in bacterial cells are commercially available. Exemplary are those corresponding to the nucleotide sequence from nucleotide 2416 to nucleotide 2914 of pCITE-1 (Novagen, Inc., Madison, Wis.).

[0264] As used herein, "interferonopathy" refers to disorders associated with upregulation of interferon due to mutations in gene products involved in pathways that regulate or induce interferon expression. Product activity is usually regulated by mediators, such as intracellular DNA, RNA, or nucleotides; when the protein product is mutated, activity is constitutive. Type I interferonopathies encompass a range of conditions, including severe forms of Aicardi-Goutières syndrome (AGS) and the milder familial lupus chilliformis (FCL). Nucleic acid molecules encoding mutant products with these properties can be generated in vitro, for example, by selecting for mutations that result in a gain of function in the product compared to products of alleles that have normal activity or that have additional gain of function compared to the disease-associated gain-of-function mutants described herein.

[0265] As used herein, a "gain-of-function mutation" is one that increases the activity of a protein compared to the same protein without the mutation.For example, if the protein is a receptor, the affinity for the ligand will be increased; if the protein is an enzyme, the activity, including constitutive activity, will be increased.In particular, for products such as STING, IRF3, IRF7, MDA5, RIG-I, etc., a constitutively active product is one that is active in the absence of its activating ligand, such as cGAS for STING, and / or in the absence of cytoplasmic nucleic acids, such as DNA, RNA, nucleotides, dinucleotides, cyclic nucleotides and / or cyclic dinucleotides or other nucleic acid molecules that cause the production of type I interferon.These nucleic acid molecules in the cytoplasm arise from viral or bacterial infection and / or radiation or other exposure, leading to the activation of the immune response in the host against such pathogens.

[0266] As used herein, an "origin of replication" is a sequence of DNA on a chromosome or plasmid, or within a virus, from which replication begins. For small DNAs, including bacterial plasmids and small viruses, a single origin is sufficient.

[0267] The origin of replication determines the vector copy number, which depends on the origin of replication selected. For example, if the expression vector is derived from the low-copy-number plasmid pBR322, the copy number may be about 15-20 copies / cell, and if it is derived from the high-copy-number plasmid pUC, the copy number may be 500-700 copies / cell. As used herein, a medium copy number of a plasmid in a cell is about 150 or less, and a low copy number is 5-30, e.g., 20 or less. A low-medium copy number is less than 150 copies / cell. A high copy number is more than 150 copies / cell.

[0268] As used herein, a "CpG motif" is a pattern of bases containing an unmethylated central CpG (where p refers to a phosphodiester bond between consecutive C and G nucleotides) surrounded by at least one base flanking (3' and 5') the central CpG. A CpG oligodeoxynucleotide is an oligodeoxynucleotide that is at least about 10 nucleotides in length and contains an unmethylated CpG. At least the C of the 5'CG3' is unmethylated.

[0269] As used herein, "RIG-I binding sequence" refers to a 5' triphosphate (5' ppp) structure synthesized directly or from a poly(dA-dT) sequence by RNA polymerase III, which can activate type I IFN through the RIG-I pathway by interacting with RIG-I. The RNA contains at least four A ribonucleotides (AAAA); it can contain 4, 5, 6, 7, 8, 9, or 10 or more. The RIG-I binding sequence is introduced into a plasmid in bacteria for transcription into poly(A).

[0270] As used herein, "cytokine" refers to a broad and vague 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 cell-to-cell communication in the immune response and stimulate cell migration toward sites of inflammation, infection, and wounds.

[0271] As used herein, "chemokine" refers to a chemoattractant (chemotactic) cytokine that binds to a chemokine receptor, and includes proteins isolated from natural sources and proteins produced synthetically by recombinant means or by chemical synthesis. Exemplary chemokines include IL-8, IL-10, GCP-2, GRO-α, GRO-β, GRO-γ, ENA-78, PBP, CTAP, and the like. III, NAP-2, LAPF-4, MIG (CXCL9), CXCL10 (IP-10), CXCL11, PF4, SDF-1α, SDF-1β, SDF-2, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1α (CCL3), MIP-1β (CCl4), MIP-1γ (CCL9), MIP-2, MIP-2α, MIP-3α, MIP-3β, MIP-4, MIP-5, MDC, HCC-1, ALP, runkin, TIM-1, eotaxin-1, eotaxin-2, I-309, SCYA17, TRAC, RANTES (CCL5), DC-CK-1, lymphotactin, and fractalkine, and others known to those of skill in the art. Chemokines are involved in the migration of immune cells to sites of inflammation, in the maturation of immune cells, and in the induction of adaptive immune responses.

