Immune stimulating bacterial delivery platform and use thereof for delivering therapeutic products

Abstract

The present invention provides attenuated immunostimulatory bacteria whose genome is modified to, for example, reduce toxicity and increase anti-tumor activity, such as by increasing accumulation in the tumor microenvironment, particularly in tumor-resident myeloid cells, increasing resistance to complement inactivation, reducing immune cell death, promoting adaptive immunity, and enhancing T cell function. The increase in phagocyte colonization improves delivery of encoded therapeutic products to the tumor microenvironment and into the tumor, and allows for routes of immunostimulatory bacteria administration such as systemic administration.

Description

Immunostimulated bacterial delivery platform and its use in delivering therapeutic products

[0001] Related applications

[0002] This application claims priority to co-pending U.S. Provisional Patent Application No. 62 / 990,404, filed March 16, 2020, entitled “TUMOR-SPECIFIC IMMUNOSTIMULATORY BACTERIA DELIVERY PLATFORM”, filed by Actym Therapeutics, Inc., and inventors Laura Hix Glickman, Christopher D. Thanos, Alexandre Charles Michel Iannello, Chris Rae, and Haixing Kehoe.

[0003] This application claims priority to co-pending U.S. Provisional Patent Application No. 62 / 962,162, filed January 16, 2020, entitled “TUMOR-SPECIFIC IMMUNOSTIMULATORY BACTERIA DELIVERY PLATFORM”, filed by Actym Therapeutics, Inc., and inventors Laura Hix Glickman, Christopher D. Thanos, Alexandre Charles Michel Iannello, Chris Rae, and Haixing Kehoe.

[0004] This application also claims priority to co-pending U.S. Provisional Patent Application No. 62 / 934,503, filed November 12, 2019, entitled “TUMOR-SPECIFIC IMMUNOSTIMULATORY BACTERIA DELIVERY PLATFORM”, filed by Actym Therapeutics, Inc., and inventors Laura Hix Glickman, Christopher D. Thanos, Alexandre Charles Michel Iannello, Chris Rae, and Haixing Kehoe.

[0005] This application relates to co-pending international patent application No. PCT / US2020 / 020240, filed on February 27, 2020, entitled “IMMUNOSTIMULATORY BACTERIA ENGINEERED TO COLONIZE TUMORS, TUMOR-RESIDENT IMMUNE CELLS, AND THE TUMOR MICROENVIRONMENT”, filed by Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, Alexandre Charles Michel Iannello, and Haixing Kehoe.

[0006] This application also relates to co-pending U.S. Provisional Application No. 62 / 962,140, ​​filed January 16, 2020, entitled “IMMUNOSTIMULATORY BACTERIA ENGINEERED TO COLONIZE TUMORS, TUMOR-RESIDENTIMMUNE CELLS, AND THE TUMOR MICROENVIRONMENT”, filed by Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, Alexandre Charles Michel Iannello, and Haixing Kehoe.

[0007] This application also relates to co-pending U.S. Provisional Application No. 62 / 934,478, filed November 12, 2019, entitled “IMMUNOSTIMULATORY BACTERIA ENGINEERED TO COLONIZE TUMORS AND THE TUMORMICROENVIRONMENT”, filed by Actym Therapeutics, Inc., and inventors Christopher D. Thanos, Laura Hix Glickman, Justin Skoble, and Alexandre Charles Michel Iannello.

[0008] This application also relates to International Patent Application No. PCT / US2018 / 041713, filed July 11, 2018, and WO 2019 / 014398, published January 17, 2019; and to co-pending U.S. Patent Application No. 16 / 033,187, filed July 11, 2018, and U.S. Patent No. 2019 / 0017050A1, published January 17, 2019, all entitled “ENGINEERED IMMUNOSTIMULATORY BACTERIAL STRAINS AND USES THEREOF”, all claiming priority to U.S. Provisional Patent Application No. 62 / 531,327, filed July 11, 2017, and U.S. Provisional Patent Application No. 62 / 648,380, filed March 26, 2018. Subject matter of each of these applications is incorporated herein by reference where permitted.

[0009] This application also relates to co-pending international patent application No. PCT / US2019 / 041489, filed July 11, 2019, and WO 2020 / 014543, published January 16, 2020, and co-pending U.S. patent application No. 16 / 520,155, filed July 23, 2019, all entitled “ENGINEERED IMMUNOSTIMULATORY BACTERIALSTRAINS AND USES THEREOF”, and all claim priority to U.S. provisional patent application No. 62 / 789,983, filed January 8, 2019, and U.S. provisional patent application No. 62 / 828,990, filed April 3, 2019.

[0010] This application also relates to priority interests in U.S. Provisional Patent Application No. 62 / 811,521, filed February 27, 2019, and No. 62 / 828,990, filed April 3, 2019.

[0011] The immunostimulatory bacteria provided in each of these applications may be modified as described in this application, and these bacteria are incorporated herein by reference. Where permitted, the subject matter of each of these applications is incorporated herein by reference in its entirety.

[0012] By incorporating into the sequence list provided electronically.

[0013] An electronic version of the sequence listing is submitted with this application, the contents of which are incorporated herein by reference in their entirety. This electronic file was created on November 11, 2020, is 687kb in size, and is named 1707SEQPC1.txt. The corrected sequence listing is submitted electronically, the contents of which are incorporated herein by reference in their entirety. This electronic file was created on March 26, 2021, is 687kb in size, and is titled 1707SEQPC2.txt. Technical Field Invention Field

[0014] We provide attenuated immunostimulatory bacteria with modified genomes to, for example, reduce toxicity and enhance antitumor activity, by increasing accumulation in the tumor microenvironment, particularly in tumor-resident bone marrow cells, by enhancing resistance to complement inactivation, by reducing immune cell death, by promoting adaptive immunity, and by enhancing T cell function. Increased phagocytic colonization improves the delivery of encoded therapeutic products to the tumor microenvironment and tumor, and allows for systemic administration of the immunostimulatory bacteria, among other pathways. Background Technology

[0015]

[0016] The clinical success of anti-CTLA-4, anti-PD-1, and anti-PD-L1 immune checkpoint antibodies demonstrates significant progress in the field of cancer immunotherapy (see, for example, Buchbinder et al. (2015) J. Clin. Invest. 125:3377-3383; Hodiet 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 an extremely immunosuppressive environment. They initiate multiple mechanisms to evade immune surveillance, reprogram anti-tumor immune cells to suppress immunity, and continuously mutate to develop resistance to the latest cancer therapies (see, for example, Mahoney et al. (2015) Nat. Rev. Drug Discov. 14(8):561-584). Designing immunotherapies and cancer treatments that overcome immune tolerance and escape while simultaneously limiting 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

[0017]

[0018] This invention provides immunostimulatory bacteria containing genomic modifications and plasmids encoding one or more therapeutic products, such as anticancer therapeutic agents or related therapeutic agents. The genomic modifications result in the accumulation of the immunostimulatory bacteria in the tumor microenvironment and tumor-resident immune cells, where the encoded therapeutic products are expressed. The immunostimulatory bacteria provided herein encode one or more complementary products that stimulate, induce, or lead to a potent anticancer response in a subject.

[0019] An immunostimulatory bacterium containing a plasmid encoding a therapeutic product or a combination of therapeutic products under the control of a eukaryotic promoter is provided. The genome of said bacteria contains modifications selected from, for example, one, two, or more of the following:

[0020] a) The bacteria are modified to produce pentanoyl lipopolysaccharide (LPS) by deleting, disrupting, or inactivating all or sufficient portions of one or more genes, thereby:

[0021] The genome of immune-stimulated bacteria is modified by deleting or disrupting all or a sufficient portion of one or more genes, thereby modifying the bacteria to produce pentanoyl lipopolysaccharides; and / or

[0022] Compared with wild-type bacteria, hexaacylated lipopolysaccharides were significantly reduced by at least 10-fold, or were absent;

[0023] b) The bacteria have a loss, disruption or inactivation of all or a sufficient portion of one or more genes, thereby giving them reduced recognition of Toll-like receptors (TLRs), namely TLR2, TLR4 and TLR5.

[0024] c) The bacteria lack, destroy, or inactivate all or a sufficient portion of one or more genes, thereby preventing the bacteria from activating the synthesis of curly pili and / or cellulose;

[0025] d) The bacteria lack, destroy, or inactivate all or a sufficient portion of one or more genes, thereby preventing the synthesis of secreted asparaginases;

[0026] e) The bacteria are auxotrophic for purines, adenosine, or ATP due to the absence, disruption, or inactivation of all or sufficient portions of one or more genes;

[0027] f) The bacteria lacks flagella due to the absence, disruption, or inactivation of all or sufficient portions of one or more genes;

[0028] g) The bacteria are modified to specifically infect tumor-resident bone marrow cells by deleting, destroying, or inactivating all or sufficient portions of one or more genes;

[0029] h) The bacteria are modified to specifically infect tumor-resident bone marrow cells and cannot replicate in them, by deleting, disrupting, or inactivating all or sufficient portions of one or more genes; and

[0030] i) Deletion, disruption, or inactivation of one or both of lppA and lppB to reduce or eliminate the expression of lipoproteins in the membrane, thereby increasing the expression of therapeutic proteins encoded by these proteins in the tumor microenvironment and / or tumor-resident immune cells.

[0031] For example, the immunostimulatory bacteria contain modifications of a), d), and f), including deletion, insertion, and substitution, or modifications of c) and d), or modifications of a), c), d), e), and f), or modifications of a), c), d), e), f), and i), or modifications of a), d), f), and i), or modifications of c), d), and i), or modifications of f), and i), or modifications of a) to i), or modifications of a), b), d), and f), or modifications of a), b), c), and d), and other combinations of a) to i).

[0032] In all embodiments, the immune-stimulating bacteria may also include, or further include, the deletion or disruption of genes encoding flagella, thereby the bacteria being flagellin. - (fliC - / fljB - And it does not produce flagella, while wild-type bacteria do have flagella. The immunostimulatory bacteria may be purine auxotrophs, such as adenosine auxotrophs, or adenosine, adenine, and / or ATP auxotrophs. The immunostimulatory bacteria may also be purI - The immune-stimulating bacteria mentioned can also be pagP. - The immune-stimulating bacteria mentioned can also be asd. - Aspartate-semialdehyde dehydrogenase - For example, when bacteria are asd - At this time, due to the complete or partial destruction or deletion of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (ASD), endogenous ASD is not expressed. The bacteria may encode aspartate-semialdehyde dehydrogenase (ASD) under the control of the bacterial promoter on a plasmid. The immune-stimulating bacteria may also be msbB. - Or it could be pagP - / msbB - For example, the immune-stimulating bacteria could be asd - purI - msbB - flagellin - (fliC - / fljB - ) and pagP- Or it could be asd - csgD - purI - msbB - flagellin - (fliC - / fljB - ) and pagP - In some embodiments, the immunostimulatory bacteria is ansB. - ,asd - csgD - purI - msbB - flagellin - (fliC - / fljB - ) and pagP - .

[0033] Provided are immunostimulatory bacteria containing plasmids encoding therapeutic products under the control of eukaryotic promoters, or encoding multiple products under the control of multiple eukaryotic promoters or a single promoter. The genome of the immunostimulatory bacteria is modified by deleting sufficient portions of genes or by disrupting genes, thereby the bacteria being ansB. - ,asd - csgD - purI - msbB - flagellin - (fliC - / fljB - ) and pagP - One or more bacteria are included. The immunostimulatory bacteria described herein also include those bacteria with deleted or disrupted genes lppA (lpp1) and / or lppB (lpp2), which encode the major outer membrane lipoproteins Lpp1 (LppA) and Lpp2 (LppB), respectively, to eliminate or substantially reduce the expression of the encoded lipoproteins. Specifically, the bacteria are lppA... - and lppB - It provides immunostimulatory bacteria containing plasmids encoding anticancer therapeutic agents under the control of eukaryotic regulatory sequences, and is lppA. - and lppB - For example, the immune-stimulating bacteria could be ansB. - ,asd - csgD - purI - msbB - flagellin - (fliC - / fljB - ), pagP - lppA - and lppB - .

[0034] In embodiments described herein, the therapeutic product is an anticancer therapeutic agent or a therapeutic agent for cancer treatment. The encoded product can be operatively linked to a nucleic acid encoding a secretion signal, whereby, when expressed, the therapeutic product is secreted, for example, from tumor-resident immune cells.

[0035] Any immunostimulatory bacterium may also have one or more genes or operators involved in Salmonella pathogenic island 1 (SPI-1) invasion deleted or inactivated, thereby preventing the immunostimulatory bacterium from invading or infecting epithelial cells. For example, the one or more genes / operators are selected from the following: 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.

[0036] Plasmids in immunostimulatory bacteria can exist at low or intermediate copy numbers. The plasmid may contain intermediate to low copy numbers of origins of replication, such as low copy numbers. In some embodiments, the plasmid exists at a higher copy number. Generally, intermediate copy numbers are less than 150 or less than about 150, greater than 20 or about 20, or between 20 or 25 and 150; low copy numbers are less than 25, or less than 20, or less than about 25, or less than about 20 copies. Specifically, low to intermediate copy numbers are less than about 150 copies, or less than 150 copies; low copy numbers are less than about 25 copies, or less than 25 copies.

[0037] Nucleic acid constructs are provided, which are nucleic acid molecules encoding products such as proteins, and are designed to be introduced into cells or plasmids to express the encoded products. The constructs contain nucleic acids encoding multiple anticancer products as polycistronic sequences under the control of a single promoter. The promoter may be a eukaryotic promoter. Other eukaryotic regulatory sequences, such as enhancers and nucleic acids encoding protein transport signals such as secretion signals, and other regulatory sequences, such as terminators, including bacterial terminators that prevent readthrough of bacterial promoters on the constructs, as well as specific conformations of elements and sequences of product-encoding nucleic acid open reading frames and / or genes, are provided and described herein. In the constructs, the polycistronic sequence may include a signal or encoding signal, such as a peptide, that results in the expression of discrete products encoded by the polycistronic construct. Examples of such peptides are the 2A family of viral peptides. The constructs include such peptides or other signals between each open reading frame encoding each product. The 2A peptides include one or more of T2A, P2A, E2A, or F2A.

[0038] Anticancer products include any product used to treat cancer or to promote, aid, stimulate, or assist an anticancer response in a subject. Anticancer products are typically proteins. Encoded products include one or more immunostimulatory proteins that confer or aid an antitumor immune response in the tumor microenvironment. Example encoded products are immunostimulatory proteins that confer or aid an antitumor immune response in the tumor microenvironment, such as those selected from any one or more of the following: IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-15, IL-2 with attenuated binding to IL-2Ra, IL-15 / IL-15Rα chain complex, IL-18, IL-21, IL-23, IL-36γ, modified IL-2 that does not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, interferon-γ, CCL3, CCL4, CCL5, which participate in or influence or enhance T cell recruitment. / Persistent proteins, co-stimulatory proteins, such as CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), including co-stimulatory proteins in the form of cytoplasmic domain deletion or truncation to eliminate immunosuppressive reverse signal transduction, with optional modifications that promote correct orientation in the cell (i.e., cytoplasmic domains in the cytoplasm); B7-CD28 family members, CD47 antagonists, anti-IL-6 antibodies or IL-6 binding decoy receptors, TGF-β peptide antagonists, including soluble TGF-β receptors and TGF-β antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily.

[0039] Other products include immunostimulatory proteins that confer or contribute to antitumor immune responses in the tumor microenvironment, selected from one or more of the following: 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-15Rα chain complex, IL-36γ, IL-2 with weakened binding to IL-2Ra, IL-2 modified to not bind to IL-2Ra, CXCL9, CXCL1 0 (IP-10), CXCL11, CCL3, CCL4, CCL5, molecules involved in potential recruitment and / or persistence of T cells, CD40, CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), 4-1BBL with missing or partially missing cytoplasmic domains (4-1BBLΔcyt), the domains being deleted or truncated to eliminate immunosuppressive reverse signaling, members of the B7-CD28 family, and members of the tumor necrosis factor receptor (TNFR) superfamily. For example, the construct may contain a nucleic acid encoding a 4-1BBL having a deleted or partially deleted cytoplasmic domain and optionally including amino acid modifications, wherein the resulting 4-1BBL is assumed to be correctly oriented when expressed in cells (see SEQ ID NO:389-392 and the detailed description below, which provides example 4-1BBL variants modified with truncated cytoplasmic domains, wherein residues are replaced with positively charged residues (i.e., K and L) to impart the correct orientation when expressed in cells). The cytoplasmic domain is truncated sufficiently to eliminate or reduce immunosuppressive reverse signaling. Thus, constructs are provided containing a nucleic acid encoding a 4-1BBL having a deleted or partially deleted cytoplasmic domain or a modified 4-1BBL encoding a truncated and modified cytoplasmic domain, wherein the sequence of 4-1BBL is shown in SEQ ID NO:389-392 and illustrated in the detailed description and examples below. The construct may also contain a nucleic acid encoding any of the following products and combinations of products:

[0040] One or more of IL-12, IL-15, IL12p70, or IL-15 / IL-15Rα chain complex;

[0041] Cytokines and STING pathway agonists;

[0042] Cytokines, STING pathway agonists and co-stimulatory molecules or immune checkpoint inhibitors;

[0043] Cytokines and STING pathway agonists, as well as TGF-β peptide antagonists;

[0044] Cytokines, STING pathway agonists, TGF-β peptide antagonists, and co-stimulatory molecules (receptors or ligands) or immune checkpoint inhibitors,

[0045] STING pathway agonists are any products that increase type I interferon expression by activating the STING pathway. Examples include constructs encoding peptides of the interferon gene stimulator (STING) or variants thereof or chimeras thereof, as described in detail herein. The constructs described herein are those encoding combinations of therapeutic products selected from the following combinations:

[0046] Anti-CTLA-4 antibody and STING peptide,

[0047] IL-15 and STING peptide,

[0048] 4-1BBL and STING peptides,

[0049] TGF-β decoy receptor or peptide antagonists, and STING peptides,

[0050] IL-12 and STING peptide,

[0051] Anti-CTLA-4 antibody, IL-15, and STING peptide,

[0052] 4-1BBL, IL-15, and STING peptides,

[0053] TGF-β decoy receptor or peptide antagonists, IL-15, and STING peptides

[0054] Anti-CTLA-4 antibody, IL-12, and STING peptide,

[0055] 4-1BBL, IL-12, and STING peptides

[0056] TGF-β decoy receptor or peptide antagonists, IL-12, and STING peptides

[0057] Anti-CTLA-4 antibody, IL-15, TGF-β decoy receptor or peptide antagonist, and STING peptide.

[0058] 4-1BBL and IL-15, TGF-β decoy receptor or peptide antagonists, and a STING peptide,

[0059] Anti-CTLA-4 antibody, and IL-12, TGF-β decoy receptor or peptide antagonist, and STING peptide,

[0060] 4-1BBL and IL-12, TGF-β decoy receptor or peptide antagonists, and STING peptide,

[0061] Anti-CTLA-4 antibody, IL-12, IL-15, and STING peptide,

[0062] 4-1BBL, IL-12, IL-15 and STING peptide,

[0063] TGF-β decoy receptor or peptide antagonists, IL-12, IL-15, and STING peptide,

[0064] TGF-β decoy receptor or peptide antagonists, IL-12, IL-15, and STING peptide,

[0065] Anti-CTLA-4 antibody, IL-12, IL-15, TGF-β decoy receptor or peptide antagonist, and STING peptide.

[0066] 4-1BBL, IL-12, IL-21, TGF-β decoy receptor or peptide antagonists and STING peptide,

[0067] Anti-CTLA-4 antibodies, IL-12, IL-15, and TGF-β decoy receptor or peptide antagonists,

[0068] 4-1BBL, IL-12, IL-21, and TGF-β decoy receptors or peptide antagonists,

[0069] IL-12, IL-15 and STING peptide,

[0070] IL-15, IL-21, and STING peptides,

[0071] IL-12, IL-21 and STING peptide,

[0072] Anti-CTLA-4 antibody, IL-15, IL-21, and STING peptide,

[0073] Anti-CTLA-4 antibody, IL-12, IL-21, and STING peptide,

[0074] 4-1BBL, IL-15, IL-21 and STING peptide,

[0075] 4-1BBL, IL-12, IL-21 and STING peptide,

[0076] Anti-CTLA-4 antibody and IL-15,

[0077] Anti-CTLA-4 antibodies, IL-15, and TGF-β decoy receptors or peptide antagonists,

[0078] 4-1BBL and IL-15,

[0079] 4-1BBL, IL-15, and TGF-β decoy receptors or peptide antagonists,

[0080] Anti-CTLA-4 antibody and IL-12,

[0081] Anti-CTLA-4 antibodies and IL-12, as well as TGF-β decoy receptors or peptide antagonists,

[0082] 4-1BBL and IL-12,

[0083] 4-1BBL, IL-12, and TGF-β decoy receptors or peptide antagonists,

[0084] Anti-CTLA-4 antibodies and TGF-β decoy receptor or peptide antagonists,

[0085] 4-1BBL and TGF-β decoy receptors or peptide antagonists,

[0086] IL-15 and TGF-β decoy receptors or peptide antagonists,

[0087] IL-12 and TGF-β decoy receptors or peptide antagonists,

[0088] IL-12, IL-15 and TGF-β decoy receptor or peptide antagonists, and

[0089] IL-15 and IL-21, as well as TGF-β decoy receptors or peptide antagonists, wherein:

[0090] Anti-CTLA-4 antibodies are scFv or scFv-Fc;

[0091] STING polypeptides include wild-type STING, or variant STING polypeptides, or chimeric STING polypeptides, and chimeric STING proteins having amino acid substitutions that confer, for example, gain-of-function; and

[0092] 4-1BBL is a 4-1BBL with a missing cytoplasmic domain, a 4-1BBL with a modified cytoplasmic domain, a 4-1BBL with a truncated cytoplasmic domain, or a 4-1BBL with both truncated and modified cytoplasmic domains.

[0093] Other constructs include those that encode combinations of the following therapeutic products:

[0094] IL-2 and IL-12p70;

[0095] IL-2 and IL-21;

[0096] IL-2, IL-12p70, and the STING gain-of-function (GOF) variant;

[0097] IL-2, IL-21, and the STING GOF variant;

[0098] IL-2, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt), where Δcyt is a missing cytoplasmic domain;

[0099] IL-2, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0100] IL-15 / IL-15Rα, and the STING GOF variant;

[0101] IL-15 / IL-15Rα, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0102] IL-15 / IL-15Rα and IL-12p70;

[0103] IL-15 / IL-15Rα and IL-21;

[0104] IL-15 / IL-15Rα, IL-12p70, and the STING GOF variant;

[0105] IL-15 / IL-15Rα, IL-21, and the STING GOF variant;

[0106] IL-15 / IL-15Rα, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0107] IL-15 / IL-15Rα, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0108] IL-12p70 and IL-21;

[0109] IL-12p70, IL-21, and the STING GOF variant;

[0110] IL-12p70, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0111] IL-12p70 and STING GOF variants;

[0112] IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0113] IL-12p70 and IL-18;

[0114] IL-12p70, IL-18, and the STING GOF variant;

[0115] IL-12p70, IL-18, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0116] TGF-β decoy receptor or antagonist peptide, IL-2, and IL-12p70;

[0117] TGF-β decoy receptor or antagonist peptides, IL-2, and IL-21;

[0118] TGF-β decoy receptor or antagonist peptide, IL-2, IL-12p70, and STING GOF variant;

[0119] TGF-β decoy receptor or antagonist peptides, IL-2, IL-21, and STING GOF variants;

[0120] TGF-β decoy receptor or antagonist peptide, IL-2, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0121] TGF-β decoy receptor or antagonist peptide, IL-2, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0122] TGF-β decoy receptor or antagonist peptide, IL-15 / IL-15Rα, and STING GOF variant;

[0123] TGF-β decoy receptor or antagonist peptide, IL-15 / IL-15Rα, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0124] TGF-β decoy receptor or antagonist peptide, IL-15 / IL-15Rα, and IL-12p70;

[0125] TGF-β decoy receptor or antagonist peptide, IL-15 / IL-15Rα, and IL-21;

[0126] TGF-β decoy receptor or antagonist peptide, IL-15 / IL-15Rα, IL-12p70, and STING GOF variant;

[0127] TGF-β decoy receptor or antagonist peptides, IL-15 / IL-15Rα, IL-21, and STING GOF variants;

[0128] TGF-β decoy receptor or antagonist peptide, IL-15 / IL-15Rα, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0129] TGF-β decoy receptor or antagonist peptide, IL-15 / IL-15Rα, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0130] TGF-β decoy receptor or antagonist peptide, IL-12p70, and IL-21;

[0131] TGF-β decoy receptor or antagonist peptide, IL-12p70, IL-21, and STING GOF variant;

[0132] TGF-β decoy receptor or antagonist peptide, IL-12p70, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0133] TGF-β decoy receptor or antagonist peptide and IL-12p70;

[0134] TGF-β decoy receptor or antagonist peptide, IL-12p70, and STING GOF variant;

[0135] TGF-β decoy receptor or antagonist peptide, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0136] TGF-β decoy receptor or antagonist peptide, IL-12p70, and IL-18;

[0137] TGF-β decoy receptor or antagonist peptide, IL-12p70, IL-18, and STING GOF variant;

[0138] TGF-β decoy receptor or antagonist peptide, IL-12p70, IL-18, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0139] TGF-β decoy receptor and STING GOF variant;

[0140] Anti-CTLA-4 antibody, IL-2, and IL-12p70;

[0141] Anti-CTLA-4 antibody, IL-2, and IL-21;

[0142] Anti-CTLA-4 antibody, IL-2, IL-12p70, and STING GOF variant;

[0143] Anti-CTLA-4 antibody, IL-2, IL-21, and STING GOF variant;

[0144] Anti-CTLA-4 antibody, IL-2, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0145] Anti-CTLA-4 antibody, IL-2, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0146] Anti-CTLA-4 antibody, IL-15 / IL-15Rα, and STING GOF variant;

[0147] Anti-CTLA-4 antibody, IL-15 / IL-15Rα, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0148] Anti-CTLA-4 antibody, IL-15 / IL-15Rα, and IL-12p70;

[0149] Anti-CTLA-4 antibody, IL-15 / IL-15Rα, and IL-21;

[0150] Anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-12p70, and STING GOF variant;

[0151] Anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, and STING GOF variant;

[0152] Anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0153] Anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0154] Anti-CTLA-4 antibody, IL-12p70, and IL-21;

[0155] Anti-CTLA-4 antibody, IL-12p70, IL-21, and STING GOF variant;

[0156] Anti-CTLA-4 antibody, IL-12p70, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0157] Anti-CTLA-4 antibody and IL-12p70;

[0158] Anti-CTLA-4 antibody, IL-12p70, and STING GOF variant;

[0159] Anti-CTLA-4 antibody, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0160] Anti-CTLA-4 antibody, IL-12p70, and IL-18;

[0161] Anti-CTLA-4 antibody, IL-12p70, IL-18, and STING GOF variant;

[0162] Anti-CTLA-4 antibody, IL-12p70, IL-18, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt);

[0163] Anti-CTLA-4 antibody and STING GOF variant;

[0164] CD40 agonists, IL-2, and IL-12p70;

[0165] CD40 agonists, IL-2, and IL-21;

[0166] CD40 agonists, IL-2, IL-12p70, and STING GOF variants;

[0167] CD40 agonists, IL-2, IL-21, and STING GOF variants;

[0168] CD40 agonists, IL-2, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt);

[0169] CD40 agonists, IL-2, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt);

[0170] CD40 agonists, IL-15 / IL-15Rα, and STING GOF variants;

[0171] CD40 agonists, IL-15 / IL-15Rα, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt);

[0172] CD40 agonists, IL-15 / IL-15Rα, and IL-12p70;

[0173] CD40 agonists, IL-15 / IL-15Rα, and IL-21;

[0174] CD40 agonists, IL-15 / IL-15Rα, IL-12p70, and STING GOF variants;

[0175] CD40 agonists, IL-15 / IL-15Rα, IL-21, and STING GOF variants;

[0176] CD40 agonists, IL-15 / IL-15Rα, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt);

[0177] CD40 agonists, IL-15 / IL-15Rα, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt);

[0178] CD40 agonists, IL-12p70, and IL-21;

[0179] CD40 agonists, IL-12p70, IL-21, and STING GOF variants;

[0180] CD40 agonists, IL-12p70, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt);

[0181] CD40 agonists and IL-12p70;

[0182] CD40 agonist, IL-12p70, and STING GOF variant;

[0183] CD40 agonists, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt);

[0184] CD40 agonists, IL-12p70, and IL-18;

[0185] CD40 agonists, IL-12p70, IL-18, and STING GOF variants;

[0186] CD40 agonists, IL-12p70, IL-18, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt); and

[0187] CD40 agonists and STING GOF variants, in which:

[0188] 4-1BBL is a 4-1BBL with a missing cytoplasmic domain (4-1BBLΔcyt), a 4-1BBL with a modified cytoplasmic domain, a 4-1BBL with a truncated cytoplasmic domain, or a 4-1BBL with both truncated and modified cytoplasmic domains; and

[0189] Anti-CTLA-4 antibody is scFv or scFv-Fc.

[0190] Examples of STING peptides include those peptides in which the STING peptide is modified to cause increased or constitutive expression of type I interferon, or a chimeric peptide comprising a human STING peptide having a C-terminal tail region from a different species, having lower NF-κB signaling activity than human STING, wherein, for example, the TRAF6 binding site in the CTT is optionally deleted; and the human STING protein has the sequence shown in SEQ ID NO:305-309. Specific combinations of encoded products include those where the encoded therapeutic protein comprises IL-12p70 and a chimeric human STING peptide having a CTT from Tasmanian devil and an amino acid substitution causing increased or constitutive expression of type I interferon, or a STING peptide having an amino acid substitution causing increased or constitutive expression of type I interferon, wherein the mutation causing increased or constitutive expression of type I interferon is a gain-of-function mutation. Examples are STING proteins or peptides having the substitutions described in the detailed description section, for example, by reference to any of SEQ ID NO:305-309 showing human STING proteins, where the amino substitution in the STING peptide corresponds to R284G or N154S / R284G. These constructs may additionally encode IL-36γ and / or immune checkpoint inhibitor antibodies. As described herein, antibodies include their antigen-binding portions, as well as any various forms of antibodies, such as, but not limited to, scFv and scFv-Fc (typically IgG Fc), wherein the presence of Fc polymerizes the resulting product, thereby giving it two chains. Targeted immune checkpoints include, but are not limited to, CTLA-4 or PD-1 or PD-L1, and their antibody forms include scFV and scFV-Fc double-chain peptides.

[0191] Provided are constructs containing the above-described constructs, as well as plasmids throughout this document. The plasmids include bacterial plasmids, wherein the constructs are operatively linked to eukaryotic transcriptional regulatory sequences.

[0192] Compositions, such as pharmaceutical compositions, are provided that contain a mixture of anticancer protein products encoded by said construct or plasmid, as the sole anticancer protein in said composition. Thus, compositions containing complementary combinations of therapeutic proteins are provided in these compositions; said mixtures comprise unique pharmaceutical combinations. These combinations include pharmaceutical combinations provided and described herein.

[0193] Immunostimulatory bacteria containing any of the said constructs and / or plasmids are also provided. The constructs and plasmids may be provided or introduced into any immunostimulatory bacteria provided herein, including those discussed above and below. The constructs and plasmids may also be introduced into suitable bacteria known in the art, such as those described in publications such as International Application Publications WO2020 / 172461 and WO2020 / 172462 and U.S. Patents 10,449,237, 10,286,051, and 9,616,114.

[0194] The immunostimulatory bacteria described in this article include genomic modifications, such as deletions, disruptions, and alterations, such as changing the orientation of all or part of the genes, thereby preventing the expression of functional gene products. The immunostimulatory bacteria provided are modified to produce msbB bacteria. - / purI - Those. In some implementations, the bacteria are msbB. - and purI - Therefore, msbB - and / or purI - At least the full length of the coding portion of the gene is deleted. It is also possible to modify the bacterial genome to make the bacteria lack flagella. This is achieved in bacteria that normally express flagella. In such bacteria, such as Salmonella, the gene is associated with fliC in other species. - / fljB - Certain genes can be deleted or otherwise modified, thus preventing the expression of functional gene products. Bacteria can also be modified to be adenosine auxotrophs and / or msbB. - / pagP - Also provided are immunostimulatory bacteria and pharmaceutical compositions containing them, wherein said bacteria do not express L-asparaginase II, and thus said bacteria are ansB. - .

[0195] Immunostimulatory bacteria containing plasmids encoding therapeutic products under the control of eukaryotic promoters are provided, wherein the genome of said immunostimulatory bacteria is modified by deletion or disruption of all or sufficient portions of one or more genes, thereby preventing said bacteria from activating the synthesis of secreted asparaginase. Examples of such bacteria are those in which the asparaginase is L-asparaginase II encoded by the gene ansB.

[0196] This article indicates that, in msbB - and purI - In the parental strain VNP20009, the gene was not completely deleted. In the provided immunostimulatory bacteria, the bacterial genome was modified, thereby enabling msbB - and purI -At least the full length of the coding portion of the gene is deleted. These strains are more adapted to grow, faster, and / or to a greater extent than the parental strain. In all embodiments described herein, bacteria may be modified to inactivate or prevent the expression of the natural asd gene product. To facilitate the generation of said strains, the asd gene is encoded on a plasmid controlled by a prokaryotic promoter such as an inducible promoter.

[0197] In the implementation scheme, the strain includes modifications that render the bacteria lacking flagella, and is pagP - ansB - and csgD - Furthermore, the bacteria in question are purI. - and asd - Therefore, strains are provided, including modified parental strains, which are already msbB. - and purI - And / or with other modifications, particularly those modifying LPS, the strain is also Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD. The strain may also be an adenosine auxotroph or an adenosine and adenine auxotroph.

[0198] The encoded therapeutic products include nucleic acids and proteins. Plasmids may encode two or more therapeutic products. Example products include, but are not limited to, cytokines, proteins that constitutively induce type I interferon (IFN), and co-stimulatory receptors or ligands. Further example combinations are described below. In some embodiments, the co-stimulatory molecule lacks all or part of the cytoplasmic domain expressed on antigen-presenting cells (APCs), thereby enabling the truncated molecule to send constitutive immunostimulatory signals to T cells via co-stimulatory receptors and preventing counter-regulatory signals to APCs due to the absence, truncation, or other modification of the cytoplasmic domain or a portion thereof. Other products include enzymes that activate therapeutic proteins, such as those that activate prodrugs. As described herein and below, the immunostimulatory bacterial-encoded anticancer therapeutic agents provided herein include combinations of therapeutic products to provide a potent anticancer response. The encoded proteins are each of the products listed above and below, as well as combinations of products from different classes. This includes co-stimulatory proteins, such as 4-1BBL, particularly those with truncated or missing cytoplasmic domains to eliminate immunosuppressive reverse signaling, and those with any compensatory mutations to ensure proper orientation of the resulting protein in the cell membrane when expressed in the cell. Other products include STING pathway agonists to induce or cause constitutive expression of type I interferon. These products include STING proteins, particularly the modified and chimeric STING proteins provided and described herein. One or more cytokines such as IL-12, IL-15, IL-21, L-12p70 (IL-12p40+IL-12p35), IL-2 with weakened binding to IL-2Ra, the IL-15 / IL-15Rα chain complex, and others such as IL-18, IL-23, and IL-36γ are also encoded on the plasmid. In addition to co-stimulatory products, STING pathway agonist proteins such as STING, cytokines, and antibodies such as checkpoint inhibitor antibodies, including anti-CTLA-4 antibodies, are also encoded on the plasmid. The antibody may be in the form of scFvs and scFvs-Fc double chains, as well as other forms. Furthermore, other products may include TGF-β antagonists and TGF-β receptor decoys.

[0199] The encoded therapeutic product can be operatively linked to a nucleic acid encoding a regulatory sequence recognized by a eukaryotic host, such as a secretion signal, to enable secretion from a cell containing the bacteria or plasmid. In embodiments where the immune-stimulated bacteria encode two or more products, the expression of each product can be controlled by a separate promoter. Alternatively, two or more products can be expressed under the control of a single promoter, and each product is separated by a nucleic acid encoding, for example, an internal ribosome entry site (IRES) or a 2A peptide, to achieve separate expression of each encoded therapeutic product. Example 2A peptides are T2A, F2A, E2A, or P2A, which can be flanked by the nucleic acid encoding the therapeutic product to achieve separate expression of the therapeutic product under the control of a single promoter. The therapeutic product is expressed under the control of a eukaryotic promoter such as an RNA polymerase II promoter or an RNA polymerase III promoter. These promoters include RNA polymerase II promoters that are viral promoters or mammalian RNA polymerase II promoters, such as, but not limited to, cytomegalovirus (CMV) promoters, SV40 promoters, Epstein-Barr virus (EBV) promoters, herpesvirus promoters, adenovirus promoters, elongation factor-1 (EF-1)α promoters, UBC promoters, PGK promoters, CAGG promoters, adenovirus 2 or 5 late promoters, EIF4A1 promoters, CAG promoters, or CD68 promoters. The plasmid may further include other eukaryotic regulatory sequences, such as terminators and / or promoters selected from SV40, human growth hormone (hGH), bovine growth hormone (BGH or bGH), MND (a synthetic promoter containing the U3 region of a MoMuLV LTR modified with a myeloproliferative sarcoma virus enhancer), chicken β-globulin, and rbGlob (rabbit globulin) genes, to control the expression of the therapeutic product. Other regulatory sequences include polyA tails, marmot hepatitis virus (WHP) post-transcriptional regulatory elements (WPREs), and hepatitis B virus post-transcriptional regulatory elements (HPREs). Additional regulatory elements may be included, such as bacterial terminators inserted into appropriate loci, as described herein, to reduce or eliminate readthroughs from bacterial promoters.

[0200] The encoded therapeutic product includes any product described herein and in the original claims, such as a nucleic acid encoding a protein that is part of a cytoplasmic DNA / RNA sensor pathway leading to type I interferon (IFN) expression, or a variant thereof. Type I interferons 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 cytoplasmic nucleic acid, nucleotide, dinucleotide, or cyclic dinucleotide to lead to type I IFN expression. Examples of such proteins are proteins selected from the following: STING, RIG-I, MDA-5, IRF-3, IRF-7, TRIM56, RIP1, Sec5, TRAF3, TRAF2, TRAF6, STAT1, LGP2, DDX3, DHX9, DDX1, DDX9, DDX21, DHX15, DHX33, DHX36, DDX60, and SNRNP200, and variants thereof that have increased activity or lead to constitutive expression of type I interferon (IFN). Variants include variants of STING, RIG-I, IRF-3, or MDA5 in which one or more serine (S) or threonine (T) residues phosphorylated due to viral infection are replaced with aspartic acid (D), resulting in variants that are constitutively induced type I IFN phosphate mimics, as well as any variants known to those skilled in the art and / or described herein.Variants include, for example, those variants in which the mutation is selected from the following: a) in STING, referring to SEQ ID NO:305-309, one or more mutations selected from the following: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q 273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A and S324A / S326A; b) In MDA5, refer to SEQ ID NO:310, selected from one or more of the following mutations: T331I, T331R, A489T, R822Q, G821S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F, and A452T; c) in RIG-I, refer to one or both of SEQ ID NO:311, E373A, and C268F; and d) in IRF-3, refer to SEQ ID NO:312, S396D, if any of the SEQ IDs are present. NO:305-309 is selected from STING variants with one or more of the following amino acid substitutions: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q 273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A, N154S / R284G and S324A / S326A, their conservative substitutions, and their combinations.

[0201] Immunostimulatory bacteria can also encode immunostimulatory proteins that confer or contribute to antitumor immune responses in the tumor microenvironment. These include, but are not limited to, cytokines, chemokines, or co-stimulatory molecules. Examples of these are proteins selected from one or more of the following: IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-36γ, IL-2 with weakened binding to IL-2Ra, the IL-15 / IL-15Rα chain complex, IL-18, IL-21, IL-23, IL-2 modified to not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, and interferon-α. CCL-γ, CCL3, CCL4, CCL5, proteins involved in or enabling or enhancing T cell recruitment and / or persistence, CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), B7-CD28 family member, CD47 antagonist, anti-IL-6 antibody or IL-6 binding decoy receptor, TGF-β peptide antagonist and member of the tumor necrosis factor receptor (TNFR) superfamily. Co-stimulatory molecules selected from CD40, CD40 ligand, CD28, OX40, OX40 ligand, 4-1BB, and 4-1BB ligand can be truncated to lack the cytoplasmic domain (or a portion thereof) expressed on antigen-presenting cells (APCs). The truncated gene product can participate in constitutive immunostimulatory signaling to T cells via co-stimulatory receptors, and cannot send counter-regulatory signals to APCs due to the missing or partially missing / truncated cytoplasmic domain, thus eliminating immunosuppressive reverse signal transduction. Other such proteins are TGF-β peptide antagonists, such as anti-TGF-β antibodies or antibody fragments, anti-TGF-β receptor antibodies or antibody fragments, soluble TGF-β antagonist peptides, or TGF-β-binding decoy receptors.

[0202] Plasmids can encode therapeutic antibodies or their antigen-binding fragments, such as Fab, Fab', F(ab')2, single-chain Fv (scFv), scFv-Fc, Fv, dsFv, nanobody, bispecific antibody 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.

[0203] In some embodiments, the immunostimulatory bacteria provided herein contain plasmids encoding two or more therapeutic proteins selected from: a) immunostimulatory proteins that confer or contribute to an antitumor immune response in the tumor microenvironment; b) one or more proteins that are part of a cytoplasmic DNA / RNA sensor pathway leading to type I interferon (IFN) expression, or variants thereof having increased activity in increasing type I interferon expression, or variants thereof leading to constitutive expression of type I IFN; and c) anticancer antibodies or their antigen-binding portions. For example, the immunostimulatory protein may be a costimulatory molecule that lacks a cytoplasmic domain or a sufficient portion thereof expressed on antigen-presenting cells (APCs), thereby truncating the costimulatory molecule to participate in constitutive immunostimulatory signaling to T cells via costimulatory receptors and preventing counterregulatory signaling to APCs. In some embodiments, the immunostimulatory bacteria encode at least two therapeutic products selected from cytokines, constitutively induced type I IFN proteins, costimulatory molecules, and anticancer antibodies or their antigen-binding portions, which can be controlled by a single promoter. For example, the expression of nucleic acids encoding at least two or all products is controlled by a single promoter, and the nucleic acid encoding each product is separated from the nucleic acid encoding the 2A polypeptide, so that each product is expressed individually during translation. The nucleic acid encoding each product can be operatively linked to a nucleic acid encoding a sequence that directs the secretion of the expressed product from the cell.

[0204] Provided are immunostimulatory bacteria encoding two or more therapeutic products, wherein at least one product is selected from a) and at least one product is selected from b), and a) is IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-23, IL-36γ, IL-2 with weakened binding to IL-2Ra, IL-15 / IL-15Rα chain complex, IL-18, IL2 modified to not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, CCL3, CCL4, CCL5, proteins involved in or affecting or enhancing T cell recruitment and / or persistence, CD40 The following are considered as possible: CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), B7-CD28 family members, TGF-β peptide antagonists, or tumor necrosis factor receptor (TNFR) superfamily members; 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 the following: TGF-β inhibitory antibodies, TGF-β binding decoy receptors, anti-IL-6 antibodies, and IL-6 binding decoy receptors.

[0205] The example coded combination of therapeutic products is any of the following combinations: IL-2 and IL-12p70; IL-2 and IL-21; IL-2, IL-12p70, and a STING GOF variant; IL-2, IL-21, and a STING GOF variant; IL-2, IL-12p70, a STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt, where Δcyt is a missing cytoplasmic domain, and 4-1BBL with a truncated cytoplasmic domain (4-1BBLcyt trunc)); IL-2, IL-21, a STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt, and 4-1BBL with a truncated cytoplasmic domain); IL-15 / IL-15Rα, and a STING GOF variant; IL-15 / IL-15Rα, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); IL-15 / IL-15Rα and IL-12p70; IL-15 / IL-15Rα and IL-21; IL-15 / IL-15Rα, IL-12p70, and STING GOF variants; IL-15 / IL-15Rα, IL-21, and STING GOF variants; IL-15 / IL-15Rα, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); IL-15 / IL-15Rα, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc). IL-12p70 and IL-21; IL-12p70, IL-21, and STING GOF variants; IL-12p70, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); IL-12p70 and STING GOF variants; IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); IL-12p70 and IL-18; IL-12p70, IL-18, and STING GOF variants; IL-12p70, IL-18, STING GOF variants. GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); 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 variants;TGF-β decoy receptors, IL-2, IL-21, and STING GOF variants; TGF-β decoy receptors, IL-2, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); TGF-β decoy receptors, IL-2, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); TGF-β decoy receptors, IL-15 / IL-15Rα, and STING GOF variants; TGF-β decoy receptors, IL-15 / IL-15Rα, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); trunc); 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 variant; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-21, and STING GOF variant; TGF-β decoy receptor, IL-15 / IL-15Rα, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt) TGF-β decoy receptors, IL-15 / IL-15Rα, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); TGF-β decoy receptors, IL-12p70, and IL-21; TGF-β decoy receptors, IL-12p70, IL-21, and STING GOF variants; TGF-β decoy receptors, IL-12p70, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); TGF-β decoy receptors and IL-12p70; TGF-β decoy receptors, IL-12p70, and STING GOF variants. GOF variants; TGF-β decoy receptor, IL-12p70, STINGGOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); TGF-β decoy receptor, IL-12p70, and IL-18; TGF-β decoy receptor, IL-12p70, IL-18, and STINGGOF variants;TGF-β decoy receptor, IL-12p70, IL-18, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); TGF-β decoy receptor and STING GOF variant; 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 variant; anti-CTLA-4 antibody, IL-2, IL-21, and STING GOF variant; anti-CTLA-4 antibody, IL-2, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); anti-CTLA-4 antibody, IL-2, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, and STING GOF variant; anti-CTLA-4 antibody, IL-15 / IL-15Rα, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBL with truncated cytoplasmic domains); 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 variant; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, and STING GOF variants; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); anti-CTLA-4 antibody, IL-12p70, and IL-21; anti-CTLA-4 antibody, IL-12p70, IL-21, and STING GOF variant; anti-CTLA-4 antibody, IL-12p70, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt) trunc); anti-CTLA-4 antibody and IL-12p70; anti-CTLA-4 antibody, IL-12p70, and STING GOF variant;Anti-CTLA-4 antibody, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); anti-CTLA-4 antibody, IL-12p70, and IL-18; anti-CTLA-4 antibody, IL-12p70, IL-18, and STING GOF variant; anti-CTLA-4 antibody, IL-12p70, IL-18, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); anti-CTLA-4 antibody and STING GOF variant; CD40 agonist, IL-2, and IL-12p70; CD40 agonist, IL-2, and IL-21; CD40 agonist, IL-2, IL-12p70, and STING GOF variants; CD40 agonists, IL-2, IL-21, and STING GOF variants; CD40 agonists, IL-2, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); CD40 agonists, IL-2, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); CD40 agonists, IL-15 / IL-15Rα, and STING GOF variants; CD40 agonists, IL-15 / IL-15Rα, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc). trunc); CD40 agonists, IL-15 / IL-15Rα, and IL-12p70; CD40 agonists, IL-15 / IL-15Rα, and IL-21; CD40 agonists, IL-15 / IL-15Rα, IL-12p70, and STING GOF variants; CD40 agonists, IL-15 / IL-15Rα, IL-21, and STING GOF variants; CD40 agonists, IL-15 / IL-15Rα, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); CD40 agonists, IL-15 / IL-15Rα, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc); CD40 agonists, IL-15 / IL-15Rα, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyt trunc). trunc); CD40 agonists, IL-12p70, and IL-21; CD40 agonists, IL-12p70, IL-21, and STING GOF variants;CD40 agonist, IL-12p70, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); CD40 agonist and IL-12p70; CD40 agonist, IL-12p70, and STING GOF variant; CD40 agonist, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); CD40 agonist, IL-12p70, and IL-18; CD40 agonist, IL-12p70, IL-18, and STING GOF variant; CD40 agonist, IL-12p70, IL-18, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt and 4-1BBLcyttrunc); and CD40 agonists and STING GOF variants.