[0272] As used herein, an "immunostimulatory protein" refers to a protein that exhibits or promotes an anti-tumor immune response in the tumor microenvironment. Examples of such proteins include cytokines, chemokines, and costimulatory molecules, such as, but not limited to, IFN-α, IFN-β, GM-CSF, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-12p70 (IL-12p40+IL-12p35), IL-15 / IL-15R alpha chain complex (also referred to herein as IL-15 / IL-15Rα, IL-15Rα-IL-15sc, IL-15 complex, and other variations described herein), IL-36 gamma, IL-2 with weakened binding to IL-2Ra, and IL-2Ra binding. These include IL-2, CXCL9, CXCL10 (IP-10), CXCL11, CCL3, CCL4, and CCL5, molecules involved in T cell recruitment and / or persistence potential, CD40, CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), 4-1BBL with a deleted cytoplasmic domain (1BBLΔcyt) or a truncated 4-1BBL with a partially deleted cytoplasmic domain, members of the B7-CD28 family, and members of the tumor necrosis factor receptor (TNFR) superfamily.

[0273] Among immune stimulatory proteins, there are truncated costimulatory molecules, such as 4-1BBL, CD80, CD86, CD27L, B7RP1, and OX40L, each of which has a complete or partial cytoplasmic domain deletion for expression on antigen-presenting cells (APCs). These truncated gene products, for example, those with a deleted or partial cytoplasmic domain deletion, are truncated so that they can transmit constitutive immune stimulatory signals to T cells through costimulatory receptor binding but cannot transmit counterregulatory signals to APCs due to the deleted or truncated cytoplasmic domain.

[0274] As used herein, a "cytoplasmic domain deletion" refers to the deletion of all or part of the amino acid residu...

Claims

1. An immunostimulatory bacterium, which is a strain of the genus Salmonella and which contains a plasmid encoding a therapeutic product, The phenotype is: YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI, or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD, or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-containing ΔpurI / ΔthyA; YS1646 is ΔpurI / ΔmsbB; and F-ΔpurI is a complete deletion of the purI coding region; Immune stimulating bacteria. Claim 2: a) the plasmid comprises the nucleotide sequence set forth in SEQ ID NO:501 or degenerate codons thereof in the protein coding region, or a sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO:501 or a plasmid containing one or more degenerate codons, wherein: the plasmid encodes a protein that is an IL-15 / IL-15R alpha chain complex or a protein having at least 95% sequence identity thereto; and the plasmid encodes a chimeric STING that constitutively induces type I interferon (IFN) activity and has reduced NF-κB signaling activity compared to human STING, or encodes a protein that has at least 95% sequence identity with the chimeric STING and has constitutive activity and reduced NF-κB signaling activity compared to human STING; or b) the plasmid comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 502-545 and degenerate sequences thereof, or comprises a nucleic acid encoding an immunostimulatory protein, a eukaryotic transcriptional and / or translational regulatory sequence, or a portion thereof comprising a sequence having at least 95% sequence identity to the coding and regulatory regions of SEQ ID NOs: 502-545; The immunostimulatory bacterium of claim 1.