[0206] In all combinations including 4-1BBL, the 4-1BBL molecule can be a full-length protein (see, for example, SEQ ID NO:389 and 393 for human and mouse 4-1BBL, respectively); a 4-1BBL variant with a missing cytoplasmic domain (4-1BBLΔcyt; see, for example, SEQ ID NO:390 and 394 for human and mouse 4-1BBLΔcyt, respectively); a 4-1BBL variant with a truncated (i.e., not completely missing) cytoplasmic domain (4-1BBLcyt trunc; see, for example, SEQ ID NO:391-392 and SEQ ID NO:395-396 for human and mouse 4-1BBLcyt trunc, respectively); or a 4-1BBL molecule with a modified cytoplasmic domain in which one or more Ser residues serving as phosphorylation sites are substituted at one or more appropriate loci, for example, for human 4-1BBL, referring to SEQ ID NO:389, Ser5 and Ser8 are substituted with residues that reduce or eliminate reverse signal transduction. In addition, all combinations including anti-CTLA-4 antibodies may include anti-CTLA-4 antibody fragments, such as anti-CTLA-4 scFv (see, for example, SEQ ID NO:403 and 404 for human and mouse anti-CTLA-4 scFv fragments, respectively), or anti-CTLA-4 scFv-Fc (see, for example, SEQ ID NO:402 and 405 for human and mouse anti-CTLA-4 scFv-Fc fragments, respectively).

[0207] Modified nonhuman interferon gene stimulating factor (STING) proteins and STING protein chimeras are also provided, along with delivery vectors, including any of those described herein, pharmaceutical compositions, cells encoding or containing these STING proteins, and their uses and methods for treating cancer. Specifically, the immunostimulatory bacteria provided herein encode modified nonhuman STING proteins, nonhuman STING proteins, and chimeras as described herein. These STING proteins encoded by immunostimulatory bacteria are provided herein and described in full. What is provided herein is:

[0208] 1. A modified non-human STING protein, wherein the non-human STING protein has lower NF-κB activation activity than human STING protein and optionally higher type I interferon activation activity compared to wild-type (WT) human STING protein. These non-human STING proteins are modified to include one or more mutations, thereby giving them increased activity or constitutively functioning in the absence of cytoplasmic nucleic acid signaling. The mutations are typically amino acid mutations occurring in human interferon diseases, such as those described above for human STING. The corresponding mutations are introduced into the non-human species STING protein, wherein the corresponding amino acid residues are identified by alignment. Furthermore, in some embodiments, the TRAF6 binding site in the C-terminal tail region (CTT) of the STING protein is deleted, thereby reducing NF-κB signaling activity.

[0209] 2. Modified STING proteins, particularly human STING proteins, which are chimeras in which the CTT (C-terminal tail) region of a STING protein from one species, such as humans, is replaced by the CTT of a STING protein from another species having lower NF-κB signaling activity and / or higher type I IFN signaling activity compared to human STING. Furthermore, the TRAF6 binding site is optionally missing in these chimeras.

[0210] 3. The STING protein modified in 2 above also includes the mutation in 1 above.

[0211] 4. Any delivery vector provided herein or known to those skilled in the art, such as immunostimulatory bacteria, including, for example, exosomes, nanoparticles, microcells, cells, liposomes, lysosomes, oncolytic viruses and other viral vectors, encoding the STING protein modified by any one of 1 to 3 above.

[0212] 5. Any delivery vector provided herein or known to those skilled in the art, such as immunostimulatory bacteria, including, for example, exosomes, nanoparticles, microcells, cells, liposomes, lysosomes, oncolytic viruses, and other viral vectors, encoding unmodified STING from a non-human species, wherein the non-human STING protein has reduced NF-κB signaling activity compared to human STING and optionally increased type I interferon stimulation / signaling activity compared to human STING.

[0213] 6. Cells (not fertilized eggs if they are human), such as cells used for cell therapy, such as T cells and stem cells, and cells used to produce the STING protein of any of 1 to 3 above.

[0214] 7. A pharmaceutical composition comprising the STING protein of any one of 1 to 3 above, or the delivery carrier of 4 and 5, or the cell of 6.

[0215] 8. The use and method of treating cancer by applying any of the above 1 to 7, as described herein with respect to immune-stimulating bacteria.

[0216] This article describes assays and methods for assessing the NF-κB activity (signaling activity) and type I interferon-stimulating or interferon-β-stimulating activity of STING, and these methods are known to those skilled in the art. Methods include, for example, those described in de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175, which specifically describes the interferon-β and NF-κB signaling activities of STING proteins from different species, including humans, thereby identifying STING proteins from different species with lower NF-κB activity than human STING, and those with interferon-β activity equivalent to or higher than human STING. de Oliveira Mann et al. (2019) provides species alignment and identification domains of STING for each species, including the CTT domain (see also the supplementary information in de Oliveira Mann et al. (2019)).

[0217] Non-human STING proteins can be, but are not limited to, STING proteins from the following species: Tasmanian devil (Sarcophilus harrisii; SEQ ID NO:349), marmoset (Callithrix jacchus; SEQ ID NO:359), cattle (Bos taurus; SEQ ID NO:360), cat (Felis catus; SEQ ID NO:356), ostrich (Struthio camelus australis; SEQ ID NO:361), crested ibis (Nipponia nippon; SEQ ID NO:362), coelacanth (Latimeria chalumnae; SEQ ID NO: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), ghost shark (Callorhinchus milii; SEQ ID NO:367). NO:368), and mouse (Mus musculus; SEQ ID NO:369). These vertebrate STING proteins readily activate immune signaling in human cells, suggesting that the molecular mechanism of STING signaling is common in vertebrates (see de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175).

[0218] This article demonstrates that by infecting bone marrow cells, such as tumor-resident and tissue-resident macrophages, and maintaining their viability for at least a limited time, and / or delivering plasmids (encoding therapeutic products and / or other immunostimulatory products that lead to type I IFN expression, e.g., gain-of-function (GOF) variants that do not require cytoplasmic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides to enable type I IFN expression), the immunostimulatory bacteria provided herein can convert macrophages with an M2 phenotype into M1 or M1-like macrophages, as well as macrophages with reduced or eliminated immunosuppressive properties and enhanced or increased immunostimulatory, antitumor, or antiviral properties. Immunostimulatory bacteria containing plasmids encoding therapeutic products are provided, wherein infection of macrophages, including human macrophages, by these bacteria converts M2 macrophages into M1 or M1-like macrophages. Immunostimulatory bacteria containing plasmids encoding therapeutic products are provided, wherein expression of the therapeutic products in macrophages leads to the conversion of M2 macrophages, such as human M2 macrophages, into M1 or M1-like phenotypes. Immunostimulatory bacteria possessing this property include any bacteria described herein that contain genomic modifications that lead to tumor-resident (in subjects with cancer) and tissue-resident myeloid cell infection. These genomic modifications include those that cause bacteria to lack flagella, where wild-type bacteria do have flagella, and other modifications, such as those that cause bacteria to be pagP. - / msbB - Those modifications. Other modifications include those that lead to the elimination of asparaginase activity, such as those that produce ansB in bacteria that infect bone marrow cells. - Bacterial modifications enhance T cell activity, as do other modifications that alter lipopolysaccharide (LPS).

[0219] This includes immunostimulatory bacteria encoding therapeutic products in macrophages that promote or cause the conversion of M2 macrophages, or convert M2 macrophages into M1 or M1-like phenotypes. Example therapeutic products are those that are part of a cytoplasmic DNA / RNA sensor pathway that leads to the expression, particularly constitutive expression, of type I interferon (IFN). This includes gain-of-function (GOF) variants of the therapeutic product that are part of the cytoplasmic DNA / RNA sensor pathway and do not require cytoplasmic nucleic acids, nucleotides, dinucleotides, or cyclic dinucleotides to lead to type I IFN expression, such as variants described and provided herein and nonhuman STING proteins. Immunostimulatory bacteria include any bacteria that can be modified as described herein, including species listed herein, such as Salmonella species and strains.

[0220] Immunostimulating bacteria are also provided, wherein the encoded therapeutic product, such as a protein, is linked to a portion conferring improved pharmacological properties, such as pharmacokinetic or pharmacodynamic properties, such as an increased serum half-life. Thus, immunostimulating bacteria are provided, wherein the encoded therapeutic product comprises an Fc domain or a half-life-extending portion, such as human serum albumin or a portion thereof. Half-life extension methods or approaches include, for example, PEGylation, glycosylation, sialylation, PASylation (modification with polymers of PAS amino acids of approximately 100-200 residues in length), ELPylation (see, for example, Floss et al. (2010) Trends Biotechnol. 28(1):37-45), HAPylation (modification with glycine homopolymers), fusion with human serum albumin, fusion with GLK, fusion with CTP, fusion with GLP, fusion with the constant fragment (Fc) domain of human immunoglobulin (IgG), fusion with transferrin, and fusion with unstructured peptides, such as XTEN (also known as rPEG, which is a gene fusion containing an inaccurate repeating peptide sequence of A, E, G, P, S, and T; see, for example, Schellenberger et al.). al. (2009) Nat. Biotechnol. 27(12): 1186-1190), as well as other such modifications and fusions such as increasing size, increasing hydrodynamic radius, changing charge or targeting receptors for recycling rather than clearance, and combinations of such modifications.

[0221] It also provides immunostimulatory bacteria, wherein the encoded therapeutic product contains a B7 protein transmembrane domain, or wherein the therapeutic product is GPI-anchored via an endogenous or added GPI anchor. The encoded therapeutic product may contain a fusion with collagen.

[0222] The immunostimulatory bacteria in any and all implementations can be any suitable species. When specific genes and gene modifications are mentioned, they are those corresponding to those mentioned in *Salmonella*, used as an example species. Species and strains include, for example, strains of the following species: *Rickettsia*, *Klebsiella*, *Bordetella*, *Neisseria*, *Aeromonas*, *Francisella*, *Corynebacterium*, *Citrobacter*, *Chlamydia*, and *Haemophilus*. (Haemophilus), Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix.Examples include Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, and Citrobacter freundii. freundii), Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, or Agrobacterium tumerfacium.

[0223] The modifications described herein can attenuate bacteria, or render them less toxic or non-toxic. Example bacteria are Salmonella species, such as *Salmonella typhimurium* strains. The immunostimulatory bacteria provided herein include those that endogenously encode and express, or are modified to encode and express, genes encoding complement killing resistance (rck) genes, such as the *Salmonella* rck gene. Therapeutic *Escherichia coli* is modified to encode rck, thereby allowing for systemic administration. As described herein and in the claims, delivery vectors, cells, pharmaceutical compositions, methods, uses, and treatments for treating cancer, particularly human cancer, are also provided. Methods for companion diagnostics and selection of treatment subjects, as well as methods for monitoring treatment, are also provided. These are described below and in the claims, all of which are incorporated herein by reference. Attached Figure Description

[0224]

[0225] Figure 1 depicts a comparison of the STING protein in wild-type human (SEQ ID NO:306) and Tasmanian devil (SEQ ID NO:349).

[0226] Figure 2 depicts a comparison of the STING protein in wild-type humans (SEQ ID NO:306) and marmosets (SEQ ID NO:359).

[0227] Figure 3 depicts a comparison of STING proteins in wild-type human (SEQ ID NO:306) and bovine (SEQ ID NO:360).

[0228] Figure 4 depicts a comparison of the STING protein in wild-type humans (SEQ ID NO:306) and cats (SEQ ID NO:356).

[0229] Figure 5 depicts a comparison of the STING protein in wild-type human (SEQ ID NO:306) and ostrich (SEQ ID NO:361).

[0230] Figure 6 depicts a comparison of the STING protein between wild-type human (SEQ ID NO:306) and crested ibis (SEQ ID NO:362).

[0231] Figure 7 depicts a comparison of STING proteins in wild-type human (SEQ ID NO:306) and coelacanth (SEQ ID NO:345).

[0232] Figure 8 depicts a comparison of STING proteins in wild-type human (SEQ ID NO:306) and zebrafish (SEQ ID NO:348).

[0233] Figure 9 depicts a comparison of STING proteins in wild-type humans (SEQ ID NO:305) and wild boars (SEQ ID NO:365).

[0234] Figure 10 depicts a comparison of the STING protein in wild-type humans (SEQ ID NO:305) and bats (SEQ ID NO:366).

[0235] Figure 11 depicts a comparison of the STING protein in wild-type human (SEQ ID NO:305) and manatee (SEQ ID NO:367).

[0236] Figure 12 depicts a comparison of the STING protein in wild-type human (SEQ ID NO:305) and ghost shark (SEQ ID NO:368).

[0237] Figure 13 depicts a comparison of STING protein in wild-type human (SEQ ID NO:305) and mouse (SEQ ID NO:369).

[0238] Figure 14 depicts an exemplary construct containing an asd expression cassette, including a bacterial promoter and any other bacterial regulatory sequences positioned opposite to the cassette encoding the payload under eukaryotic promoter control and including bacterial terminators flanking the nucleic acids encoding the payload, and in the direction of terminating any readthrough transcripts from a prokaryotic promoter. Detailed Implementation

[0239]

[0240] Outline

[0241] A. Definition

[0242] Overview of immune-stimulating bacteria used in cancer treatment

[0243] 1. Bacterial cancer immunotherapy

[0244] 2. Existing therapies targeting the tumor microenvironment

[0245] a. Limitations of autologous T-cell therapy

[0246] b. Virus vaccine platform

[0247] c. Bacterial cancer therapy

[0248] i. Listeria

[0249] ii. Salmonella

[0250] iii.VNP20009

[0251] iv. Wild-type strain

[0252] 3. Limitations of existing bacterial cancer immunotherapies

[0253] C. Modification and enhancement of immune-stimulating bacteria to increase the therapeutic index and increase the accumulation of tumor-resident bone marrow cells.

[0254] 1. Gene deletions in the LPS biosynthetic pathway

[0255] a.msbB is missing.

[0256] b. PagP deficiency

[0257] 2. Nutritional Deficiency Type

[0258] a.purI missing / damaged

[0259] b. Adenosine deficiency

[0260] 3. Plasmid maintenance and delivery

[0261] a.asd missing

[0262] b.endA is missing / damaged

[0263] 4. Flagellin knockout strains

[0264] 5. Engineered bacteria to promote adaptive immunity and enhance T cell function

[0265] L-asparaginase II (ansB) deficiency / disruption

[0266] 6. Deletion / damage in Salmonella genes required for expression of curly pili

[0267] 7. Improved resistance to complement

[0268] Rck expression

[0269] 8. Deletion of genes required for lipoprotein expression in Salmonella and other Gram-negative bacteria.

[0270] 9. A potent immunostimulatory bacterium whose genome is modified to be optimized for anti-tumor therapy and encodes therapeutic products, including a variety of therapeutic products.

[0271] 10. M2 phenotype macrophages transform into M1 and M1-like phenotype macrophages.

[0272] D. Immunostimulatory bacteria with an enhanced therapeutic index that encode genetic payloads that stimulate immune responses in the tumor microenvironment.

[0273] 1. Immune-stimulating proteins

[0274] a. Cytokines and chemokines

[0275] b. Co-stimulatory molecules

[0276] 2. Activate prodrug molecules

[0277] 3. Constitutive active proteins that stimulate immune responses and / or type I IFN, non-human STING proteins, chimeric and modified forms.

[0278] a. Constitutive STING expression and function gain mutations

[0279] b. Constitutive IRF3 expression and function gain mutations

[0280] c. Non-human STING proteins and their variants with enhancing or constitutive activity, as well as STING chimeras and their variants with enhancing or constitutive activity.

[0281] d. Other gene products and their constitutive variants that serve as cytoplasmic DNA / RNA sensors

[0282] i.RIG-I

[0283] ii.MDA5 / IFIH1

[0284] iii.IRF7

[0285] e. Other type I IFN regulatory proteins

[0286] 4. Antibodies and antibody fragments

[0287] a.TGF-β

[0288] b. Bispecific scFvs and T cell adaptor proteins

[0289] c. Anti-PD-1 / anti-PD-L1 antibody

[0290] d. Anti-CTLA-4 antibody

[0291] e. Other examples of checkpoint targets

[0292] 5. Combinations of immunomodulatory proteins can have synergistic and / or complementary effects.

[0293] 6. Immunostimulating bacteria for delivering combination therapy

[0294] E. Constructing example plasmids encoding therapeutic products for bacterial delivery

[0295] 1. Constitutive promoters for heterologous protein expression

[0296] 2. Multiple therapeutic product expression kits

[0297] a. Single promoter construct

[0298] b. Dual / Multiple Promoter Builders

[0299] 3. Adjustment element

[0300] a. Post-transcriptional regulatory elements

[0301] b. Polyadenylation signal sequence and terminator

[0302] c. Enhancer

[0303] d. Secretion signals

[0304] e. Improve bacterial adaptability

[0305] 4. Origin of replication and plasmid copy number

[0306] 5. CpG motifs and CpG islands

[0307] 6. Plasmid maintenance / selection components

[0308] 7. DNA nuclear targeting sequence

[0309] F. Pharmaceutical preparation, compositions and formulations

[0310] 1. Preparation

[0311] a. Cell bank preparation

[0312] b. Preparation of active pharmaceutical ingredient (API)

[0313] c. Pharmaceutical product preparation

[0314] 2. Composition

[0315] 3. Formulation

[0316] a. Fluids, injections, emulsions

[0317] b. Dry, heat-stable preparations

[0318] 4. Compositions for use via other routes of administration

[0319] 5. Dosage and administration

[0320] 6. Packaging and product preparation

[0321] G. Treatment methods and uses

[0322] 1. Selecting patients for treatment and monitoring of the diagnosis.

[0323] a. Patient selection

[0324] b. Diagnostic assessment or detection of immune-stimulating bacterial activity indicates the effectiveness of treatment.

[0325] 2. Tumor

[0326] 3. Application

[0327] 4. Monitoring

[0328] H. Example

[0329] A. Definition

[0330] Unless otherwise defined, 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 pertains. Unless otherwise stated, all patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other publicly available material referred to throughout this disclosure are incorporated herein by reference in their entirety. Where terms herein have multiple definitions, the definition set forth in this section shall prevail. Where URLs or other such identifiers or addresses are referenced, it should be understood that such identifiers are changeable and specific information on the Internet may change, but equivalent information can be found by searching the Internet. References to such URLs confirm the availability and public dissemination of this information.

[0331] As used in this article, "therapeutic bacteria" refers to bacteria that, when administered to a subject such as a human, achieve therapeutic effects such as anticancer or antitumor therapy.

[0332] As used herein, “immunostimulatory bacteria” are therapeutic bacteria that, when introduced into a subject, accumulate in immune-exempt tissues and cells, such as tumors, tumor microenvironments, and tumor-resident immune cells, and replicate and / or express immunostimulatory or immunostimulatory products. For example, because immunostimulatory bacteria cannot replicate and / or express products outside of immune-exempt environments (or have reduced replication / product expression), they are attenuated in the host due to reduced toxicity or pathogenicity and / or due to reduced toxicity or pathogenicity of the encoded products. The immunostimulatory bacteria described herein are modified to encode one or more products or exhibit traits or properties that make them immunostimulatory. Such products, properties, and characteristics include, but are not limited to, at least one of the following: immunostimulatory proteins, such as cytokines, chemokines, or costimulatory molecules; cytoplasmic DNA / RNA sensors or gain-of-function or constitutive active variants thereof (e.g., STING, IRF3, IRF7, MDA5, RIG-I); RNAi, such as siRNA (shRNA and microRNA), CRISPR, which target, disrupt, or inhibit immune checkpoint genes such as TREX1, PD-1, CTLA-4, and / or PD-L1; antibodies and fragments thereof, such as anti-immune checkpoint antibodies, anti-IL-6 antibodies, anti-VEGF antibodies, or TGF-β inhibitory antibodies; and other antibody constructs, such as bispecific T cell adaptor proteins. Soluble TGF-β receptors, which act as decoys to bind TGF-β or TGF-β antagonistic peptides; and IL-6-binding decoy receptors. Immunostimulatory bacteria may also include modifications that render the bacteria auxotrophic to immunosuppressive metabolites or metabolites in immunosuppressive pathways, such as adenosine.

[0333] As used herein, the strain name VNP20009 (see, for example, International PCT Application Publication No. WO 99 / 13053, and also US Patent No. 6,863,894) and YS1646 and 41.2.9 are used interchangeably and refer to the strain deposited at the American Center for Type Culture Collection (ATCC) with accession number 202165. VNP20009 is a modified attenuated strain of Salmonella typhimurium containing deletions of msbB and purI, produced by the wild-type strain ATCC#14028.

[0334] As used herein, the strain names YS1456 and 8.7 are used interchangeably and refer to the strain deposited at the American Center for Type Culture Collection (ATCC) with accession number 202164 (see US Patent No. 6,863,894).

[0335] As used in this article, mentioning that bacteria are “derived from” a specific strain means that such a strain can be used as starting material and can be modified to produce a specific bacterium.

[0336] As used herein, an "expression cassette" refers to a nucleic acid construct comprising a regulatory sequence for gene expression, operatively linked to a nucleic acid encoding an open reading frame (ORF) that encodes a payload, such as a therapeutic product or other protein.

[0337] As used herein, 2A peptides are viral oligopeptides of 18 to 22 amino acids (aa) in length that mediate polypeptide cleavage during translation in eukaryotic cells. The name "2A" refers to a specific region of the viral genome, and different viral 2A peptides are usually named after the virus 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). See, for example, Liuet al. (2017) Scientific Reports 7:2193, Figure 1, coding sequence. Also see SEQ ID NO:327-330. These peptides typically share the core sequence motif of DxExNPGP and are present in a large number of viral families. They help break down multiproteins by preventing ribosomes from forming peptide bonds. 2A peptides are provided to polycistronic vectors, in which multiple proteins are expressed from a single open reading frame (ORF). For the purposes of this document, 2A peptides include those naturally occurring peptides and any modified forms thereof, such as any peptide having 97%, 98%, or 99% sequence identity with any naturally occurring 2A peptide, including those disclosed herein, which result in transcription and translation of a single polypeptide from a transcript containing multiple (two or more) open reading frames.

[0338] As used herein, interferon disorders are conditions associated with the upregulation of interferon due to mutations in gene products involved in pathways that regulate or induce interferon expression. The activity of these products is typically regulated by mediators such as cytoplasmic DNA, RNA, or nucleotides; when the protein product is mutated, the activity is constitutive. Type I interferon disorders include a range of conditions, including a severe form of Aicardi-Goutières syndrome (AGS) and a milder form of familial chilblain lupus (FCL). Nucleic acid molecules encoding mutant products with these properties can be generated in vitro, for example by selecting mutations that produce gain-of-function products compared to alleles with normal activity, or further gain-of-function products compared to gain-of-function mutants associated with the diseases described herein.

[0339] As used herein, a “gain-of-function mutation” is a mutation that increases the activity of a protein compared to the same protein without the mutation. For example, if the protein is a receptor, its affinity for a ligand is increased; if it is an enzyme, it will have increased activity, including constitutive activity.

[0340] As used in this article, an "origin of replication" is the DNA sequence in a chromosome, plasmid, or virus where replication begins. For small DNA sequences, including bacterial plasmids and small viruses, a single origin of replication is sufficient.

[0341] The origin of replication determines the vector copy number, which depends on the chosen origin of replication. For example, if the expression vector originates from the low copy number plasmid pBR322, its copy number is between approximately 15 and 20 copies per cell, while if it originates from the high copy number plasmid pUC, it can be between 500 and 700 copies per cell.

[0342] As used in this article, a moderate copy number of plasmids in cells is approximately 150 or less, and a low copy number is 15 to 30, for example, 20 or less. Low to moderate copy numbers are less than 150 copies / cell. High copy numbers are greater than 150 copies / cell.

[0343] As used herein, a “CpG motif” is a base pattern comprising an unmethylated central CpG (“p” refers to the phosphodiester bond between consecutive C and G nucleotides) surrounded by at least one base flanking the central CpG (on its 3' and 5' sides). A CpG oligodeoxynucleotide is an oligodeoxynucleotide of at least about 10 nucleotides in length, including an unmethylated CpG. At least one C in the 5'CG 3' is unmethylated.

[0344] As used herein, “RIG-I binding sequence” refers to a direct 5' triphosphate (5'ppp) structure, or a 5' triphosphate (5'ppp) structure synthesized from a poly(dA-dT) sequence by RNA pol III, which can activate type I IFN via the RIG-I pathway through interaction with RIG-I. The RNA comprises at least four A ribonucleotides (AAAA); it may contain 4, 5, 6, 7, 8, 9, 10, or more ribonucleotides. The RIG-I binding sequence is introduced into a bacterial plasmid for transcription into polyA.

[0345] As used in this article, "cytokine" is a broad category of small proteins (approximately 5-20 kDa) that are important in cell signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines are cell signaling molecules that facilitate cell communication in immune responses and stimulate cells to migrate to sites of inflammation, infection, and trauma.

[0346] As used herein, "chemokine" refers to a chemically induced (chemokinetic) cytokine that binds to a chemokine receptor, including proteins isolated from natural sources and those synthesized, such as through recombinant methods or chemical synthesis. Examples of chemokines include, but are not limited to, IL-8, IL-10, GCP-2, GRO-α, GRO-β, GRO-γ, ENA-78, PBP, CTAPIII, 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), M... IP-1γ (CCL9), MIP-2, MIP-2α, MIP-3α, MIP-3β, MIP-4, MIP-5, MDC, HCC-1, ALP, lungkine, Tim-1, eosinophil activation chemokine-1, eosinophil activation chemokine-2, I-309, SCYA17, TRAC, RANTES (CCL5), DC-CK-1, lymphocyte chemokines, and fractal chemokines, as well as other chemokines known to those skilled in the art. Chemokines are involved in the migration of immune cells to sites of inflammation, as well as in the maturation of immune cells and the generation of adaptive immune responses.

[0347] As used herein, “immunostimulatory proteins” are proteins that present or promote antitumor immune responses in the tumor microenvironment. Examples of such proteins include cytokines, chemokines, and co-stimulatory 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), the IL-15 / IL-15Rα chain complex, IL-36γ, IL-2 with weakened binding to IL-2Ra, IL-2 modified to not bind to IL-2Ra, CXCL9, and CXCL10 (I P-10), CXCL11, CCL3, CCL4, CCL5, molecules involved in potential T cell recruitment / persistence, CD40, CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), 4-1BBL with a missing cytoplasmic domain (4-1BBLΔcyt) or 4-1BBL with a partially missing (truncated) cytoplasmic domain, members of the B7-CD28 family, and members of the tumor necrosis factor receptor (TNFR) superfamily.

[0348] The immunostimulatory proteins are truncated costimulatory molecules, such as 4-1BBL, CD80, CD86, CD27L, B7RP1, and OX40L, each with a complete or partial deletion of a cytoplasmic domain expressed on antigen-presenting cells (APCs). These truncated gene products, such as those lacking or partially lacking the cytoplasmic domain, can participate in constitutive immunostimulatory signals to T cells via costimulatory receptors, but cannot emit counterregulatory signals to APCs due to the truncated or missing cytoplasmic domains.

[0349] As used herein, “cytoplasmic domain deletion” is the deletion of all or part of the amino acid residues of a protein’s cytoplasmic or intracellular domains, wherein the deletion is sufficient to participate in constitutive immunostimulatory signaling to T cells via co-stimulatory receptors and sufficient to inhibit counterregulatory signaling to APCs. For example, the cytoplasmic domain of human 4-1BBL (also known as TNFSF9) contains amino acid residues 1 to 28 of SEQ ID NO:342. The cytoplasmic domain of human CD80 contains amino acid residues 264 to 288 of the protein; the cytoplasmic domain of human CD86 contains amino acid residues 269 to 329 of the protein; the cytoplasmic domain of human CD27L (also known as CD70) contains amino acid residues 1 to 17 of the protein; the cytoplasmic domain of human B7RP1 (also known as ICOSLG or ICOS ligand) contains amino acid residues 278 to 302 of the protein; and the cytoplasmic domain of human OX40L (also known as TNFSF4 or CD252) contains amino acid residues 1 to 23 of the protein.

[0350] As used in this article, a "decoy receptor" is a receptor that can effectively and specifically bind to a particular growth factor or cytokine, but is structurally unable to signal or activate the intended receptor complex. Decoy receptors exert their inhibitory effect by binding to a ligand and preventing it from binding to its homologous receptor.

[0351] For example, TGF-β family receptors include cell surface serine / threonine kinase receptors type I (TβRI or TGFβR1) and type II (TβRII or TGFβR2), which form heteropolymeric complexes in the presence of dimerizing ligands, and type III receptors including β-glycans (TβRIII or TGFβR3). Soluble decoy receptors for TGF-β that prevent TGF-β from binding to its receptor include soluble extracellular domains (TGF-β binding regions) of TβRI, TβRII, or TβRIII (β-glycans), which can fuse with other molecules such as Fc domains. Furthermore, BAMBI (a bone morphogenetic protein (BMP) and activin membrane-binding inhibitor) is structurally associated with type I receptors and acts as a decoy to inhibit receptor activation. Dominant-negative TGFβR2 (DN-TGFβR2), which contains the extracellular domain and transmembrane region of TGFβR2 but lacks the cytoplasmic domain required for signal transduction, can also be used as a TGF-β decoy receptor (see, for example, International Application Publication No. WO 2018 / 138003).

[0352] As used herein, a co-stimulatory molecule agonist is a molecule that activates or increases the activity of a co-stimulatory molecule upon binding. For example, the agonist may be an agonist antibody. CD40 agonist antibodies include, for example, CP-870,893, dacetuzumab, ADC-1013 (mitazalimab), and Chi Lob 7 / 4.

[0353] As used herein, the cytoplasmic DNA / RNA sensor pathway is a pathway initiated by the presence of DNA, RNA, nucleotides, dinucleotides, cyclic nucleotides and / or cyclic dinucleotides or other nucleic acid molecules, leading to the production of type I interferon. The nucleic acid molecules in the cytoplasm originate from viruses or bacteria, or from radiation or other such exposures, resulting in the activation of the host's immune response.

[0354] As used in this article, “type I interferon pathway protein” is a protein that induces innate immune responses, such as inducing type I interferon.

[0355] As used herein, a “cytoplasmic DNA / RNA sensor” is a protein that is part of the cytoplasmic DNA / RNA sensor pathway that leads to the expression of immune response mediators such as type I interferon. The “cytoplasmic DNA / RNA sensor” includes proteins in the type I interferon pathway. For example, as described herein and known to those skilled in the art, cytoplasmic DNA is sensed by cGAS, leading to the production of cGAMP and subsequent STING / TBK1 / IRF3 signaling and type I interferon production. Bacterial cyclic dinucleotides (such as bacterial cyclic di-AMP) also activate STING. Thus, STING is an immunostimulatory protein that induces type I interferon. 5'-triphosphate RNA and double-stranded RNA are sensed by RIG-I and MDA-5 or MDA-5 / LGP2 alone. This leads to the polymerization of mitochondrial MAVS (mitochondrial antiviral signaling protein) and also activates TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Proteins in this pathway are immunostimulatory and lead to the expression of innate immune response mediators such as type I interferon. It can modify immunostimulatory proteins in the DNA / RNA sensor pathway, giving them increased activity or constitutive effects in the absence of cytoplasmic nucleic acids, thereby leading to immune responses, such as the expression of type I interferon.

[0356] As used herein, the "C-terminal tail" or "C-terminal tail" (CTT) of the innate immune protein STING refers to the C-terminal portion of the STING protein, which in wild-type STING is linked to the cGAMP-binding domain via a flexible linker region. The CTT includes an IRF3 binding site, a TBK1 binding site, and a TRAF6 binding site. STING promotes the induction of interferon-β (IFN-β) production by phosphorylating the CTT of the STING protein through TANK-binding kinase 1 (TBK1). The interaction between STING and TBK1 is mediated by an evolutionarily conserved 8-amino acid residue segment in the STING CTT. TRAF6 catalyzes the formation of a K63-linked pervasive protein chain on STING, leading to activation of the transcription factor NF-κB and induction of another STING-dependent gene expression program. Deletion or disruption of the TRAF6 binding site in the CTT reduces the activation of NF-κB signaling. Replacing human STING CTT (or a portion thereof) with a CTT (or a corresponding portion thereof) from a STING protein from a species with low NF-κB activation reduces the NF-κB activation of the resulting modified human STING protein. The STING CTT is an unstructured sequence of approximately 40 amino acids containing the sequence motif required for STING phosphorylation and IRF3 recruitment (see de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175). Human STING residue S366 has been identified as a major TBK1 phosphorylation site, part of the LxIS motif common to innate immune aptamer proteins that activate interferon signaling (see de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175). The human STING CTT contains a second PxPLR motif, which includes the residue L374 required for TBK1 binding; the LxIS and PxPLR sequences are conserved in vertebrate STING alleles (see de Oliveira Mann et al. (2019) Cell Reports 27:1165-1175). Example STING CTT sequences and IRF3, TBK1, and TRAF6 binding sites are listed in the table below:

[0357]

[0358]

[0359] As used herein, a “STING pathway agonist” is any product that increases type I interferon (IFN) expression by activating the STING pathway. Examples of such agonists are the gain-of-function STING peptide variants provided herein, as well as gain-of-function variants of other cytoplasmic DNA / RNA sensor and type I IFN pathway proteins, such as variants of IRF-3, IRF-7, MDA5, and RIG-I, which increase or constitutively express type I IFN via the STING pathway.

[0360] As used herein, bacteria modified to "induce less cell death in tumor-resident immune cells" or "induce less cell death in immune cells" are bacteria that are less virulent than unmodified bacteria, or bacteria with reduced virulence compared to unmodified bacteria. Examples of such modifications are those that eliminate pyroptosis in phagocytes and alter the lipopolysaccharide (LPS) profile on bacteria. These modifications include disruption or deletion of flagellin genes, pagP, one or more components of the SPI-1 pathway such as hilA, rod proteins (e.g., pragJ), needle proteins (e.g., pragI), and QseC.

[0361] As used herein, bacteria that are "modified to preferentially infect tumor-resident immune cells" or "modified to preferentially infect immune cells" have modifications in their genome that reduce their ability to infect cells other than immune cells. Examples of such modifications are modifications that disrupt type 3 or type 4 secretion systems or other genes or systems that affect the bacteria's ability to invade non-immune cells. For example, modifications include the disruption / deletion of the SPI-1 component, which is required for Salmonella to infect cells such as epithelial cells, but does not affect Salmonella's infection of immune cells such as phagocytes.

[0362] As used herein, “modification” refers to the modification of the amino acid sequence of a polypeptide or the nucleotide sequence of a nucleic acid molecule, including the deletion, insertion, and substitution of amino acids or nucleotides, respectively. Methods for modifying polypeptides are routine to those skilled in the art, for example, through the use of recombinant DNA methods.

[0363] As used in this article, modifications to bacterial genomes, plasmids, or genes include deletions, substitutions, and insertions of nucleic acids.

[0364] As used in this article, RNA interference (RNAi) is a biological process in which RNA molecules suppress gene expression or translation by neutralizing target mRNA molecules to inhibit translation and thereby suppress the expression of the target gene.

[0365] As used in this article, the RNA molecules that function via RNAi refer to those that silence or repress the expression of target genes. Silencing expression means that the expression of the target gene is reduced, blocked, or suppressed.

[0366] As used in this article, gene silencing via RNAi is referred to as repression, inhibition, disruption, or silencing of the expression of a target gene. The target gene contains a nucleotide sequence that corresponds to a sequence in the repressive RNA, thereby silencing the expression of the target mRNA.

[0367] As used herein, inhibition, repression, disruption, or silencing of a target gene refers to altering the expression of the target gene, such as the translation process, thereby reducing the activity or expression of the product encoded by the target gene. Reduction includes complete or partial knockout, thereby achieving therapeutic effects with reference to the immunostimulatory bacteria described and applied herein.

[0368] As used in this article, small interfering RNA (siRNA) is a small, double-stranded (ds) RNA fragment, typically about 21 nucleotides in length, with a 3' overhang (2 nucleotides) at each end. It "interferes" with protein translation by binding at specific sequences and promoting the degradation of messenger RNA (mRNA). In doing so, siRNA prevents the production of specific proteins based on the nucleotide sequence of its corresponding mRNA. This process is called RNA interference (RNAi), also known as siRNA silencing or siRNA knockdown.

[0369] As used in this article, short hairpin RNA or small hairpin RNA (shRNA) is an artificial RNA molecule with tight hairpin turns that can be used to silence target gene expression via RNA interference (RNAi). shRNA expression in cells is typically accomplished through plasmid delivery or via viral or bacterial vectors.

[0370] As used in this article, the tumor microenvironment (TME) is the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). Presence of tumors includes, but is not limited to, increased angiogenesis, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure, and metabolite or metabolic alterations, such as elevated adenosine levels.

[0371] As used in this article, "bactofection" refers to the transfer of bacterial-mediated gene or plasmid DNA into eukaryotic cells, such as mammalian cells.

[0372] As used herein, human type I interferon (IFN) is a subgroup of interferon proteins that regulate the activity of the immune system. All type I IFNs bind to specific cell surface receptor complexes, such as the IFN-α receptor. Type I interferons include IFN-α and IFN-β, among others. Bone marrow cells are the primary producers of IFN-α and IFN-β, possessing antiviral activity that is primarily involved in the innate immune response. The two types of IFN-β are IFN-β1 (IFNB1) and IFN-β3 (IFNB3).

[0373] As used in this article, M1 and M2 macrophage phenotypes refer to the two main categories of macrophage phenotypes: M1 (classical activated macrophages) and M2 (alternative activated macrophages). M1 macrophages secrete pro-inflammatory cytokines and chemokines, and present antigens, thereby participating in the positive immune response and acting as immune surveillance. Their main pro-inflammatory cytokines are IL-6, IL-12, and TNF-α. M2 macrophages mainly secrete anti-inflammatory cytokines such as arginase-I, IL-10, and TGF-β, which have functions of reducing inflammation, promoting tumor growth, and immunosuppression. Macrophages with an M1-like phenotype secrete pro-inflammatory cytokines but do not possess the immunosuppressive activity of M2 macrophages. Converting M2 macrophages into macrophages with an M1 or M1-like phenotype will transform them into macrophages that do not have immunosuppressive activity but participate in anti-tumor responses. M2 macrophages that transform into macrophages with an M1 or M1-like phenotype exhibit increased levels of pro-inflammatory cytokines / chemokines and receptors, such as CD80 and CCR7, as well as chemokines, such as IFNγ and CXCL10. M1 phenotype markers include, but are not limited to, one or more of CD80, CD86, CD64, CD16, and CD32. Nitric oxide synthase (iNOS) expression in M1 macrophages can also serve as a phenotype marker. CD163 and CD206 are major markers for identifying M2 macrophages. Other surface markers for M2-type cells include CD68. A decrease or elimination of any M2 marker, and an increase in cytokines / chemokines indicative of M1 macrophages, reflects a transition from the M2 phenotype to the M1 or M1-like phenotype. The following sections, along with examples of work on M2-to-M1-like or M1-like phenotype transitions, describe example cytokine profiles and induced markers.

[0374] As used herein, the expression of a nucleic acid or its encoded RNA targeting gene refers to the suppression, repression, or silencing of gene expression through any mechanism. Typically, such a nucleic acid includes at least a portion complementary to the target gene, wherein this portion is sufficient to form a hybrid with the complementary portion.

[0375] As used in this article, “deletion” when referring to a nucleic acid or polypeptide sequence means the absence of one or more nucleotides or amino acids compared to a sequence such as a target polynucleotide or polypeptide or a natural or wild-type sequence.

[0376] As used herein, “insertion” when referring to a nucleic acid or amino acid sequence describes the inclusion of one or more additional nucleotides or amino acids within the target, native, wild-type, or other relevant sequence. Therefore, a nucleic acid molecule containing one or more inserts has one or more additional nucleotides within the linear length of the sequence compared to a wild-type sequence.

[0377] As used in this article, “addition” to nucleic acid and amino acid sequences describes the addition of a nucleotide or amino acid to either end compared to another sequence.

[0378] As used herein, “substitution” or “replacement” refers to the substitution of one or more nucleotides or amino acids in a natural, target, wild-type, or other nucleic acid or polypeptide sequence with an optional nucleotide or amino acid, without altering the length of the molecule (as described by the number of nucleotides or residues). Therefore, one or more substitutions in a molecule do not change the number of nucleotide or amino acid residues in the molecule. Amino acid substitutions compared to a specific polypeptide can be expressed based on the number of amino acid residues along the length of the polypeptide sequence.

[0379] As used herein, the expressions “at the position corresponding to…” or “the nucleotide or amino acid position “corresponds to” the nucleotide or amino acid position in the published sequence as described in the sequence listing refer to the nucleotide or amino acid position identified when aligned with the published sequence using a standard alignment algorithm such as the GAP algorithm to maximize similarity. By aligning the sequences, those skilled in the art can, for example, use conserved and identical amino acid residues as guidance to identify the corresponding residues. Typically, to identify the corresponding position, the amino acid sequence is aligned to obtain the highest-order match (see, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAM J. Applied Math 48:1073).

[0380] As used herein, sequence alignment refers to the alignment of two or more nucleotide or amino acid sequences using homology. Typically, two or more sequences with 50% or more similarity are aligned. An aligned sequence set refers to two or more sequences aligned at the corresponding position and may include alignment sequences derived from RNA (e.g., ESTs and other cDNAs) aligned to genomic DNA sequences. Related or variant polypeptide or nucleic acid molecules can be aligned using any method known to those skilled in the art. This method generally maximizes matching and includes, for example, manual alignment as well as methods using many available alignment programs (e.g., BLASTP), and other methods known to those skilled in the art. By aligning polypeptide or nucleic acid sequences, those skilled in the art can use conserved and identical amino acid residues as a guide to identify similar portions or positions. Furthermore, those skilled in the art can use conserved amino acid or nucleotide residues as a guide to find corresponding amino acid or nucleotide residues between and within human and non-human sequences. Corresponding positions can also be based on structural alignment, for example, by using computer-simulated protein structure alignment. In other cases, corresponding regions can be identified. Those skilled in the art can also use conserved amino acid residues as a guide to find corresponding amino acid residues between and within human and non-human sequences.

[0381] As used herein, the “property” of a polypeptide, such as an antibody, refers to any property exhibited by the polypeptide, including but not limited to binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, heat resistance, and tolerance to pH conditions. Changes in properties can alter the “activity” of a polypeptide. For example, changes in the binding specificity of an antibody polypeptide can alter the polypeptide’s ability to bind to antigens and / or various binding activities, such as affinity or affinity, or in vivo activity.

[0382] As used herein, the “activity” or “functional activity” of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. These activities can be determined empirically. Example activities include, but are not limited to, the ability to interact with biomolecules, such as through antigen binding, DNA binding, ligand binding, or dimerization, or enzymatic activity such as kinase activity or proteolytic activity. For antibodies (including antibody fragments), the activity includes, but is not limited to, the ability to specifically bind to a particular antigen, the affinity of antigen binding (e.g., high or low affinity), the affinity of antigen binding (e.g., high or low affinity), the binding rate (on-rate), the dissociation rate (off-rate), effector functions such as the ability to promote antigen neutralization or clearance, viral neutralization, and in vivo activities such as the ability to prevent pathogen infection or invasion, or promote clearance, or penetrate specific tissues or fluids or cells in the body. Activity can be assessed in vitro or in vivo using recognized assays such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays to measure binding or dissociation rates, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, and binding assays (e.g., panning assays).

[0383] As used herein, “binding,” “bound,” or any grammatical variation thereof, refers to any attractive interaction between a molecule and another molecule that results in a stable association of the two molecules in close proximity. Binding includes, but is not limited to, non-covalent binding, covalent binding (e.g., reversible and irreversible covalent binding), and interactions between molecules such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules such as drugs.

[0384] As used herein, “antibody” refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetic, such as recombinant, including any fragment containing at least a portion of the variable heavy chain and light chain regions of an immunoglobulin molecule, sufficient to form an antigen-binding site and sufficient to specifically bind an antigen upon assembly. Therefore, antibodies include any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). For example, an antibody is defined as an antibody containing two heavy chains (which may be denoted as H and H') and two light chains (which may be denoted as L and L'), wherein each heavy chain may be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen-binding site (e.g., the heavy chain includes, but is not limited to, V). H Chain, V H -C H 1 chain and V H -C H 1-C H 2-C H 3 light chains), each light chain can be a full-length light chain or a portion thereof sufficient to form an antigen-binding site (e.g., light chains include, but are not limited to, V).L Chain and V L -C L Each heavy chain (H and H') pairs with a light chain (L and L', respectively). Typically, antibodies consist of at least a variable heavy chain (V). H ) and / or variable light chains (V L Antibodies may include all or at least a portion of a constant region.