3. A plasmid encoding a bispecific T cell engager antibody that binds to DLL3 and CD3, wherein the bispecific T cell engager antibody is one of the following: a) to f): a) a light chain comprising amino acid residues 154-260 of SEQ ID NO: 487, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and b) a heavy chain comprising the sequence of amino acid residues set forth as amino acid residues 22 to 138 of SEQ ID NO: 487, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and c) a light chain comprising the sequence of amino acid residues set forth as amino acid residues 155-261 of SEQ ID NO: 489, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and d) a heavy chain comprising the sequence of amino acid residues set forth as amino acid residues 22 to 139 of SEQ ID NO: 489, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and e) a heavy chain and a light chain, the light chain comprises the sequence of amino acid residues set forth as amino acid residues 155-261 of SEQ ID NO:485, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and A heavy chain and a light chain, wherein the heavy chain comprises the sequence of amino acid residues set forth as amino acid residues 22 to 139 of SEQ ID NO: 485, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and f) the heavy and light chains of an anti-CD3 antibody, the light chain of the anti-CD3 antibody comprises the sequence of amino acid residues set forth as amino acid residues 398-504 of SEQ ID NO:485, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto; and The heavy and light chains of an anti-CD3 antibody, wherein the heavy chain of the anti-CD3 antibody comprises the sequence of amino acid residues set forth as amino acid residues 267 to 382 of SEQ ID NO: 485, or a humanized variant thereof, or a variant having at least 95% sequence identity thereto.

2. The immunostimulatory bacterium of claim 1, comprising a combination of:

4. The immunostimulatory bacterium described in claim 1, wherein the plasmid encodes a tumor-associated antigen.

5. The immunostimulatory bacterium of claim 4, wherein the tumor-associated antigen is a carcinoembryonic antigen, a cancer viral antigen, an overexpressed / accumulated antigen, a cancer testis antigen, a lineage-restricted antigen, a mutated antigen, a post-translationally altered antigen, or an idiotypic antigen.

6. The method of claim 1, wherein the carcinoembryonic antigen is carcinoembryonic antigen (CEA), immature laminin receptor, or tumor-associated glycoprotein 72 (TAG-72); The cancer virus antigen is HPV E6 or HPV E7; the overexpressed / accumulated antigen is BING-4, epidermal growth factor receptor (EGFR), Wilms tumor protein, calcium-activated chloride channel 2, cyclin B1, 9D7, delta-like ligand 3 (DLL3), epithelial cell adhesion molecule (EpCAM), ephrin type A receptor 3 (EphA3), human epidermal growth factor receptor 2 (HER2 / Neu), telomerase, mesothelin, gastric cancer-associated protein tyrosine phosphatase 1 (SAP-1), or survivin; the cancer-testis antigen is any of the BAGE (B melanoma antigen) family, CAGE (cancer / testis antigen) family, GAGE ​​(G antigen 1) family, MAGE (melanoma antigen) family, SAGE (sarcoma antigen) family, PAGE (P antigen) family, XAGE (X antigen) family, CT9, CT10, New York esophageal squamous cell carcinoma 1 (NY-ESO-1), LAGE-1, antigen preferentially expressed in melanoma (PRAME), or synovial sarcoma / X breakpoint 2 (SSX-2); the lineage-restricted antigen is T-cell-recognized melanoma antigen 1 (Melan-A / MART-1), gp100 / Pmel17, tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, P. polypeptide, melanocortin 1 receptor (MC1R), or prostate-specific antigen (PSA); the mutant antigen is β-catenin, BRCA1, BRCA2, cyclin-dependent kinase 4 (CDK4), chronic myeloid leukemia tumor antigen 66 (CML66), fibronectin, T-cell-recognized melanoma antigen 2 (MART-2), p53, Ras, or TGF-β receptor type II (TGF-βRII); the post-translationally modified antigen is mucin 1 cell surface-associated antigen (MUC1); and The idiotype antigen is an immunoglobulin (Ig), a T cell receptor (TCR), The immunostimulatory bacterium of claim 5.