[0385] For the purposes of this document, the term antibody includes full-length antibodies and portions thereof, including antibody fragments, such as anti-CTLA-4 antibody fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), and scFv-Fc fragments (where the V in scFv is...). H The domain is attached to an Fc, such as human IgG1 Fc, a single-chain Fab (scFab), a bispecific antibody, an anti-idiotype (anti-Id) antibody, or an antigen-binding fragment of any of the above antibodies. Antibodies also include synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intracellular antibodies. The antibodies described herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or sub-subclass (e.g., IgG2a and IgG2b). Antibodies used for human treatment are typically human antibodies or humanized antibodies.

[0386] As used herein, “antibody fragment” means (i) a monovalent and monospecific antibody derivative containing a variable heavy chain and / or light chain, or a functional fragment of an antibody lacking the Fc moiety; and (ii) (tandem scFv), DART, biantibodies, and single-chain biantibodies (scDb). Therefore, antibody fragments include: Fab, Fab', scFab, scFv, scFv-Fc, Fv fragments, nanobodies (see, for example, antibodies derived from Bactrian camels (Camelus bactriamus), dromedary camels (Camelus dromedarius), or alpacas (Lama pacco)) (see, for example, U.S. Patent No. 5,759,808; and Stijlemans et al. (2004) J. Biol. Chem. 279:1256-1261), V HH dAb (single-domain antibody), minimum recognition unit, single-chain biantibody (scDb) And DART. The antibody fragment has a molecular weight of less than 60 kDa.

[0387] As used herein, “nucleic acid” refers to at least two linked nucleotides or nucleotide derivatives typically linked together by phosphodiester bonds, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term “nucleic acid” also includes analogs of nucleic acids, such as peptide nucleic acids (PNA), phosphorothioated DNA, and other such analogs and derivatives or combinations thereof. Nucleic acids also include DNA and RNA derivatives containing, for example, nucleotide analogs or “backbone” bonds instead of phosphodiester bonds, such as phosphotriester bonds, aminophosphate bonds, thiophosphate bonds, thioester bonds, or peptide bonds (peptide nucleic acids). The term also includes RNA or RNA equivalents, derivatives, variants, and analogs made from nucleotide analogs and single-stranded (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For RNA, the uracil base is uridine.

[0388] As used herein, isolated nucleic acid molecules are nucleic acid molecules separate from other nucleic acid molecules present in the natural sources of nucleic acid molecules. When produced by recombinant technology, “isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium, or when chemically synthesized, may be substantially free of chemical precursors or other chemicals. The isolated nucleic acid molecules provided as examples herein include isolated nucleic acid molecules encoding antibody or antigen-binding fragments provided herein.

[0389] As used herein, “operably linked” in relation to nucleic acid sequences, regions, elements, or domains means that nucleic acid regions are functionally related to each other. It refers to a juxtaposition such that the components described are in a relationship that allows them to function in the intended manner. For example, a promoter is operably linked to a coding sequence if it influences or enables transcription or expression of that sequence. For instance, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, thereby allowing transcription and translation of said nucleic acids to express a functional fusion protein, wherein the leader peptide enables the secretion of the fusion polypeptide. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to a nucleic acid encoding a second polypeptide, and the nucleic acids are transcribed into a single mRNA transcript, but translation of the mRNA transcript can produce one of the two expressed polypeptides. For example, an amber stop codon can be located between the nucleic acids encoding the first polypeptide and the second polypeptide, such that when introduced into partially amber-repressed cells, the resulting single mRNA transcript can be translated to produce a fusion protein containing both the first and second polypeptides, or can be translated to produce only the first polypeptide. In another instance, a promoter can be operatively linked to a nucleic acid encoding a polypeptide, thereby regulating or mediating the transcription of the nucleic acid.

[0390] As used herein, when referring to, for example, synthetic nucleic acid molecules, synthetic genes, or synthetic peptides, "synthetic" means nucleic acid molecules, genes, or polypeptide molecules produced by recombination and / or by chemical synthesis.

[0391] As used in this article, naturally occurring α-amino acid residues are the 20 α-amino acid residues found in nature that are incorporated into proteins through the specific recognition of charged tRNA molecules with their homologous mRNA codons in the human body.

[0392] As used herein, a polypeptide refers to two or more amino acids covalently linked together. The terms "polypeptide" and "protein" are used interchangeably in this document.

[0393] As used in this article, "peptide" refers to a polypeptide with a length of 2 to approximately 40 amino acids.

[0394] As used herein, “amino acid” is an organic compound containing an amino and a carboxylic acid group. A polypeptide contains two or more amino acids. For the purposes of this document, the amino acids contained in the antibodies and immunostimulatory proteins provided include twenty naturally occurring amino acids (see the table below), non-natural amino acids, and amino acid analogs (e.g., amino acids in which the α-carbon has a side chain). As used herein, amino acids in the various amino acid sequences of the polypeptides appearing herein are identified by their well-known three-letter or one-letter abbreviations (see the table below). Nucleotides present in various nucleic acid molecules and fragments are named with standard one-letter names conventionally used in the art.

[0395] As used herein, “amino acid residue” refers to the amino acid formed by the chemical digestion (hydrolysis) of a polypeptide at its peptide bond. The amino acid residues referred to herein are typically in the “L” isomer form. The “D” isomer form residues can be substituted by any L-amino acid residue, provided the polypeptide retains the desired functional properties. NH2 refers to the free amino group present at the amino terminus of the polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of the polypeptide. The abbreviations for amino acid residues are shown in the table below, according to the standard polypeptide nomenclature described in J. Biol. Chem., 243:3557-59 (1968) and accepted under 37 C. FR § § 1.821-1.822:

[0396] Correspondence table

[0397]

[0398]

[0399] In this article, all sequences of amino acid residues represented by the formula are arranged from left to right in the conventional direction from the amino terminus to the carboxyl terminus. The phrase "amino acid residue" is defined as the amino acids included in the above correspondence table, as well as modified, non-natural, and uncommon amino acids. A dash at the beginning or end of an amino acid residue sequence indicates a peptide bond with another sequence of one or more amino acid residues or with an amino-terminal group such as NH2 or a carboxyl-terminal group such as COOH.

[0400] In peptides or proteins, suitable conserved substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that, generally, substitutions of a single amino acid in a non-essential region of a polypeptide do not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0401] This substitution can be performed based on the examples listed in the table below:

[0402] Example of conserved amino acid substitution

[0403]

[0404]

[0405] Other substitutions are also permissible and can be determined empirically or based on other known conservative or non-conservative substitutions.

[0406] As used in this article, “naturally occurring amino acids” refers to the 20 L-amino acids present in polypeptides.

[0407] As used herein, the term "non-natural amino acid" refers to an organic compound having a structure similar to that of a natural amino acid but which has been structurally modified to mimic the structure and reactivity of a natural amino acid. Therefore, non-natural amino acids include, for example, amino acids or amino acid analogs other than the 20 naturally occurring amino acids, including but not limited to D-stereoisomers of amino acids. Examples of non-natural amino acids are known to those skilled in the art, including but not limited to 2-aminohexanoic acid (Aad), 3-aminohexanoic acid (bAad), β-alanine / β-aminopropionic acid (Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid / piperidinic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosin (Des), 2,2'-diaminopimelic acid (Dpm), 2,3-diaminobutyric acid (Dbu), and others. Aminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), allohydroxylysine (Ahyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesin (Ide), alloisoleucine (Aile), N-methylglycine, sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), novovaline (Nva), novoleucine (Nle), and ornithine (Orn).

[0408] As used herein, DNA constructs are single-stranded or double-stranded, linear or circular DNA molecules containing DNA segments arranged and juxtaposed in ways not found in nature. DNA constructs exist as a result of artificial manipulation, including cloning and other copies of the manipulated molecule.

[0409] As used herein, a DNA segment is a portion of a larger DNA molecule that has specific properties. For example, a DNA segment encoding a specific polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5' to 3' direction, encodes the amino acid sequence of the specific polypeptide.

[0410] As used herein, the term polynucleotide refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to 3' ends. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated as "nt") or base pairs (abbreviated as "bp"). Where the context permits, the term nucleotide refers to both single-stranded and double-stranded molecules. When the term is applied to a double-stranded molecule, it is used to denote the entire length and should be understood to be equivalent to the term base pair. Those skilled in the art will recognize that the two strands of a double-stranded polynucleotide may differ slightly in length, and their ends may be staggered; therefore, all nucleotides in a double-stranded polynucleotide molecule may not pair. Such unpaired ends will typically not exceed 20 nucleotides in length.

[0411] As used in this article, production via recombination refers to the expression of proteins encoded by cloned DNA using methods well-known in molecular biology.

[0412] As used herein, a "heterologous nucleic acid" is a nucleic acid that encodes a product (i.e., RNA and / or protein) that is not normally produced in vivo by the cell in which the product is expressed, or a nucleic acid located at a locus in which it is not normally present, or a nucleic acid that mediates or encodes a mediator that alters the expression of an endogenous nucleic acid, such as DNA, by influencing transcription, translation, or other regulated biochemical processes. Heterologous nucleic acids, such as DNA, are also called foreign nucleic acids. Any nucleic acid, such as DNA, that is heterologous or foreign to the cell in which the nucleic acid is expressed is covered herein as heterologous nucleic acid; heterologous nucleic acids include nucleic acids that are also exogenously added after endogenous expression. Heterologous nucleic acids are generally not endogenous to the cell in which they are introduced, but have been obtained or synthesized from another cell, or have been introduced into a genomic locus in which they are not naturally present, or whose expression is under the control of a regulatory sequence or a sequence different from the natural regulatory sequence.

[0413] Examples of heterologous nucleic acids in this article include, but are not limited to, nucleic acids encoding proteins of the DNA / RNA sensor pathway or gain-of-function or constitutive active variants thereof, or nucleic acids encoding immunostimulatory proteins such as cytokines, chemokines, or co-stimulatory molecules that confer or contribute to antitumor immunity in the tumor microenvironment. Other products, such as antibodies and fragments thereof, are also included. Decoy receptors, antagonistic peptides, and RNAi confer or contribute to antitumor immunity in the tumor microenvironment. In immunostimulatory bacteria, heterologous nucleic acids are typically encoded on introduced plasmids, but can be introduced into the bacterial genome, for example, by altering promoters that express bacterial products. Heterologous nucleic acids, such as DNA, include nucleic acids that can mediate the expression of DNA encoding therapeutic products in some way, or that can encode products, such as peptides or RNA, that directly or indirectly mediate the expression of therapeutic products in some way.

[0414] As used herein, cell therapy involves delivering cells to a subject to treat a disease or condition. The cells may be allogeneic or autologous cells, modified in vitro, for example by infecting them with immunostimulatory bacteria as described herein, to deliver or express products upon introduction into the subject.

[0415] As used herein, gene therapy involves transferring heterologous nucleic acids, such as DNA, into certain cells, such as target cells, of mammals, particularly humans, suffering from a disease or condition for which such treatment is sought. The nucleic acid, such as DNA, is introduced into the selected target cells in a manner that causes the heterologous nucleic acid, such as DNA, to be expressed and to produce a therapeutic product encoded therefrom. Gene therapy can also be used to deliver nucleic acids encoding gene products that replace defective genes or supplement gene products produced by the introduced mammal or cell. The introduced nucleic acid may encode therapeutic compounds, such as growth factors or inhibitors thereof, or tumor necrosis factor or inhibitors thereof, such as their receptors, which are typically not produced normally by the mammalian host or not produced at therapeutically effective amounts or for therapeutically effective times. The heterologous nucleic acid, such as DNA, encoding the therapeutic product may be modified prior to introduction into the cells of the diseased host to enhance or otherwise alter the product or its expression. Gene therapy may also include the delivery of inhibitors or antagonists or other regulators of gene expression.

[0416] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for determining the amount of polypeptide produced by the host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates using ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0417] As used herein, a “host cell” is a cell used to receive, maintain, regenerate, and / or amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When a host cell divides, the nucleic acids contained in the vector are replicated, thereby amplifying the nucleic acids.

[0418] As used herein, a “vector” is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those that typically allow the introduction of nucleic acids encoding polypeptides or fragments thereof through restriction digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides, such as modified anti-CTLA-4 antibodies. Vectors are used to introduce nucleic acids encoding polypeptides into host cells to amplify the nucleic acid or to express / display the polypeptide encoded by the nucleic acid. Vectors are typically kept in a free state but can be designed to enable the integration of genes or portions thereof into a genomic chromosome. Vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also considered. The selection and use of these vectors are well known to those skilled in the art. Vectors also include “viral vectors” or “viral vectors.” A viral vector is an engineered virus operatively linked to a foreign gene to transfer the foreign gene into a cell (as a vector or delivery medium).

[0419] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences such as promoter regions, which enable the expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector that, when introduced into a suitable host cell, results in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, including those that are reproducible in eukaryotic and / or prokaryotic cells, as well as those that remain free or are integrated into the host cell genome.

[0420] As used in this article, "primary sequence" refers to the amino acid residue sequence of a polypeptide or the nucleotide sequence of a nucleic acid molecule.

[0421] As used herein, “sequence identity” refers to the number of identical or similar amino acids or nucleotide bases between the compared test and reference polypeptides or polynucleotides. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify similar or identical regions. For the purposes of this article, sequence identity is typically determined by alignment to identify identical residues. Alignment can be local or global. Matches, mismatches, and vacancies can be identified between the compared sequences. A vacancy is an empty amino acid or nucleotide inserted between residues in the aligned sequences to arrange identical or similar characters. Typically, internal and terminal vacancies may be present. When using vacancy penalties, sequence identity can be determined without penalizing terminal vacancies (e.g., no penalty for terminal vacancies). Alternatively, sequence identity can be determined without considering vacancies as the number of identical positions in the total aligned sequences / length × 100.

[0422] As used herein, “global alignment” compares two sequences from beginning to end, with each letter in each sequence being compared only once. Alignment is performed regardless of whether there is similarity or identity between the sequences. For example, a 50% sequence identity based on “global alignment” means that 50% of the residues in each 100-nucleotide sequence are identical in the full sequence alignment of the two compared sequences. It should be understood that global alignment can be used to determine sequence identity even if the compared sequences are of different lengths. Differences at the ends of sequences are considered when determining sequence identity unless “no penalty for terminal gaps” is selected. Typically, global alignment is used for sequences that are significantly similar over most of their length. An example algorithm used for global alignment includes the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48:443). Exemplary procedures for performing global alignment are publicly available, including a global sequence alignment tool available on the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ) and a procedure available at deepc2.psi.iastate.edu / aat / align / align.html.

[0423] As used herein, "local alignment" is the alignment of two sequences, but only of the parts of the sequences that are similar or identical. Therefore, local alignment determines whether a segment of one sequence exists in another. If no similarity exists, no alignment information is returned. Local alignment algorithms include BLAST or the Smith-Waterman algorithm (Adv. Appl. Math. 2:482 (1981)). For example, a 50% sequence identity based on "local alignment" means that in a full sequence alignment of two compared sequences of any length, regions of 100 nucleotides of similarity or identity have 50% of the same residues in those regions.

[0424] For the purposes of this paper, sequence identity can be determined using a standard alignment algorithm procedure with a default gap penalty established by each vendor. The default parameters of the GAP procedure may include: (1) a univariate comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix as described in Gribskov et al. (1986) Nucl. Acids Res. 14: 6745-6763, as described in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for terminal gaps. Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% “identical” or other similarly expressed percentage identical, or any two polypeptides have amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% “identical” or other similarly expressed percentage identical, they can be determined using known computer algorithms based on local or global alignment (see, for example, wikipedia.org / wiki / Sequence_alignment_software, which provides links to dozens of known and publicly available alignment databases and programs). For the purposes of this paper, sequence identity is typically determined using computer algorithms based on global alignment, such as the Needleman-Wunsch global sequence alignment tool from NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (William Pearson implementing the Huang and Miller algorithm (Adv.Appl.Math.(1991)12:337-357)); and Xiaoqui Huang's program, available at deepc2.psi.iastate.edu / aat / align / align.html. Typically, in global alignment, the full-length sequence of each compared polypeptide or nucleotide is aligned to the full length of each sequence. Local alignment can also be used when the compared sequences are substantially the same length.

[0425] Therefore, as used herein, the term "identity" refers to a comparison or alignment between a test and a reference polypeptide or polynucleotide. In a non-limiting example, "at least 90% identical to" means having 90% to 100% identity relative to a reference polypeptide or polynucleotide. Identity at 90% or higher indicates that, for illustrative purposes, a test and a reference polypeptide or polynucleotide of 100 amino acids or nucleotides in length are compared, and no more than 10% (i.e., 10 out of 100) of the amino acids or nucleotides in the test polypeptide or polynucleotide differ from those in the reference polypeptide or polynucleotide. Similar comparisons can be made between the test and the reference polynucleotide. This difference can be represented as point mutations randomly distributed across the entire length of the amino acid sequence, or it can cluster at one or more positions of varying lengths up to the maximum permissible length, such as a 10 / 100 amino acid difference (approximately 90% identity). Differences can also be due to the deletion or truncation of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. Depending on the length of the sequences being compared, at a homology or similarity level of approximately 85-90%, the results can be independent of the procedure and space parameter settings; such a high level of similarity can usually be easily assessed without relying on software.

[0426] As used herein, “disease or symptom” means a pathological condition in an organism caused by certain causes or conditions, including but not limited to infection, acquired disease and genetic disease, and is characterized by identifiable symptoms.

[0427] As used in this article, “treatment” for a subject with a disease or condition means that the subject’s symptoms are partially or completely reduced or remain stable after treatment.

[0428] As used herein, “treatment” means any action that improves the symptoms of a disease or condition. Treatment includes prevention, treatment, and / or cure. Treatment also covers any pharmaceutical use of any immunostimulatory bacteria or compositions provided herein.

[0429] As used in this article, “prophylaxis” refers to the prevention of underlying disease and / or the prevention of the worsening or progression of disease symptoms.

[0430] As used in this article, “prevention” or “prophylaxis” and its grammatical equivalents refer to methods that reduce the risk or likelihood of progression to a disease or condition.

[0431] As used herein, “pharmaceuticalally effective substance” includes any therapeutic or bioactive agent, including but not limited to, anesthetics, vasoconstrictors, dispersants, and conventional therapeutic agents, including small molecule drugs and therapeutic proteins.

[0432] As used in this article, "therapeutic effect" refers to the change produced by treating a subject, usually by improving or alleviating the symptoms of a disease or condition, or by curing a disease or condition.

[0433] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, is at least sufficient to produce a therapeutic effect. Therefore, the amount is the amount required to prevent, cure, improve, suppress, or partially suppress the symptoms of a disease or condition.

[0434] As used herein, “therapeutic efficacy” means the ability of a substance, compound, material, or composition comprising a compound to produce a therapeutic effect in a subject who has been administered the substance, compound, material, or composition comprising a compound.

[0435] As used herein, "preventive effective amount" or "preventive effective dose" refers to an amount that, when administered to a subject, has the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom, or reducing the incidence of viral infection. A single dose is not required to achieve complete preventive effect, and the effect may occur after a series of doses. Therefore, a preventive effective amount can be administered in one or more doses.

[0436] As used herein, "improvement of symptoms of a particular disease or condition by treatment, such as by application of a pharmaceutical composition or other therapeutic agent," means any relief of symptoms attributable to or related to the application of the composition or therapeutic agent, whether permanent or temporary, continuous or transient.

[0437] As used herein, "anticancer agent" or "anticancer therapeutic agent" means any substance or therapeutic agent that is directly or indirectly destructive or toxic to malignant cells and tissues. For example, anticancer agents include substances that kill cancer cells or otherwise inhibit or impair the growth of tumors or cancer cells. Example anticancer agents are chemotherapeutic agents and immunotherapeutic agents.

[0438] As used in this article, "therapeutic activity" refers to the in vivo activity of therapeutic products such as peptides, nucleic acid molecules, and other therapeutic molecules. Typically, therapeutic activity is activity associated with the treatment of a disease or condition.

[0439] As used in this article, the term "subject" refers to an animal, including mammals such as humans.

[0440] As used in this article, "patient" refers to a human subject.

[0441] As used herein, "animal" includes any animal, such as, but not limited to, primates, including humans, gorillas, and monkeys; rodents, such as mice and rats; birds, such as chickens; ruminants, such as goats, cattle, deer, and sheep; and pigs and other animals. Non-human animals are excluded; humans are the intended animal. The peptides provided herein are from any source, including animals, plants, prokaryotes, and fungi. Most peptides are of animal origin, including those from mammals.

[0442] As used herein, “composition” means any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous phase, non-aqueous phase, or any combination thereof.

[0443] As used herein, “combination” refers to any association between or among two or more items. A combination can be two or more individual items, such as two compositions or collections, mixtures thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are typically functionally related or associated.

[0444] As used herein, “combination therapy” refers to the administration of two or more different therapeutic agents. These different therapeutic agents may be provided and administered individually, sequentially, intermittently, or in a single composition.

[0445] As used herein, a “kit” is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or its elements, for purposes including but not limited to activation, administration, diagnosis and evaluation of biological activity or properties.

[0446] As used herein, “unit dosage form” means a physically separate unit suitable for human and animal subjects, as well as individually packaged units as known in the art.

[0447] As used in this article, "single-dose formulation" refers to a formulation that is administered directly.

[0448] As used herein, a "multi-dose formulation" refers to a formulation containing multiple doses of a therapeutic agent and that can be directly applied to provide several single doses of the therapeutic agent. The doses can be administered over a period of minutes, hours, weeks, days, or months. Multi-dose formulations may allow for dose adjustment, dose combining, and / or dose splitting. Because multi-dose formulations are used over time, they typically contain one or more preservatives to prevent microbial growth.

[0449] As used herein, “manufactured product” means a product that is prepared and sold. As used throughout this application, the term is intended to cover any composition provided herein contained in packaged articles.

[0450] As used herein, “fluid” means any composition that can flow. Therefore, fluid encompasses compositions in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0451] As used herein, isolated or purified polypeptides or proteins (e.g., isolated antibodies or their antigen-binding fragments) or their biologically active portions (e.g., isolated antigen-binding fragments) are substantially free of cellular material or other contaminating proteins from cells or tissues from which said polypeptides or proteins are derived, or substantially free of chemical precursors or other chemicals used in chemical synthesis. A preparation is substantially free of these substances if, as determined by standard analytical methods used by those skilled in the art to assess such purity, such as thin-layer chromatography (TLC), gel electrophoresis, and high-performance liquid chromatography (HPLC), the preparation exhibits the absence of readily detectable impurities, or if the preparation is pure enough that further purification does not detectably alter the physical and chemical properties of the substance, such as enzymatic and biological activities. Methods for purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, substantially chemically pure compounds can be mixtures of stereoisomers. In such cases, further purification may enhance the specific activity of the compound.

[0452] As used herein, “cell extract” or “lysate” refers to a preparation or fraction made from lysed or destroyed cells.

[0453] As used in this article, a "control" is a sample that is substantially the same as the test sample, except that it is not processed using the test parameters, or, if it is a plasma sample, it may be derived from healthy volunteers unaffected by the condition of interest. A control can also be an internal control.

[0454] As used herein, the singular forms “an,” “a,” and “the” include their plural indicators unless the context clearly indicates otherwise. Thus, for example, with respect to a polypeptide containing an “immunoglobulin domain,” this includes polypeptides having one or more immunoglobulin domains.

[0455] As used herein, unless explicitly stated otherwise, the term "or" is used to mean "and / or".

[0456] As used in this article, ranges and quantities can be expressed as “about” a specific value or range. “About” also includes precise quantities. Therefore, “about 5 amino acids” means “about 5 amino acids” as well as “5 amino acids”.

[0457] As used herein, “optional” or “optionally” means that the event or situation subsequently described occurs or does not occur, including both the scenario where the event or situation occurs and the scenario where it does not occur. For example, an “optionally variant” section means that the section is a variant or a non-variant.

[0458] Unless otherwise stated, abbreviations for any protecting group, amino acid and other compound used herein shall be consistent with their usual usage, recognized abbreviations or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 11(9): 1726-1732).

[0459] For the sake of clarity and disclosure rather than restriction, the detailed description of the invention is divided into the following paragraphs and sections.

[0460] B. Overview of Immunostimulatory Bacteria Used in Cancer Treatment

[0461] The understanding of bacteria's anti-cancer activity dates back to the 19th century, when several doctors observed tumor regression in patients infected with Streptococcus pyogenes. William Coley initiated the first research into using bacteria to treat terminal cancer and developed a vaccine composed of Streptococcus pyogenes and Serratia marcescens, which was successfully used to treat a variety of cancers, including sarcoma, carcinoma, lymphoma, and melanoma. Since then, many bacterial species, including Clostridium, Mycobacterium, Bifidobacterium, Listeria monocytogenes, and Escherichia, have been studied as sources for anticancer vaccines (see, for example, international PCT application publications WO 1999 / 013053 and WO 2001 / 025399; Bermudes et al. (2002) Curr. Opin. Drug Discov. Devel. 5:194-199; Patyar et al. (2010) Journal of Biomedical Science 17:21; and Pawlek et al. (2003) Lancet Oncol. 4:548-556).

[0462] As a therapeutic platform, bacteria offer several advantages over other therapies such as oncolytic viruses. Some bacterial species can be engineered for oral and systemic (intravenous; IV) administration, are readily propagated in vitro and in vivo, and can be stored and transported in a lyophilized state. Bacterial chromosomes are easily manipulated due to the lack of exons, and the complete genomes of many strains have been well identified (Felgner et al. (2016) mBio 7(5):e01220-16). Many types of bacteria are cheaper and easier to produce than viruses, and the appropriate delivery of engineered bacteria is more advantageous than viral delivery because they do not permanently integrate into the host cell genome, preferentially infect bone marrow cells rather than epithelial cells, and can be rapidly eliminated with antibiotics if necessary, making them safer.

[0463] Immunostimulatory bacteria modified to utilize these advantageous properties are provided. The bacteria provided herein are modified to infect and accumulate in the tumor microenvironment, particularly in tumor-resident immune cells (bone marrow cells), such as tumor-associated macrophages (TAMs), dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs), and are also engineered to express and deliver high levels of therapeutic proteins and combinations thereof, particularly complementary combinations. The immunostimulatory bacteria provided herein possess advantageous properties compared to existing bacterial therapies, as well as cell therapies, oncolytic virus therapies, and existing bacterial treatments. While the immunostimulatory bacteria provided herein can be administered via any suitable route, they are suitable for systemic administration, such as intravenous administration. As shown and described herein, the immunostimulatory bacteria provided herein can target major immune pathways.

[0464] The bacteria presented in this article were designed and engineered to retain beneficial scaffold properties while possessing virus-like immune characteristics. This is advantageous for use as an anticancer therapeutic agent. The table below summarizes some of the immune and scaffold properties of bacteria and viruses; the immunostimulatory bacteria presented in this article retain the viability of the bacterial scaffold but elicit a virus-like immune response in treated subjects (discussed in more detail in Section C below).

[0465]

[0466] In Salmonella and other bacterial species, flagella contribute to TRL5-mediated inflammation, LPS leads to TLR4-mediated inflammatory responses, and sticky, curly pili lead to TLR2-mediated inflammatory responses. The genome of the immunostimulatory bacteria described herein is modified to lack flagella and sticky, curly pili, and to possess modified LPS, thereby resulting in a reduction or elimination of the TLR4-mediated inflammatory response. As a result, the immunostimulatory bacteria described herein induce a virus-like antitumor immune response. The elimination or modification of these components confers other advantageous properties, such as those discussed in detail below. The immunostimulatory bacteria deliver therapeutic products, such as anticancer agents, particularly complementary combinations of products. The immunostimulatory bacteria described herein deliver encoded genetic payloads to tumor-resident bone marrow cells in a tumor-specific manner.

[0467] This provides anticancer therapeutic products and immunostimulatory bacteria that deliver genetic payloads encoding one or more therapeutic products. These include truncated costimulatory molecules (receptors or ligands; e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, OX40L) with complete or partial deletion of cytoplasmic domains expressed on antigen-presenting cells (APCs), wherein the truncated gene product is capable of participating in constitutive immunostimulatory signaling to T cells via costimulatory receptors and is unable to send counterregulatory signals to APCs due to the truncated or deleted cytoplasmic domains.

[0468] Immunostimulatory bacteria can encode and express one or more of the following: IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-12, IL-15, IL-15 / IL-15Rα chain complex, IL-18, IL-21, IL-23, IL-36γ, interferon-α, interferon-β, IL-2 with weakened binding to IL-2Ra, modified IL-2 that does not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, cytoplasmic D. NA / RNA sensors or type I IFN pathway proteins, such as gain-of-function or constitutively active STING, IRF3, IRF7, MDA5, or RIG-I variants (inducing type I IFN), TGF-β inhibitors, such as TGF-β inhibitory antibodies, TGF-β peptide antagonists, and TGF-β binding decoy receptors, antibodies and fragments thereof, such as those targeting immune checkpoints and other anticancer targets, such as VEGF and IL-6, co-stimulatory receptors / molecules, such as 4-1BBL, including 4-1BBL with missing or truncated cytoplasmic domains, etc. The immunostimulatory bacteria may also encode and express truncated costimulatory molecules (e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, OX40L) with partial or complete deletion of cytoplasmic domains expressed on antigen-presenting cells (APCs). The truncated gene products are capable of constitutive immunostimulatory signals to T cells via costimulatory receptors and cannot send counterregulatory signals to APCs due to the deletion or truncation of the cytoplasmic domains. This combination of therapeutic products and agents can be expressed in a single therapeutic composition. Through modification of the bacterial genome, the immunostimulatory bacteria exhibit tumor-specific localization and enrichment, and provide intravenous (IV) administration to activate antitumor immune pathways that would otherwise be toxic if systemically activated.

[0469] The immunostimulatory bacteria described herein are genetically engineered to be safe and target tumors, the tumor microenvironment, and / or tumor-resident immune cells. The immunostimulatory bacteria described herein comprise combinations of genomic modifications and other modifications, and encoded therapeutic products, which synergistically provide immunostimulatory bacteria that accumulate in tumor-resident immune cells and persist for a sufficient duration to deliver the therapeutic product, particularly combinations that induce or promote anticancer immunostimulation in tumors and the tumor microenvironment, with little or no toxic side effects. When delivered systemically, such as intravenously (IV), the immunostimulatory bacteria are enriched in tumors, including metastatic lesions; they provide efficient genetic transfer of the immune payload, particularly to tumor-resident bone marrow cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and dendritic cells (DCs); they induce a potent local immune response, destroying the tumor and vaccinating against future recurrence; and upon completion of treatment, they are naturally eliminated, for example through phagocytosis and destruction by infected cells, or rapidly destroyed by a course of antibiotics.

[0470] Due to their designed purine / adenosine nutritional deficiency, the immunostimulatory bacteria presented herein exhibit preferential accumulation in the tumor microenvironment and / or tumor-resident immune cells and demonstrate an inability to replicate within phagocytes. These immunostimulatory bacteria, which avoid inactivation by serum complement, allow for the direct delivery of various high concentrations of immunotherapeutic agents and therapeutic products within the tumor microenvironment while minimizing toxicity to normal tissues, and are provided herein.

[0471] For example, as described in more detail in Section C.8, the immunostimulatory bacteria presented herein include genomic modifications to make them msbB - / pagP - This alters lipid A in LPS, leading to pentacylation (wild-type lipid A has 6-7 fatty acid chains), and reduces TLR4 affinity; it is an adenosine / adenine auxotroph, such as purI. - It is asparaginase II. - (ansB - This can improve T cell quality; it is csgD - Among other properties, it also removes the curled pili; and includes other optional genomic modifications, such as insertions, deletions, disruptions, and any other modifications, such that the encoded product is not produced in an active form, as discussed in detail herein. The immunostimulatory bacteria include plasmids encoding one or more therapeutic products, particularly anticancer products, under the control of eukaryotic promoters.

[0472] The immunostimulatory bacteria described herein deliver therapeutic products (such as constitutively active STING variants and other immunomodulatory proteins and products) to tumor-resident bone marrow cells, promoting adaptive immunity and enhancing T cell function. These immunostimulatory bacteria lead to a complete remodeling of the immunosuppressive tumor microenvironment, shifting towards an adaptive antitumor phenotype, away from bacterial phenotypes characterized by promoting innate immunity and suppressing adaptive immunity.

[0473] The immunostimulatory bacteria described herein include genomic modifications that allow them to target or accumulate in tumor-resident immune cells, particularly tumor-resident bone marrow cells such as macrophages, MDSCs (myeloid-derived suppressor cells), and DCs (dendritic cells). In these cells, they deliver payloads of encoded therapeutic products expressed under the control of regulatory sequences recognized by host cell (eukaryotic) transcription / translation mechanisms. The encoded products are expressed in bone marrow cells and appropriately delivered into the tumor microenvironment. These bacteria generate antitumor immunity and can also deliver antitumor products that directly treat tumors, as well as products that can activate prodrugs.

[0474] Compared to unmodified bacteria, the immunostimulatory bacteria described herein exhibit at least approximately 100,000 times higher tumor infiltration and enrichment. These immunostimulatory bacteria are consumed by tumor-resident immune cells and deliver plasmids encoding therapeutic products that are expressed and generated in both immune cells and the tumor microenvironment to induce antitumor immunity.

[0475] 1. Bacterial cancer immunotherapy

[0476] Many solid tumor types have evolved a highly immunosuppressive microenvironment, making them highly resistant to approved checkpoint therapies, such as anti-CTLA-4, anti-PD-1, and anti-PD-L1 therapies. One mechanism by which tumors gradually develop resistance to checkpoint therapies is the lack of intratumoral T cells and tumor antigen cross-presenting dendritic cells (DCs), described as T cell exclusion, non-inflammatory, or “cold tumors” (Sharma et al. (2017) Cell 168(4):707-723). For a small number of patients with T-cell inflammatory tumors who respond to checkpoint immunotherapy, they often experience severe autoimmune toxicity, and many eventually relapse and become checkpoint refractory (see, for example, 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 initiate multiple mechanisms to evade immune surveillance, reprogram anti-tumor immune cells to suppress immunity, exclude and inactivate anti-tumor T cells, and develop resistance to targeted cancer therapies (see, for example, Mahoney et al. (2015) Nat. Rev. Drug Discov. 14(8):561-584). Solving this problem will require immunotherapies capable of appropriately inducing inflammation in these tumors and generating anti-tumor immunity that can provide long-term tumor regression. Furthermore, intratumoral therapy is challenging and significantly limited in metastatic disease cases. Systemic administration therapies that appropriately induce inflammation in each individual metastatic lesion and overcome multiple immunosuppressive pathways are needed. With their ability to specifically target tumor-resident immune cells and express multiple complementary genetic payloads / therapeutic products, the immunostimulatory bacteria presented in this article aim to address these issues.

[0477] 2. Existing therapies targeting the tumor microenvironment

[0478] Numerous therapies have been developed that target the tumor microenvironment (TME) and attempt to boost anti-tumor immunity. Each therapy has its own challenges and limitations, and the immunostimulatory bacteria presented in this article address these challenges and limitations.

[0479] a. Limitations of autologous T-cell therapy

[0480] Several systemically administered therapeutic platforms have been investigated clinically with the aim of penetrating the highly immunosuppressive tumor microenvironment and inducing an appropriate immune response to inflammatory tumors, thereby promoting antitumor immunity. These platforms include chimeric antigen receptor T cells (CAR-T cells), which are generated by harvesting T cells from a patient and reengineering them to fuse T cell receptors to a variable extracellular domain of antibody Ig specific to a particular tumor antigen. This confers antigen-recognizing properties to the cells, as well as the cytolytic properties of activated T cells (see, for example, Sadelain et al. (2015) J. Clin. Invest. 125(9):3392-3400). Despite the promising nature and efficacy of this technology, such as the FDA approval of CD19 CAR-T tisagenlecleucel (e.g., trademarked as...),... ) and axicabtagene ciloleucel (trademarked as However, success was limited to CD19. + Hematopoietic malignancies, and at the cost of fatal immune-related adverse events (see, for example, Jackson et al. (2016) Nat. Rev. Clin. Oncol. 13(6):370-383). Tumors can rapidly mutate to downregulate the target tumor antigens of solid tumors, including the antigen CD19, thereby promoting immune escape (see, for example, Mardiana et al. (2019) Sci. Transl. Med. 11(495):eaaw2293). There are not many tumor-specific target antigens. Solid tumor targets not expressed in healthy tissues are a major obstacle to CAR-T therapy. In addition, there are other obstacles to CAR-T therapy entering the solid tumor microenvironment due to the lack of a sufficient gradient of T cell chemokines, which is necessary for proper T cell infiltration into the tumor. Furthermore, once it infiltrates the tumor, it is rapidly inactivated (see, for example, Brown et al. (2019) Nat. Rev. Immunol. 19(2):73-74). Even if the safety of CAR-T cells is significantly improved and their efficacy is extended to solid tumors, the feasibility and cost associated with these laborious therapies will still limit their wider adoption.

[0481] b. Virus vaccine platform

[0482] Oncolytic viruses (OVs) possess both natural and engineered properties that can induce tumor cell lysis, recruit T cells to the tumor, and deliver genetic material that can be read by tumor cells to produce immunomodulatory proteins. For example, an oncolytic virus named Talimogenelaherparepvec (T-VEC) is a modified herpes simplex virus that encodes an anti-melanoma antigen and the cytokine GM-CSF (granulocyte-macrophage colony-stimulating factor) and can be administered intratumorally. It has been approved by the FDA for metastatic melanoma (see, for example, Bastin et al. (2016) Biomedicines 4(3):21). T-VEC has been shown to have clinical benefits for some melanoma patients and to have lower immunotoxicity than immune checkpoint antibodies or FDA-approved systemically administered cytokines such as IL-2 and interferon-α (see, for example, Kim et al. (2006) Cytokine Growth Factor Rev. 17(5):349-366; and Paul et al. (2015) Gene 567(2):132-137).

[0483] Oncolytic viruses (OVs) have several limitations as an anticancer therapy. First, oncolytic viruses are rapidly inactivated by the body's complement system in the bloodstream. It has been shown that it is difficult to deliver sufficient virus to produce the desired therapeutic effect through systemic administration. Intratumoral administration is limited in metastatic cases (lesions spread throughout the body), is refractory to most solid tumor types (e.g., lung and visceral lesions), and requires invasive, radioguided injection, which limits repeated administration. Viruses are difficult to produce and store on a commercial scale. Most OV-based vaccines, such as those based on paramyxoviruses, reoviruses, and microRNAs, have similar limitations (see, for example, Chiocca et al. (2014) Cancer Immunol. Res. 2(4):295-300). Oncolytic viruses possess inherent immunogenicity and can be rapidly cleared from the human bloodstream. T cells entering tumors exhibit a much higher affinity for viral antigens than for weaker tumor neoantigens (see, for example, Aleksic et al. (2012) Eur. J. Immunol. 42(12): 3174-3179). Therefore, in addition to the well-known limitations of platform technology, the ability of OV to stimulate durable antitumor immunity has been limited to date.

[0484] c. Bacterial Cancer Treatment

[0485] In preclinical animal studies, many bacterial species have been shown to preferentially replicate within solid tumors when injected from distal sites. These bacterial species include, but are not limited to, *Salmonella*, *Bifodobacterium*, *Clostridium*, and *Escherichia*. The tumor-homing nature of bacteria, combined with the host's innate immune response to bacterial infection, can mediate antitumor responses. This tumor tropism reduces tumor size to varying degrees. One contributing factor to the tumor tropism of these bacterial species is their ability to replicate in hypoxic and hypoxic environments. Many of these naturally occurring tumor-loving bacteria have been further engineered to enhance the potency of antitumor responses (Zu et al. (2014) Crit. Rev. Microbiol. 40(3):225–235; and Felgner et al. (2017) Microbial Biotechnology 10(5):1074–1078). Despite proof-of-concept studies in animal studies, the complement factor in human serum, which is absent in animal models, can inactivate bacteria, thus limiting its use as a treatment for cancer.

[0486] For oral or systemic administration, the bacterial strain is attenuated to prevent it from causing systemic disease and / or septic shock, but retains a degree of infectivity to effectively colonize tumors and resist complement inactivation. Many different bacterial species have been investigated as potential drugs for cancer treatment. These bacterial species include: *Clostridium* (see, for example, Dang et al. (2001) Proc. Natl. Acad. Sci. USA 98(26):15155-15160; U.S. Patent Publications 2017 / 0020931 and 2015 / 0147315; and U.S. Patents 7,344,710 and 3,936,354), *Mycobacterium* (see, for example, U.S. Patent Publications 2015 / 0224151 and 2015 / 0071873), *Bifidobacterium* (see, for example, Dang et al. (2001); and Kimura et al. (1980) Cancer Res. 40:2061-2068), and *Lactobacillus* (see, for example, Dang et al.). Al. (2001)), Listeria monocytogenes (see, for example, Le et al. (2012) Clin. Cancer Res. 18(3): 858-868; Starks et al. (2004) J. Immunol. 173: 420-427; and U.S. Patent Publication No. 2006 / 0051380) and Escherichia coli (see, for example, U.S. Patent No. 9,320,787).

[0487] The immunostimulatory bacteria described herein include genomic modifications that address problems of existing bacteria developed for tumor treatment. These modifications improve bacterial targeting or accumulation in the tumor microenvironment, particularly by designing the bacteria to infect tumor-resident immune cells rather than healthy tissue, thereby reducing toxicity and improving the delivery of encoded products. The immunostimulatory bacteria are also designed to deliver therapeutic products, including combinations thereof, aimed at eliminating the immunosuppressive effects of tumors, enhancing the host's anti-tumor response, and providing anti-tumor products.

[0488] i. Listeria

[0489] Listeria monocytogenes is a live, attenuated intracellular bacterium capable of inducing strong CD8 mutations. +T-cell activation to express tumor antigens in mouse cancer models has also been explored as a bacterial cancer vector (see, for example, Le et al. (2012) Clin. Cancer Res. 18(3):858-868). In a clinical trial of a Listeria monocytogenes-based vaccine, combined with the tumor antigen mesothelin and an allogeneic pancreatic cancer-based GVAX vaccine, using a primary-booster approach, median survival was shown to be 6.1 months in patients with advanced pancreatic cancer, compared to 3.9 months in patients treated with GVAX alone (see, for example, Le et al. (2015) J. Clin. Oncol. 33(12):1325-1333). However, these results were not replicated in a larger phase 2b study, suggesting that it is difficult to subvert peripheral immune surveillance to low-affinity tumor neoantigens in humans. Because bacterial lysis after phagocytosis is a prerequisite for effective plasmid transfer, Listeria monocytogenes also exhibits a limited immune response to encoded tumor antigens, and this has not been demonstrated in human macrophages.

[0490] ii. Salmonella species

[0491] Salmonella enterica serotype *Salmonella typhimurium* is an example of a bacterial species used as an anticancer therapeutic agent. *Salmonella typhimurium* is a Gram-negative facultative anaerobe that preferentially accumulates in hypoxic and necrotic areas due to its ability to utilize nutrients from necrotic, leaking tumor vascular systems and its increased likelihood of survival in the immunosuppressive tumor microenvironment (see, for example, Baban et al. (2010) Bioengineered Bugs 1(6):385-394). As a facultative anaerobe, *Salmonella typhimurium* can grow under both aerobic and anaerobic conditions, thus enabling it to colonize both small tumors with less hypoxia and large tumors with more hypoxia.

[0492] Salmonella Typhimurium is transmitted via the fecal-oral route, causing localized gastrointestinal infections. This bacterium can also enter the bloodstream and lymphatic system, infecting systemic tissues such as the liver, spleen, and lungs. Systemic administration of wild-type Salmonella Typhimurium overstimulates TNF-α and IL-6, leading to a cytokine cascade and septic shock, which can be fatal if left untreated. Therefore, attenuation of pathogenic bacterial strains such as Salmonella Typhimurium is necessary to prevent systemic infection, rather than completely inhibiting its ability to effectively colonize tumor tissue. Attenuation is typically achieved by mutating cellular structures that can trigger an immune response through pathogen pattern recognition, such as the bacterial outer membrane, or by limiting the bacteria's ability to replicate without supplemental nutrients.

[0493] Salmonella typhimurium is an intracellular pathogen that can be rapidly absorbed by phagocytic bone marrow cells such as macrophages. It can also directly invade non-phagocytic cells, such as epithelial cells, through its Salmonella pathogenic island 1 (SPI-1) encoded type III secretion system (T3SS1). Once inside the cell, it can replicate in Salmonella-containing vacuoles (SCVs) through SPI-2 regulation, or escape into the cytosol of some epithelial cells (see, for example, Agbor et al. (2011) Cell Microbiol. 13(12): 1858-1869; and Galan and Wolf-Watz (2006) Nature 444: 567-573). Genetically modified strains of Salmonella typhimurium have been described as antitumor agents to induce direct tumor killing and / or deliver tumor-killing molecules (see, for example, Clairmont et al. (2000) J. Infect. Dis. 181: 1996-2002; Bermudes, D. et al. (2002) Curr. Opin. Drug Discov. Devel. 5: 194-199; Zhao, M. et al. (2005) Proc. Natl. Acad. Sci. USA 102: 755-760; and Zhao, M. et al. (2006) Cancer Res. 66: 7647-7652).

[0494] Various methods for attenuating virulent bacterial pathogens are known in the art. For example, auxotrophic mutations prevent bacteria from synthesizing essential nutrients, and deletions / mutations in genes such as aro, pur, gua, thy, nad, and asd are employed (see, for example, U.S. Patent Publication No. 2012 / 0009153). Nutrients produced by biosynthetic pathways involving these genes are typically unavailable in host cells, thus challenging bacterial survival. For instance, attenuation of Salmonella and other species can be achieved by deleting or disrupting the aroA gene, which is part of the shikimate pathway, linking glycolysis to aromatic amino acid biosynthesis (see, for example, Felgner et al. (2016) mBio7(5):e01220-16). The absence or disruption of aroA leads to bacterial auxotrophy of aromatic amino acids and subsequent attenuation (see, for example, U.S. Patent Publications 2003 / 0170276, 2003 / 0175297, 2012 / 0009153 and 2016 / 0369282; and International Application Publications WO 2015 / 032165 and WO 2016 / 025582). Similarly, other enzymes involved in the biosynthetic pathway of aromatic amino acids, including aroC and aroD, have been absent to achieve attenuation (see, for example, U.S. Patent Publication 2016 / 0369282; and International Application Publication WO 2016 / 025582). For example, Salmonella Typhimurium strain SL7207 is an aromatic amino acid auxotroph (aroA...). - (Mutants), strains A1 and A1-R are leucine-arginine auxotrophs.