7. The plasmid comprising the following combination of therapeutic products: IL-2 and IL-12p70; IL-2 and IL-21; IL-2, IL-12p70 and STING GOF mutants; IL-2, IL-21 and STING GOF mutants; IL-2, IL-12p70, STING GOF mutant, and 4-1BBL (including 4-1BBLΔcyt), in which Δcyt is a deletion of the cytoplasmic domain; IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα and STING GOF mutants; IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα and IL-12p70; IL-15 / IL-15Rα and IL-21; IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; IL-15 / IL-15Rα, IL-21, and STING GOF mutants; IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-21; IL-12p70, IL-21, and STING GOF mutants; IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and STING GOF mutants; IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-18; IL-12p70, IL-18, and STING GOF mutants; IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-2, and IL-12p70; TGF-β decoy receptor, IL-2, and IL-21; TGF-β decoy receptor, IL-2, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-2, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, and IL-12p70; TGF-β decoy receptor, IL-15 / IL-15Rα, and IL-21; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-12p70, and IL-21; TGF-β decoy receptor, IL-12p70, IL-21, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor and IL-12p70; TGF-β decoy receptor, IL-12p70, and STING GOF mutant; TGF-β decoy receptor, IL-12p70, STING GOF mutant, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor, IL-12p70, and IL-18; TGF-β decoy receptor, IL-12p70, IL-18, and STING GOF mutants; TGF-β decoy receptor, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptor and STING GOF mutant; anti-CTLA-4 antibody, IL-2, and IL-12p70; anti-CTLA-4 antibodies, IL-2, and IL-21; anti-CTLA-4 antibody, IL-2, IL-12p70, and STING GOF mutant; anti-CTLA-4 antibodies, IL-2, IL-21, and STING GOF mutants; anti-CTLA-4 antibody, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-15 / IL-15Rα, and STING GOF mutants; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, and IL-12p70; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, and IL-21; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; anti-CTLA-4 antibodies, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-12p70, and IL-21; anti-CTLA-4 antibodies, IL-12p70, IL-21, and STING GOF mutants; anti-CTLA-4 antibodies, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody and IL-12p70; anti-CTLA-4 antibody, IL-12p70, and STING GOF mutant; anti-CTLA-4 antibody, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-12p70, and IL-18; anti-CTLA-4 antibodies, IL-12p70, IL-18, and STING GOF mutants; anti-CTLA-4 antibodies, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibodies and STING GOF mutants; CD40 agonist, IL-2, and IL-12p70; CD40 agonist, IL-2, and IL-21; CD40 agonist, IL-2, IL-12p70, and STING GOF mutants; CD40 agonist, IL-2, IL-21, and STING GOF mutants; CD40 agonist, IL-2, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-2, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, and IL-12p70; CD40 agonist, IL-15 / IL-15Rα, and IL-21; CD40 agonist, IL-15 / IL-15Rα, IL-12p70, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, IL-21, and STING GOF mutants; CD40 agonist, IL-15 / IL-15Rα, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-15 / IL-15Rα, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-12p70, and IL-21; CD40 agonist, IL-12p70, IL-21, and STING GOF mutants; CD40 agonist, IL-12p70, IL-21, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist and IL-12p70; CD40 agonist, IL-12p70, and STING GOF mutant; CD40 agonist, IL-12p70, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist, IL-12p70, and IL-18; CD40 agonist, IL-12p70, IL-18, and STING GOF mutants; CD40 agonist, IL-12p70, IL-18, STING GOF mutants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonist and STING GOF mutant; Bispecific T cell engagers (BiTe) targeting DLL3, EGFR, Her2, CEA, mesothelin, PSMA, EpCAM, CD74, folate receptor, nectin-4, EphA2, CA-IX, B7H3, Siglec-15, Mucl, or Lewis Y antigens; BiTe+STING protein, BiTe+IL-15, BiTe+IL-15+STING protein; Tumor antigen(s) + STING gain-of-function mutant; Therapeutic compositions of tumor antigen(s) and IL-15; Therapeutic compositions of tumor antigen(s) + IL-15 + STING gain-of-function mutant; one or more antigens and IFN; one or more antigens and IFNα; one or more antigens, and IFNα2 or IFNα1-16; one or more antigens and any of IFNα1-16; one or more antigens and IFN-β; one or more antigens, IFNα2, and IFN-β; IRF3 GOF mutant with one or more antigens and mutation S396D; one or more antigens, IFNα2 or IFNα1-16, and an IRF3 GOF mutant with mutation S396D; Two different interferon type I proteins; Interferon-a and / or interferon-b; IFN alpha 2 + IRF3-S396D; IFNα1-16+IRF3-S396D; IFN alpha 2 + IFN-beta; IFNα1-16+IFN-beta; FLT-3L, sialidase, or IL-12p35, or azurin, or membrane-anchored IL-2, IL-12, IL-12p35, IL-21, IL-15, FLT-3L, alone or in combination with other immunostimulatory proteins; and TLR8 agonists, either alone or in combination with other immune stimulating proteins, wherein the agonist is polyU or polyU / G, microRNA, or miR-21. The immunostimulatory bacterium of claim 1 , comprising nucleic acids encoding one or more of:

8. An immunostimulatory bacterium as described in any of claims 1 to 7 for use in treating a subject having or at risk of having a benign nervous system tumor, optionally in combination with an immune checkpoint inhibitor and / or an angiogenesis inhibitor, and optionally encoding at least two anti-cancer therapeutic agents.

9. An immunostimulatory bacterium described in any of claims 1 to 7, wherein the plasmid comprises a sequence of nucleotides set forth as SEQ ID NOs: 502 to 545 and degenerate sequences or portions thereof, including nucleic acid(s) encoding an immunostimulatory protein, eukaryotic transcriptional and / or translational regulatory sequences, or a sequence having at least 95% sequence identity to the coding portions and regulatory regions of SEQ ID NOs: 502 to 545.

10. A plasmid comprising the sequence of nucleotides set forth in SEQ ID NO:501 or degenerate codons thereof in the protein coding region, or a sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO:501 or to a plasmid comprising one or more degenerate codons, a plasmid encoding a protein that is an IL-15 / IL-15R alpha chain complex or a protein that has at least 95% sequence identity thereto; and A plasmid encoding a chimeric STING that constitutively induces type I interferon (IFN) activity and has reduced NF-κB signaling activity compared to human STING, or a plasmid encoding a protein that has at least 95% sequence identity to the chimeric STING and has constitutive activity and reduced NF-κB signaling activity compared to human STING.

11. 1. A method for identifying a subject who is likely to respond or predicted to respond to treatment with a therapeutic agent comprising a delivery vehicle containing a non-integrated nucleic acid encoding one or more immunostimulatory proteins, the method comprising testing a tumor or body fluid sample to detect specific markers indicative of proliferating macrophages, wherein: Treatment-responsive and / or proliferative macrophages are identified by a combination of markers detectable by immunohistochemistry (IHC) and genetic markers for a particular tumor type; and The combination of IHC detectable markers and tumor type genetic markers is as follows: SPP1+ and NRF2 pathway alterations in tumor biopsies or body fluid samples from subjects with squamous cell carcinoma; SPP1+ and TP53 mutations in breast cancer (BRCA), SPP1+ and PI3K mutations in prostate cancer (PRAD), SPP1+ and BRAF mutations in cutaneous melanoma (SKCM), C1QC+ and HIPPO pathway mutations, C1QC+ in uterine corpus endometrial cancer (UCEC); C1QC+ and KMT2A mutations in bladder cancer (BLCA), and C1QC+ and TP53 pathway mutations in breast cancer (BRCA) A method selected from the following.

12. The proliferative macrophages a) biopsy surface markers: CD68+KI67 and / or PCNA, presence of MERTK and / or gene expression of the G2M module where half or more than half (≥ or > 14 genes of the set) are expressed; and / or b) tumor gene expression of the G2M module (>14 genes in the set) alone or plus stathmin 1 (STMN1); and / or c) the markers CD68, MERTK and K167 and / or PCNA, and / or d) Biopsy surface markers: CD68+KI67 and / or PCNA, MERTK, and / or e) SPP1 in lung or gastric tumors, and / or f) C1QC in colon and breast cancer The method of claim 11, wherein the genus is identified by:

13. 1. A method for identifying a therapeutic agent that converts macrophages to an M1 / M2 hybrid phenotype, comprising: a) preparing one or more candidate therapeutics comprising a delivery vehicle and a nucleic acid encoding an immunostimulatory protein, one of which induces an anti-viral or anti-cancer immune response and another of which induces type I IFN, and wherein TLR2 or TLR4 or TLR2 and TLR4 or 5 or TLR2 / 4 / 5 of the delivery vehicle are attenuated, whereby the therapeutic does not inhibit type I IFN in macrophages when introduced into or infecting macrophages; b) introducing the candidate therapeutic agent(s) into the proliferating macrophages; c) determining the phenotype of the resulting macrophages; and d) selecting candidate therapeutic(s) if the resulting macrophages have an M1 / M2 hybrid phenotype; The method of claim 11 , comprising:

14. Macrophages are converted to an M1 / M2 hybrid phenotype; and The M1 / M2 hybrid phenotype is characterized by the expression of at least two of the following markers in a tumor or body fluid sample: Hybrid markers (lower than M2, higher than M1): SPP1, CD209, CD206; and Inducible markers: MERTK, C1QC, IFN-α2a, IFNβ1, CXCL10, 4-1BBL (TNFSF9), and MYC The method of claim 13, wherein the gene is identified by a marker comprising:

15. the macrophages contain M1 and M2 markers whose levels change after treatment to levels indicative of an M1 / M2 hybrid phenotype as follows: the M1 phenotype markers CD80, CD86, CCR7, CXCL10, and CXCL11 are upregulated compared to pre-treatment levels; the M2 phenotype markers CD206 and CD209 are downregulated relative to M2 macrophages but upregulated relative to M1 macrophage phenotype markers; and the M1 and M2 markers CD14, CD68, and CD163 are upregulated after treatment; or The method of claim 13 or 14, wherein the markers that are upregulated in the resulting macrophages after treatment are costimulatory molecules CD80, CD86, chemokine signaling CCR7, CXCL10, CXCL11, PRRs (pattern recognition receptors), CD206, CD209, which are upregulated for M1 and downregulated for M2 macrophages, scavenger receptors CD68, CD163, which are upregulated.

16. A therapeutic regimen or combination for increasing the therapeutic effect of an immunostimulatory bacterium in a subject having a tumor, comprising: an anti-PD-1 antibody or other PD-1 antagonist for pre-treatment of a subject to suppress PD-1 expression on macrophages in the subject's tumor, thereby promoting their phagocytic ability; an immunostimulatory bacterium for use in treating a subject, the bacterium encoding one or more immunostimulatory proteins; and Anti-PD-L1 Agents for Use After Treatment With Immunostimulatory Bacteria - Patent application A therapeutic regimen or combination comprising:

17. a) the therapeutic agent is an immunostimulatory bacterium that is a gram-positive bacterium; or b) the therapeutic agent is an immune-stimulating bacterium that is a strain of Shigella, E. coli, Listeria, or Salmonella; or c) the therapeutic agent is an immunostimulatory bacterium that is a strain of Salmonella typhimurium; The method of claim 11.

18. A method for treating a patient with a delivery vehicle, comprising: (a) suppressing PD-1 expression in macrophages, thereby promoting the phagocytic activity of macrophages; and (b) inhibiting PD-1 expression in macrophages prior to treatment with the delivery vehicle. b) a delivery vehicle comprising a nucleic acid encoding an anti-cancer product, the delivery vehicle being targeted to or capable of being phagocytosed by phagocytic macrophages; 10. An anti-cancer treatment regimen comprising:

19. 19. The regimen of claim 18, further comprising an anti-PD-L1 agent for use in treatment after the anti-cancer product encoded in the delivery vehicle is expressed, resulting in the induction of PD-L1 in macrophages.

20. a) anti-PD-1 antibody; b) a delivery vehicle for use in the subsequent treatment of a), comprising: The delivery vehicle may be selected from the group consisting of: YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-ΔpurI, or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD, or YS1646Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / F-containing ΔpurI / ΔthyA; YS1646 is ΔmsbB / ΔpurI, and F-ΔpurI represents the strain with purI deletion; a delivery vehicle comprising a plasmid encoding an IL-15 / IL-15R alpha chain complex and a chimeric STING having CTT and substitutions N154S / R284G from Tasmanian devil, or a derivative thereof having additional genomic modifications; and c) immunotherapy for administration after a) and b).

19. The regimen of claim 18, comprising:

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