[0495] Mutations in attenuated bacteria also include, but are not limited to, mutations in genes that alter lipopolysaccharide (LPS) biosynthesis, such as rfaL, rfaG, rfaH, rfaD, rfaP, rFb, rfa, msbB, htrB, firA, pagL, pagP, lpxR, arnT, eptA, and lpxT; mutations introducing suicide genes, such as sacB, nuk, hok, gef, kil, or phlA; mutations introducing bacterial lysis genes, such as hly and cly; and mutations in genes encoding virulence factors, such as IsyA. pag, prg, iscA, virG, plc, and act; gene mutations that modify stress responses, such as recA, htrA, htpR, hsp, and groEL; gene mutations that disrupt the cell cycle, such as min; and gene mutations that disrupt or inactivate regulatory functions, such as cya, crp, phoP / phoQ, and ompR (see, for example, U.S. Patent Publications 2012 / 0009153, 2003 / 0170276, and 2007 / 0298012; U.S. Patent No. 6,190,657; International Application Publication No. WO 2015 / 032165; Felgner et al. (2016) Gut Microbes 7(2):171-177; Broadway et al. (2014) J. Biotechnology 192:177-178; Frahm et al. (2015) mBio 6(2):e00254-15; Kong et al. (2011) Infection and Immunity 79(12):5027-5038; and Konget et al. (2012) Proc. Natl. Acad. Sci. USA 109(47):19414-19419). Generally, attenuated mutations are gene deletions to prevent spontaneous compensatory mutations that could lead to a reversal to a virulence phenotype.

[0496] Another safe method for attenuating Salmonella typhimurium is to use the PhoP / PhoQ operon system, a typical bacterial two-component regulatory system consisting of a membrane-associated sensor kinase (PhoQ) and a cytoplasmic transcription regulator (PhoP) (see, for example, Miller, SI et al. (1989) Proc. Natl. Acad. Sci. USA 86:5054-5058; and Groisman, Ea et al. (1989) Proc. Natl. Acad. Sci. USA 86:7077-7081). PhoP / PhoQ is essential for virulence; its absence results in poor survival of this bacterium in macrophages and significant attenuation in mice and humans (see, for example, Miller, SI et al. (1989) Proc. Natl. Acad. Sci. USA 86: 5054-5058; Groisman, EA et al. (1989) Proc. Natl. Acad. Sci. USA 86: 7077-7081; Galan, JE and Curtiss, R. III. (1989) Microb. Pathog. 6: 433-443; and Fields, PI et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5189-5193). PhoP / PhoQ deletion strains have been used as vaccine delivery vectors (see, for example, Galan, JE and Curtiss, R.III. (1989) Microb. Pathog. 6:433-443; Fields, P.I et al. (1986) Proc. Natl. Acad. Sci. USA 83:5189-5193; and Angelakopoulos, H. and Hohmann, EL (2000) Infect. Immun. 68:2135-2141). However, as discussed herein, the limited survival of the strain in macrophages is disadvantageous if the bacteria are not subjected to plasmid transfer.

[0497] These attenuated bacterial strains have been found to be safe in mice, pigs, and monkeys when administered intravenously (IV) (see, for example, Zhao, M. et al. (2005) Proc. Natl. Acad. Sci. USA 102:755-760; Zhao, M. et al. (2006) Cancer Res. 66:7647-7652; Tjuvajev J. et al. (2001) J. Control. Release 74:313-315; and Zheng, L. et al. (2000) Oncol. Res. 12:127-135), and some live attenuated Salmonella strains have shown good tolerability after oral administration in human clinical trials (see, for example, Chatfield, S. Net et al. (1992) Biotechnology 10:888-892; DiPetrillo, MD et al. (1999) Vaccine 18:449-459; Hohmann, E et al. (1996) J. Infect. Dis. 173:1408-1414; and Sirard, JC et al. (1999) Immunol. Rev. 171:5-26).

[0498] Other attenuated Salmonella strains used for treatment include, for example, leucine-arginine auxotrophic A-1 (see, for example, Zhao et al. (2005) Proc. Natl. Acad. Sci. USA 102(3):755-760; Yu et al. (2012) Scientific Reports 2:436; US Patent No. 8,822,194; and US Patent Publication No. 2014 / 0178341), and its derivative AR-1 (see, for example, Yu et al. (2012) Scientific Reports 2:436; Kawaguchi et al. (2017) Oncotarget 8(12):19065-19073; Zhao et al. (2006) Cancer Res. 66(15):7647-7652; Zhao et al. (2012) Cell Cycle 11(1):187-193; Tome et al. (2013) Anticancer Research 33:97-102; Murakami et al. (2017) Oncotarget 8(5):8035-8042; Liu et al. (2016) Oncotarget7(16):22873-22882; and Binder et al. al.(2013)Cancer Immunol.Res.1(2):123-133); aroA - The mutant Salmonella typhimurium strain SL7207 (see, for example, Guo et al. (2011) Gene Therapy 18:95-105; and US Patent Publications 2012 / 0009153, 2016 / 036928 and 2016 / 0184456) and its obligate anaerobic derivative YB1 (see, for example, International Application Publication WO 2015 / 032165; Yu et al. (2012) Scientific Reports 2:436; and Leschner et al. (2009) PLoSONE 4(8):e6692); aroA - / aroD - The mutant Salmonella typhimurium strain BRD509 is a derivative of the wild-type strain SL1344 (see, for example, Yoon et al. (2017) Eur. J. Cancer 70: 48-61); asd - / cya - / crp -The mutant Salmonella typhimurium strain χ4550 (see, for example, Sorenson et al. (2010) Biologics: Targets & Therapy 4:61-73), and phoP - / phoQ - Salmonella typhimurium strain LH430 (see, for example, International Application Publication No. WO 2008 / 091375).

[0499] However, attenuation can affect the bacteria's ability to accumulate in tumor-resident immune cells, the tumor microenvironment, and tumor cells. This problem is addressed here. Immunostimulatory bacteria, such as the Salmonella strains illustrated herein, are attenuated through modifications that may include some of those described above, but also possess other modifications and properties described herein that enhance their effectiveness as cancer therapeutic agents.

[0500] Attenuated strains of Salmonella typhimurium possess an innate ability to deliver DNA after phagocytosis and degradation (see, for example, Weiss et al. (2003) Int. J. Med. Microbiol. 41(7): 3413-3414). They have been used as vectors for gene therapy. For example, *Salmonella typhimurium* strains have been used to deliver and express a variety of genes, including those encoding cytokines, angiogenesis inhibitors, toxins, and prodrug-converting enzymes (see, for example, U.S. Patent Publication No. 2007 / 0298012; Loeffler et al. (2008) Cancer Gene Ther. 15(12):787-794; Loeffler et al. (2007) Proc. Natl. Acad. Sci. USA 104(31):12879-12883; Loeffler et al. (2008) J. Natl. Cancer Inst. 100:1113-1116; Clairmont, C. et al. (2000) J. Infect. Dis. 181:1996-2002; Bermudes, D. et al. (2002) Curr. Opin. Drug Discov.Devel.5:194-199; Zhao, M.et al. (2005) Proc.Natl.Acad.Sci.USA102:755-760; Zhao, M.et al. (2006) CancerRes.66:7647-7652; and Tjuvajev J.et al. (2001) J.Control.Release 74:313-315).

[0501] Salmonella typhimurium has been modified to deliver tumor-associated antigen (TAA) survivin (SVN) to antigen-presenting cells (APCs) to elicit adaptive immunity (see, for example, U.S. Patent Publication No. 2014 / 0186401, and Xu et al. (2014) Cancer Res. 74(21):6260-6270). SVN is an inhibitor of apoptosis protein (IAP), which prolongs cell survival and provides cell cycle control; it is overexpressed in all solid tumors but poorly expressed in normal tissues. This technique utilizes SPI-2 and its type III secretion system to deliver TAA into the cytoplasm of APCs, where it is then activated to induce TAA-specific CD8. + T cells and antitumor immunity (see, for example, Xu et al. (2014) Cancer Res. 74(21): 6260-6270). Similar to Listeria-based TAA vaccines, this approach has shown promise in mouse models, but effective tumor antigen-specific T cell activation has not yet been demonstrated in humans.

[0502] In addition to delivering protein-coding DNA, Salmonella typhimurium has been used to deliver small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) for cancer therapy. For example, attenuated Salmonella typhimurium has been modified to express certain shRNAs, such as those targeting the immunosuppressive gene indoleamine dioxygenase (IDO). Silencing IDO expression in a mouse melanoma model resulted in tumor cell death and significant neutrophil tumor infiltration (see, for example, Blache et al. (2012) Cancer Res. 72(24):6447-6456; International Application Publication No. WO 2008 / 091375; and U.S. Patent No. 9,453,227). Co-administration of this vector with hyaluronidase showed positive results in the treatment of pancreatic ductal adenocarcinoma in mice (see, for example, Manuel et al. (2015) Cancer Immunol. Res. 3(9): 1096-1107; and US Patent Publication No. 2016 / 0184456). In another study, Salmonella Typhimurium strains attenuated due to phoP / phoQ deficiency and expressed STAT3-specific shRNA inhibited tumor growth, reduced the number of metastatic organs, and prolonged the lifespan of C57BL / 6 mice (see, for example, Zhang et al. (2007) Cancer Res. 67(12): 5859-5864). In another instance, Salmonella Typhimurium strain SL7207 has been used to deliver shRNA targeting CTNNB1, the gene encoding β-catenin (see, for example, Guo et al. (2011) Gene Therapy 18:95-105; and U.S. Patent Publications 2009 / 0123426 and 2016 / 0369282). Salmonella Typhimurium strain VNP20009 has been used to deliver shRNA targeting STAT3 (see, for example, Manuel et al. (2011) Cancer Res. 71(12):4183-4191; U.S. Patent Publications 2009 / 0208534, 2014 / 0186401 and 2016 / 0184456; and International Application Publications WO 2008 / 091375 and WO 2012 / 149364). siRNAs targeting the autophagy genes Atg5 and Beclin1 have been delivered to tumor cells using Salmonella Typhimurium strains A1-R and VNP20009 (see, for example, Liu et al. (2016) Oncotarget 7(16):22873-22882).

[0503] However, these strains have been found to be ineffective at stimulating antitumor immune responses or at colonizing tumors to deliver therapeutic doses of encoded products. There is a need to improve these strains to make them more effective at stimulating antitumor immune responses, such as the immunostimulatory bacteria described herein. Further and alternative modifications to various bacteria are described in published International PCT application WO 2019 / 014398 and US Publication No. 2019 / 0017050A1. The bacteria described in each of these publications, and also described herein, can be modified as described herein to further improve their immunostimulatory and tumor-targeting properties.

[0504] iii.VNP20009

[0505] An example of a therapeutic bacterium that can be used as a starting strain for the modifications described herein is a strain called VNP20009 (ATCC#202165, YS1646). This virus is a clinical candidate virus. By deleting the msbB and purI genes, VNP20009 (ATCC#202165, YS1646) has a safety attenuation of at least 50,000-fold (see, for example, Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002; Low et al. (2003) Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy: Methods and Reviews, pp. 47-59; and Lee et al. (2000) International Journal of Toxicology 19:19-25). Preventing the loss or disruption of msbB gene expression alters the composition of the lipid A domain of lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria (see, for example, Lowet et al. (1999) Nat. Biotechnol. 17(1):37-41). This can prevent LPS-induced septic shock, attenuate bacterial strains and reduce systemic toxicity, while reducing the production of potentially harmful TNFα (see, for example, Dinarello, CA (1997) Chest 112(6Suppl):321S-329S; and Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Deletion or disruption of purI gene expression renders bacteria auxotrophic to purines, which further attenuates the bacteria and enriches them in the tumor microenvironment (see, for example, Pawelek et al. (1997) Cancer Res. 57:4537-4544; and Broadway et al. (2014) J. Biotechnology 192:177-178). As shown in this paper, VNP20009 is also auxotrophic to the immunosuppressive nucleoside adenosine. Adenosine can accumulate to pathologically high levels in tumors and contribute to the formation of an immunosuppressive tumor microenvironment (see, for example, Peter Vaupel and Arnulf Mayer, Oxygen Transport to Tissue XXXVII, Advances in Experimental Medicine and Biology 876 chapter 22, pp.177-183).

[0506] When VNP20009 was administered to mice carrying syngeneic or human xenograft tumors, the bacteria preferentially accumulated in the extracellular components of the tumor at a ratio of more than 300-1000 to 1 compared with control mice, and exhibited tumor growth inhibition and prolonged survival (see, for example, Clairmont et al. (2000) J. Infect. Dis. 181: 1996-2002). VNP20009 has been demonstrated in animal models to successfully target and inhibit tumor growth while inducing very low toxicity (see, for example, Broadway et al. (2014) J. Biotechnology 192:177-178; Loeffler et al. (2007) Proc. Natl. Acad. Sci. USA 104(31):12879-12883; Luo et al. (2002) Oncology Research 12:501-508; and Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002).

[0507] Phase 1 clinical trial results in human metastatic melanoma showed that while VNP20009 was relatively safe and well-tolerated, its observed antitumor activity was very limited (see, for example, Toso et al. (2002) J. Clin. Oncol. 20(1):142-152). Although the use of VNP20009 did not result in a change in metastatic disease burden, it did demonstrate evidence of tumor colonization at the maximum tolerated dose (MTD). Higher doses were not possible to achieve any antitumor activity due to toxicity associated with high levels of pro-inflammatory cytokines.

[0508] The immunostimulatory bacteria described herein offer numerous improvements and advantages lacking in strain VNP20009. These stimulatory bacteria deliver encoded genetic payloads to tumor-resident bone marrow cells in a tumor-specific manner. Through genomic modifications, such as gene deletion or disruption, and other genomic modifications, these stimulatory bacteria exhibit TLR2, TLR4, and TLR5-mediated reductions in inflammation, such as flagellar ablation, LPS modification, and the elimination of curly pili and reduction in biofilm formation. These immunostimulatory bacteria enhance T cell function, for example by eliminating L-asparaginase II expression, and promote, provide, allow, and support plasmid maintenance. The bacteria accumulate (or target) only, or essentially only, bone marrow cells, particularly tumor-resident bone marrow cells, providing highly efficient plasmid delivery following phagocytosis. The immunostimulatory bacteria described herein colonize the tumor microenvironment and can be administered systemically. Compared to VNP20009, the immunostimulatory bacteria described herein exhibit at least a 15-fold improvement in LD50. 50Therefore, higher doses of the immunostimulatory bacteria provided herein can be administered without toxicity, if necessary, compared to VNP20009 (see the table in section F.5 below describing dosage and administration).

[0509] This document shows and describes immunostimulatory bacteria modified as described herein, including flagellar ablation, LPS modification, and other modifications, which preferentially accumulate in or target bone marrow cells, particularly tumor-resident bone marrow cells. Examples of this document demonstrate the accumulation of immunostimulatory bacteria in these cells following systemic administration, such as intravenous administration. Examples also describe and demonstrate plasmid transfer from immunostimulatory bacteria to tumor-resident bone marrow cells, and persistent protein expression after bacterial cell death, thereby delivering therapeutic products, including those leading to anticancer responses and phenotypes.

[0510] iv. Wild-type strain

[0511] The accumulation of VNP20009 in tumors is caused by a combination of factors, including the inherent invasiveness of the parental strain ATCC 14028, its ability to replicate in hypoxic environments, and its need for high concentrations of purines present in the tumor stroma. As described herein, it is not necessary to use an attenuated strain such as VNP20009 as the starting bacterial strain. The bacteria are rendered non-virulent or attenuated through modifications as described herein. The parental strain ATCC 14028 or another wild-type strain can be used as the starting strain and modified as described herein.

[0512] 3. Limitations of existing bacterial cancer immunotherapies

[0513] Many classes of immunotherapies have significant limitations that restrict their safety and efficacy, as well as complex platforms that are unlikely to be widely used. Compared to, for example, oncolytic viruses, bacteria possess many advantageous properties for use as anticancer therapeutic agents. These properties include the ease with which bacteria can be spread, manufactured, stored, and eliminated from the host after treatment. However, viruses also possess advantageous properties, including host response. The response to bacterial infection is an innate inflammatory response, which is detrimental to anticancer therapy. The response to viral infection is similar to an anticancer response. The following table summarizes this (see also the overview above):

[0514]

[0515] Therefore, the limitations of bacteria as a microbial anticancer platform, compared to viral sensing pathways which are more similar to anticancer pathways, stem from specific immune programs that are initiated when the immune system senses bacteria, even intracellular bacteria. Viral sensing programs allow for highly effective vaccines and durable adaptive immunity. However, vaccination against bacteria has achieved limited success. For example, FDA-approved vaccines against typhoid fever are only 55% effective (see, for example, Hart et al. (2016) PLoSONE 11(1):e0145945), but Salmonella typhi, which contains highly immunogenic Vi capsules and O:9 antigens, does not appear in less immunogenic bacterial strains such as Listeria monocytogenes and Salmonella typhimurium, for which there are currently no vaccines.

[0516] Bacteria and viruses contain conserved structures called pathogen-associated molecular patterns (PAMPs), which are sensed by host cell pattern recognition receptors (PRRs). PRR recognition of PAMPs triggers a downstream signaling cascade, leading to the induction of cytokines and chemokines and initiating specific immune responses (see, for example, Iwasaki and Medzhitov (2010) Science 327(5963):291-295). The way PAMPs participate in the innate immune system and the type of infectious agent from which they originate determine whether an appropriate innate or adaptive response is generated to combat the invading pathogen.

[0517] A class of PRRs called Toll-like receptors (TLRs) recognize PAMPs derived from bacteria and viruses and are located in various compartments within the cell. TLRs can recognize a variety of ligands, including lipopolysaccharide (TLR4), lipoprotein (TLR2), flagellin (TLR5), unmethylated CpG motifs in DNA (TLR9), double-stranded RNA (TLR3), and single-stranded RNA (TLR7 and TLR8) (see, for example, Akira et al. (2001) Nat. Immunol. 2(8):675-680; and Kawai and Akira (2005) Curr. Opin. Immunol. 17(4):338-344). DNA- and RNA-based viruses can be sensed in the host cytoplasmic compartments after phagocytosis or directly in the cytoplasm. Type I interferons (IFN-α, IFN-β) are characteristic cytokines induced by host recognition of single- and double-stranded DNA and RNA (virally derived or derived from the uptake of damaged host cell DNA). For example, the synthetic dsRNA analog polyinosinic acid:polycytidylic acid (poly(I:C)) is an agonist of endosome TLR3 and a potent inducer of type I IFN, and its more stable version, polyICLC (e.g., under the trademark...), is also available. (Sales), and has been in clinical development (see, for example, Caskey et al. (2011) J.Exp.Med.208(12):2357-2366). Similarly, single-stranded RNA (ssRNA) in the body is sensed by TLR7 and TLR8 (in humans only), whose known synthetic ligands resiquimod and imiquimod are FDA-approved local cancer immunotherapies.

[0518] In the cytoplasm, double-stranded RNA (dsRNA) is sensed by RNA helicases, such as retinoic acid-induced gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5), thereby inducing type I interferon (see, for example, Ireton and Gale (2011) Viruses 3(6):906-919). Cytoplasmic sensors of dsDNA are mediated by interferon gene-stimulating factor (STING), an ER-resident connector that is a major mediator for sensing cytoplasmic dsDNA from infectious pathogens or abnormal host cell damage (see, for example, Barber (2011) Immunol. Rev. 243(1):99-108). STING signaling activates the TANK-binding kinase 1 (TBK1) / interferon regulatory factor 3 (IRF3) axis and the NF-κB signaling axis, thereby inducing IFN-β and other potent pro-inflammatory cytokines and chemokines that activate innate and adaptive immunity (see, for example, Burdette et al. (2011) Nature 478(7370):515-518). Sensing cytoplasmic dsDNA via STING requires circular GMP-AMP synthase (cGAS), a host cell nucleotide transferase that directly binds to dsDNA, which in response synthesizes the cyclic dinucleotide (CDN) second messenger circular GMP-AMP (cGAMP), which binds to and activates STING (see, for example, Sun et al. (2013) Science 339(6121):786-791; and Wu et al. (2013) Science 339(6121):826-830).

[0519] STING can also bind to bacterial-derived CDNs, such as c-di-AMP produced by *Listeria monocytogenes* or c-di-GMP produced by *Salmonella typhimurium*. It was later discovered that non-canonical CDNs produced by cGAS can activate human STING alleles that are unresponsive to bacterial-derived canonical CDNs. Unlike bacterial CDNs (where two purine nucleosides are linked by phosphate bridges with 3'-3' bonds), the nucleotide phosphate bridges in cGAMP synthesized by cGAS are linked by non-canonical 2'-3' bonds. These 2'-3' molecules have an affinity for STING that is 300 times higher than that of bacterial 3'-3' c-di-GMP, and are therefore more effective physiological ligands for STING (see, for example, Civril et al. (2013) Nature 498(7454):332-337; Diner et al. (2013) Cell Rep. 3(5):1355-1361; Gao et al. (2013) Sci. Signal 6(269):pl1; and Ablasser et al. (2013) Nature 503(7477):530-534). The cGAS / STING signaling pathway in humans appears to have evolved to preferentially respond to viral pathogens rather than bacterial pathogens.

[0520] Therefore, viral PRRs and TLRs, such as STING, RIG-I, TLR3, and TLR7 / 8, induce type I IFN, along with cytokines and chemokines, leading to effective T cell-mediated adaptive immunity. In the tumor setting, type I IFN signaling is required to induce T cell transport of chemokines, such as CXCL10, and also to activate DC cross-presentation of tumor antigens to elicit CD8+. + T cells (see, for example, Diamond et al. (2011) J. Exp. Med. 208 (10): 1989–2003; and Fuertes et al. (2011) J. Exp. Med. 208 (10): 2005–2016).

[0521] In contrast, host surveillance of bacteria such as Salmonella typhimurium is primarily mediated by TLR2, TLR4, and TLR5 (see, for example, Arpaia et al. (2011) Cell 144(5):675-688). These TLRs signal via the MyD88 (myeloid differentiation primary reactive protein 88) and TRIF (interferon-β induced by the Toll / interleukin-1 receptor (TIR) ​​domain containing an interferon-β connector) connector molecules to mediate the induction of NF-κB-dependent pro-inflammatory cytokines TNF-α and IL-6 (see, for example, Pandeye et al. (2015) Cold Spring Harb. Perspect. Biol. 7(1):a016246). Salmonella typhimurium has been shown to activate the NLRP3 inflammasome pathway, leading to the cleavage of caspase-1 and the induction of pro-inflammatory cytokines IL-1β and IL-18, thereby resulting in pyroptosis. The involvement of TLR2, TLR4, and TLR5, along with the activation of inflammasomes, induces chemokines and cytokines, leading to the clearance of bacteria by neutrophils and macrophages. For example, there is limited evidence of T cell clearance of Salmonella typhimurium, and antibodies against it are non-neutralizing (see, for example, McSorley (2014) Immunol. Rev. 260(1):168-182). Furthermore, Salmonella typhimurium possesses mechanisms that directly suppress T cell function, thereby weakening the generation of any potential antitumor T cell responses (see, for example, Kullas et al. (2012) Cell Host Microbe. 12(6) 791-798). As a result, bacterial cancer therapies such as Salmonella typhimurium lead to the recruitment and clearance by neutrophils and macrophages, which are not the T cells required to generate adaptive antitumor immunity. This article describes how these differences may explain why previous bacterial anticancer vaccines, even those containing host tumor antigens, have been poor T cell initiation vectors in humans.

[0522] These problems fall under the category of those addressed by the immunostimulatory bacteria described in this paper. The immunostimulatory bacteria presented in this paper are engineered to possess advantageous properties previously available only through viral therapy, while also retaining the advantageous properties of bacterial therapy. The bacteria presented in this paper can be administered systemically, targeting tumors, tumor-resident immune cells, and / or the tumor microenvironment, overcoming immunosuppression, and appropriately activating anti-tumor immunity, while also limiting the autoimmune-related toxicities of existing systemic immunotherapies. The immunostimulatory bacteria presented in this paper effectively target tumor-resident immune cells and encode therapeutic anticancer products, and can encode multiple such products. For example, the bacteria presented in this paper can encode complementary therapeutic products.

[0523] This article provides a superior microbial anticancer platform engineered to retain the beneficial properties of bacteria while inducing a virus-like immune response that elicits effective adaptive immunity. As described herein, bacteria, such as Salmonella and other bacterial strains, can be modified to have reduced inflammatory effects and thus lower toxicity. As a result, higher doses can be administered, for example. Any of these Salmonella strains, as well as strains of other bacterial species known to those skilled in the art and / or listed above and herein, can be modified as described herein. The immunostimulatory bacteria provided herein are modified to have increased tumor microenvironment, tumor-resident immune cells, and tumor colonization. The bacteria are engineered to have reduced toxicity and other properties that allow them to target the tumor microenvironment, including adenosine auxotrophy. The strains provided herein are also engineered to prevent complement inactivation.

[0524] An anticancer therapeutic product is provided that delivers a genetic payload encoding a truncated costimulatory molecule (receptor or ligand; e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, OX40L) expressed on antigen-presenting cells (APCs) with intact, truncated, or partially missing cytoplasmic domains. The truncated gene product is capable of participating in constitutive immunostimulatory signaling to T cells via the costimulatory receptor, and cannot generate counterregulatory signals to APCs due to the missing or truncated cytoplasmic domains. The costimulatory molecule can also be modified to include residues (e.g., positive residues) in the truncated cytoplasmic domains to ensure its correct orientation in the cell membrane (this is described in more detail in the following examples; see, for example, Example 19).

[0525] The bacterial strains described in this article are engineered to deliver therapeutic products. These strains deliver immunostimulatory proteins, including cytokines, chemokines, and co-stimulatory molecules, as well as modified gain-of-function cytoplasmic DNA / RNA sensors that can constitutively induce or elicit type I IFN expression, and other therapeutic products, such as, but not limited to, antibodies and their fragments, TGF-β and IL-6 binding decoy receptors, TGF-β peptide antagonists, and bispecific T-cell adaptor agents. RNAi, and its complementary combinations, promote tumor immune responses in the anti-tumor microenvironment. Bacterial strains also include genomic modifications that reduce phagocytic pyroptosis, thereby providing a more potent immune response and / or reducing or eliminating the ability to infect / invade epithelial cells while retaining the ability to infect / invade phagocytes, thus accumulating more effectively in tumors, the tumor microenvironment, and tumor-resident immune cells. The bacterial strains can also be modified to resist inactivation of complement factors in human serum. The bacterial strains can also be modified to encode therapeutic products, including, alone or in combination, such as cytokines, chemokines, co-stimulatory molecules, constitutive activity inducers of type I IFNs and immune checkpoints, and other monoclonal antibodies (and fragments thereof) targeting such sites.

[0526] C. Modification and enhancement of immune-stimulating bacteria to increase the therapeutic index and increase the accumulation of tumor-resident bone marrow cells.

[0527] This article provides methods for enhancing, including modifying bacterial genomes or immunostimulating bacteria to reduce toxicity and improve antitumor activity, for example by increasing accumulation in tumor-resident bone marrow cells, enhancing resistance to complement inactivation, reducing immune cell death, promoting adaptive immunity, and enhancing T cell function. The modifications described are specific to Salmonella, particularly Salmonella Typhimurium; it should be understood that similar enhancements / modifications can be achieved in other bacterial species and other Salmonella strains by those skilled in the art. Examples of such enhancements / modifications are given below.

[0528] 1. Gene deletions in the LPS biosynthetic pathway

[0529] Lipopolysaccharide (LPS) of Gram-negative bacteria is a major component of the bacterial exocellular membrane. It consists of three main parts: lipid A, a non-repeating core oligosaccharide, and the O antigen (or O polysaccharide). The O antigen is the outermost part of the LPS and acts as a protective layer against bacterial penetration; however, the sugar composition of the O antigen varies considerably between strains. Lipid A and the core oligosaccharide vary less and are more generally conserved within strains of the same species. Lipid A is the part of the LPS that contains endotoxin activity. It is typically a disaccharide decorated with various fatty acids. These hydrophobic fatty acid chains anchor the LPS to the bacterial membrane, while the remaining LPS protrudes from the cell surface. The lipid A domain is responsible for much of the virulence in Gram-negative bacteria. Typically, LPS in the blood is considered an important pathogen-associated molecular pattern (PAMP) and induces a significant pro-inflammatory response. LPS is a ligand for a membrane-bound receptor complex containing CD14, MD2, and TLR4. TLR4 is a transmembrane protein that signals via the MyD88 and TRIF pathways to stimulate the NF-κB pathway and lead to the production of pro-inflammatory cytokines such as TNF-α and IL-1β, potentially resulting in fatal endotoxic shock. LPS in the cytoplasm of mammalian cells can directly bind to the CARD domains of caspases 4, 5, and 11, leading to autoactivation and pyroptosis (see, for example, Hagar et al. (2015) Cell Research 25:149-150). The composition of lipid A and the toxicity of lipid A variants are well documented. For example, monophosphorylated lipid A is much less inflammatory than lipid A with multiple phosphate groups. The number and length of acyl chains on lipid A also greatly influence the degree of toxicity. Canonical lipid A from *E. coli* has six acyl chains, and this hexaacylation is highly toxic. Salmonella typhimurium lipid A is similar to that of Escherichia coli; it is a glucosamine disaccharide with four primary and two secondary hydroxyl acyl chains (see, for example, Raetz et al. (2002) Annu. Rev. Biochem. 71: 635-700).

[0530] a.msbB is missing.

[0531] The myristyltransferase enzyme, encoded by the msbB gene in *Salmonella typhimurium*, catalyzes the addition of a terminal myristyl group to the lipid A domain of lipopolysaccharide (LPS) (see, for example, Low et al. (1999) Nat. Biotechnol. 17(1):37-41). Therefore, the deletion of msbB alters the acyl composition of the lipid A domain of LPS, a major component of the outer membrane of Gram-negative bacteria. For instance, the deletion of msbB in *Salmonella typhimurium* strain VNP20009 results in the production of predominantly pentacetylated lipid A, which is less toxic than the naturally occurring hexaacylated lipid A and allows for systemic delivery without inducing toxic shock (see, for example, Lee et al. (2000) International Journal of Toxicology 19:19-25). This modification significantly reduces the ability of LPS to induce septic shock and attenuated bacterial strains, thereby improving the therapeutic index of Salmonella-based immunotherapies (see, for example, U.S. Patent Publications 2003 / 0170276, 2003 / 0109026, 2004 / 0229338, 2005 / 0255088, and 2007 / 0298012). Importantly, msbB mutants that do not express the msbB product cannot replicate intracellularly, as illustrated herein (see, for example, Example 2), which is a requirement for Salmonella virulence (see, for example, Leung et al. (1991) Proc. Natl. Acad. Sci. USA 88:11470-11474).

[0532] Other LPS mutations that alter LPS expression, including substitutions, deletions, or insertions, can be introduced into the bacterial strains provided herein, including Salmonella strains, thereby significantly reducing virulence, providing lower toxicity, and allowing for the administration of higher doses.

[0533] Genes encoding homologs or orthologs of myristoyltransferases, which are involved in lipid A biosynthesis in other bacterial species, can also be deleted or disrupted to achieve similar results. These genes include, but are not limited to, lpxM, which encodes a myristoyl-acyltransferase-dependent acyltransferase in *Escherichia coli*, and msbB, which encodes a lipid A acyltransferase in *Salmonella typhi*.

[0534] b. PagP deficiency

[0535] As mentioned above, the msbB mutant of *Salmonella typhimurium* cannot undergo terminal myristoylation of LPS and primarily produces pentacylated lipid A, which is far less toxic than hexaacylated lipid A. Modification of lipid A with palmitate is catalyzed by the enzyme lipid A palmityltransferase (PagP). Transcription of the pagP gene is controlled by the PhoP / PhoQ system, which is activated by low concentrations of magnesium, for example, within the SCV. Therefore, the acyl content of *Salmonella typhimurium* lipid A is variable; for wild-type bacteria, it can be hexaacylated or pentacylated. The ability of *Salmonella typhimurium* to palmitate its lipid A increases resistance to antimicrobial peptides secreted into phagolysosomes.

[0536] In wild-type Salmonella Typhimurium, expression of pagP leads to heptaacylated lipid A. In the msbB mutant (in which the terminal acyl chain of lipid A cannot be added), pagP induction leads to hexaacylated lipid A (see, for example, Kong et al. (2011) Infection and Immunity 79(12):5027–5038). Hexaacylated lipid A has been shown to be the most pro-inflammatory. Although the research team attempted to utilize this pro-inflammatory signal, for example by deleting or disrupting pagP to produce only hexaacylated lipid A (see, for example, Felgner et al. (2016) Gut Microbes 7(2):171-177; and Felgner et al. (2018) Oncoimmunology 7(2):e1382791), this could lead to poor tolerability and less adaptive immunity due to the TNF-α-mediated pro-inflammatory nature of LPS (see, for example, Kocijancic et al. (2017) Oncotarget 8(30):49988-50001).

[0537] LPS is a potent TLR4 agonist that induces TNF-α and IL-6. At 1E9 CFUs / m 2 Dose-limiting toxicities in intravenous VNP20009 clinical trials (see, for example, Toso et al. (2002) J. Clin. Oncol. 20(1):142-152) were cytokine-mediated (fever, hypotension), with serum TNF-α levels >100,000 pg / ml and IL-6 levels >10,000 pg / ml at 2 hours. Despite the loss of msbB and reduced pyrogenicity in VNP20009, LPS may still be toxic at high doses, possibly due to the presence of hexaacylated lipid A. Therefore, pagP - / msbB -The strain cannot produce hexaacylated lipid A, only pentacylated lipid A, resulting in lower induction of pro-inflammatory cytokines, better tolerability at higher doses, and allowing for induction at or above 1E9 CFUs / m³. 2 The dosage is administered to the human body. Higher doses lead to increased colonization of tumors, tumor-resident immune cells, and the tumor microenvironment, thereby enhancing the therapeutic efficacy of the immunostimulatory bacteria. Because of the resulting changes in the bacterial membrane and structure, host immune responses such as complement activity are altered, thus the bacteria are not eliminated upon systemic administration. For example, pagP is shown herein (see Example 5). - / msbB - The mutant strain exhibits increased resistance to complement inactivation and increased stability in human serum.

[0538] This article presents immunostimulatory bacteria, such as live attenuated Salmonella strains, such as the example Salmonella Typhimurium strain, that produce only LPS with pentacylated lipid A, containing a deletion of the msbB gene, and further modified by deletion / disruption of pagP. As mentioned above, the deletion of msbB expression prevents terminal myristylation of lipid A, while the deletion of pagP expression prevents palmitoylation. Strains modified to produce LPS with pentacylated lipid A, when further modified to express heterogeneous genetic payloads that stimulate immune responses in the tumor microenvironment, exhibit reduced levels of pro-inflammatory cytokines, increased blood stability, complement fixation resistance, increased sensitivity to antimicrobial peptides, enhanced tolerance, and increased antitumor immunity.

[0539] Genes encoding homologs and orthologs of lipid A palmityltransferase (PagP) in other bacterial species can also be deleted or disrupted to achieve similar results. These genes include, but are not limited to, pagP encoding lipid IVA palmityltransferase in Escherichia coli; and pagP encoding antimicrobial peptide resistance and lipid A acylation protein in Salmonella typhi.

[0540] 2. Nutritional Deficiency Type

[0541] The immunostimulatory bacteria described herein can be attenuated by making them auxotrophic to one or more essential nutrients, such as purines (e.g., adenine), nucleosides (e.g., adenosine), amino acids (e.g., aromatic amino acids, arginine, and leucine), adenosine triphosphate (ATP), or other nutrients known and described in the art.

[0542] a.purI missing / damaged

[0543] Phosphoribose-aminoimidazole synthase is an enzyme encoded by the purI gene (synonymous with the purM gene) that participates in the biosynthesis of purines. Destruction, deletion, or inactivation of the purI gene renders bacteria purine-deficient. Besides being attenuated, purI... - The mutants are enriched in the tumor environment and exhibit significant antitumor activity (see, for example, Paweleket et al. (1997) Cancer Research 57:4537-4544). This colonization has previously been described as being due to high concentrations of purines in the tumor interstitial fluid, resulting from rapid tumor cell turnover. Due to purI... - The bacteria cannot synthesize purines and require adenine from external sources, which is thought to limit their growth in purine-rich tumor microenvironments (see, for example, Rosenberg et al. (2002) J. Immunotherapy 25(3):218-225). Although the VNP20009 strain was initially reported to contain a deletion of the purI gene (see, for example, Low et al. (2003) Methods in Molecular Medicine Vol.90, Suicide Gene Therapy: Methods and Reviews, pp.47-59), subsequent studies of the entire VNP20009 genome showed that the purI gene was not deleted, but rather disrupted by a chromosomal inversion (see, for example, Broadway et al. (2014) Journal of Biotechnology 192:177-178). The entire purI gene is contained in two parts of the VNP20009 chromosome, flanked by insertion sequences, one of which contains an active transposase. While disruption of the purI gene restricts replication within the tumor tissue / microenvironment, it still allows for intracellular replication and virulence. As illustrated herein (see Example 2), deletion or disruption of each of the msbB and purI genes is essential for limiting extracellular growth within tumor tissue and preventing intracellular replication. This document provides strains in which the coding portions of these genes are completely deleted to eliminate any possibility of reverting to wild-type via recombination.

[0544] Besides the deletion or disruption of the purI gene, nutrient auxotrophy can be introduced into immunostimulatory bacteria through deletions / mutations of genes such as aro, gua, thy, nad, and asd. Nutrients produced by biosynthetic pathways involving these genes are generally unavailable in host cells, thus posing a challenge to bacterial survival. For example, attenuation of Salmonella and other bacterial substances can be achieved by deletion of the aroA gene, which is part of the shikimic acid pathway that links glycolysis with the biosynthesis of aromatic amino acids (see, for example, Felgner et al. (2016) mBio7(5):e01220-16). Deletion of aroA results in bacterial auxotrophy of aromatic amino acids and subsequent attenuation (see, for example, US Patent Publications 2003 / 0170276, 2003 / 0175297, 2012 / 0009153 and 2016 / 0369282; and International Application Publications WO2015 / 032165 and WO2016 / 025582). Similarly, other enzymes involved in the biosynthesis of aromatic amino acids, including aroC and aroD, have been missing to achieve attenuation (see, for example, US Patent Publication No. 2016 / 0369282; and International Application Publication No. WO2016 / 025582). For example, Salmonella Typhimurium strain SL7207 is an aromatic amino acid auxotroph (aroA...). - (Mutants); A1 and A1-R strains are leucine-arginine auxotrophs; VNP20009 / YS1646 is a purine auxotroph (purine-arginine auxotroph). - (Mutant). As shown in this article, VNP20009 / YS1646 is also an auxotroph for the immunosuppressive nucleoside adenosine and ATP (see Example 1).

[0545] The corresponding genes encoding homologs or orthologs of phosphoribosylaminoimidazolium synthase (PurI), as well as other genes required for purine synthesis in other bacterial species, can also be deleted or disrupted to obtain similar results. These genes include, but are not limited to, for example, purM, which encodes phosphoribosylglycine cyclic ligase in *Escherichia coli*; purM, which encodes phosphoribosylglycine cyclic ligase in *Salmonella typhi*; purA, which encodes adenosine succinate synthase; purQ, which encodes phosphoribosylglycine synthase II; and purS, which encodes the phosphoribosylglycine synthase subunit PurS in *Listeria monocytogenes*; purM(BL1122), which encodes phosphoribosylglycine cyclic ligase in *Bifidobacterium longum*; and NT01CX_RS09765, which encodes AIR synthase; and NT01CX_RS07625(purM), which encodes phosphoribosylglycine cyclic ligase in *Clostridium novyi*.

[0546] b. Adenosine auxotrophic type

[0547] Metabolites derived from the tryptophan and adenosine triphosphate (ATP) / adenosine pathway are key drivers of the immunosuppressive environment within the tumor / tumor microenvironment (TME). Adenosine, existing in free form both intracellularly and extracellularly, is an effector of immune function. Adenosine reduces T cell receptor-induced NF-κB activation and inhibits IL-2, IL-4, and IFN-γ. Adenosine reduces T cell cytotoxicity, increases T cell immunodeficiency, and increases T cell differentiation into Foxp3. + Or Lag-3 + Regulatory T cells (T-reg cells, T-regs, or Tregs). For natural killer (NK) cells, adenosine reduces IFN-γ production and inhibits NK cell cytotoxicity. Adenosine blocks neutrophil adhesion and extravasation, reduces phagocytosis, and attenuates superoxide and nitric oxide levels. Adenosine also reduces the expression of TNF-α, IL-12, and MIP-1α (CCL3) on macrophages, attenuates the expression of major histocompatibility complex (MHC) class II, and increases the levels of IL-10 and IL-6. Adenosine's immunomodulatory activity occurs after it is released into the extracellular space of the tumor and activates adenosine receptors (ADRs) on the surface of target immune cells, cancer cells, or endothelial cells. High adenosine levels in the tumor microenvironment lead to local immunosuppression, which limits the immune system's ability to eliminate cancer cells.

[0548] Extracellular adenosine is produced by the successive activity of membrane-associated extracellular enzymes CD39 (extracellular nucleoside diphosphate hydrolase 1, or NTPDase 1) and CD73 (extracellular 5'-nucleotidase), which are expressed on tumor stromal cells and co-produce adenosine through the phosphorylation of ATP or ADP produced by dead or dying cells. CD39 converts extracellular ATP (or ADP) into 5'AMP, which is then converted into adenosine by CD73. The expression of CD39 and CD73 on endothelial cells increases under hypoxic conditions in the tumor microenvironment, thereby increasing adenosine levels. Tumor hypoxia can be caused by insufficient blood supply and tumor vascular system disturbances, thereby impairing oxygen delivery (see, for example, Carroll and Ashcroft (2005) Expert. Rev. Mol. Med. 7(6), DOI: 10.1017 / S1462399405009117). Hypoxia occurring in the tumor microenvironment also inhibits adenosine kinase (AK), which converts adenosine to AMP, resulting in very high extracellular adenosine concentrations. Extracellular adenosine concentrations in hypoxic tumor microenvironments have been measured at 10–100 μM, which is approximately 100–1000 times higher than the typical extracellular adenosine concentration of about 0.1 μM (see, for example, Vaupel et al. (2016) Adv. Exp. Med. Biol. 876:177–183; and Antonioli et al. (2013) Nat. Rev. Can. 13:842–857). Because hypoxic areas within a tumor are far from microvessels, localized adenosine concentrations in certain regions of the tumor may be higher than in others.

[0549] To guide the effects of suppressing the immune system, adenosine can also control the growth and spread of cancer cells by influencing their proliferation, apoptosis, and angiogenesis. For example, adenosine can primarily stimulate A... 2A and A 2B Receptors promote angiogenesis. Stimulation of receptors on endothelial cells can regulate the expression of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on endothelial cells, maintaining vascular integrity and promoting angiogenesis (see, for example, Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Adenosine activates A receptors on various cells. 2A A 2B One or more of A3 can stimulate the production of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), or angiopoietin-2 (see, for example, Antonioli et al. (2013) Nat. Rev. Can. 13: 842-857).

[0550] Adenosine can also directly regulate tumor cell proliferation, apoptosis, and metastasis by interacting with receptors on cancer cells. For example, studies have shown that A1 and A... 2A Activation of receptors promotes tumor cell proliferation in certain breast cancer cell lines, and A 2B Activation of the receptor has a cancer-promoting property in colon cancer cells (see, for example, Antonioli et al. (2013) Nat. Rev. Can. 13:842-857). Adenosine can also trigger apoptosis in cancer cells, and various studies have linked this activity to activation via the extrinsic apoptosis pathway through A3 or via A... 2A and A 2B It is associated with the activation of the intrinsic apoptosis pathway (see, for example, Antonioli et al. (2013)). Adenosine can promote the migration and metastasis of tumor cells by increasing cell motility, adhesion to the extracellular matrix, and the expression of cell adhesion proteins and receptors to promote cell movement and motility.

[0551] Extracellular release of adenosine triphosphate (ATP) occurs in stimulated immune cells as well as damaged, dying, or stressed cells. Upon stimulation by this ATP release, the inflammasome containing the NLR family pyrin domain 3 (NLRP3) activates caspase-1, leading to the secretion of the cytokines IL-1β and IL-18, which in turn activate innate and adaptive immune responses (see, for example, Stagg and Smyth (2010) Oncogene 29:5346–5358). ATP can accumulate in tumor tissues at concentrations exceeding 100 mM, while levels found in healthy tissues are very low, around 1–5 μM (see, for example, Song et al. (2016) Am.J. Physiol. Cell Physiol. 310(2):C99–C114). ATP is metabolized to adenosine by the enzymes CD39 and CD73. Activated adenosine acts as a highly immunosuppressive metabolite through a negative feedback mechanism and exhibits pleiotropic effects on multiple immune cell types in the hypoxic tumor microenvironment (see, for example, Stagg and Smyth (2010) Oncogene 29:5346–5358). Adenosine receptor A 2A and A 2BAdenosine is expressed on a variety of immune cells and stimulated by adenosine to promote cAMP-mediated signaling changes, resulting in an immunosuppressive phenotype in T cells, B cells, NK cells, dendritic cells (DCs), mast cells, macrophages, neutrophils, and natural killer (NKT) cells. Consequently, adenosine levels can accumulate to more than 100 times their normal concentration in pathological tissues such as solid tumors, which have shown overexpression of extracellular nucleotidases such as CD73. Adenosine has also been shown to promote tumor angiogenesis and development. Therefore, engineered bacteria that are auxotrophic to adenosine will exhibit enhanced tumor targeting and colonization.

[0552] Immunostimulatory bacteria, such as *Salmonella typhi*, can be made auxotrophic to adenosine by, for example, by deleting the tsx gene (see, for example, Bucarey et al. (2005) Infection and Immunity 73(10):6210–6219) or by deleting purD (see, for example, Husseiny (2005) Infection and Immunity 73(3):1598–1605). In the Gram-negative bacterium *Xanthomonas oryzae*, purD gene knockout strains have shown auxotrophic symptom to adenosine (see, for example, Park et al. (2007) FEMS Microbiol. Lett. 276:55–59). As exemplified in this paper, *Salmonella typhimurium* strain VNP20009 is auxotrophic to adenosine due to the deletion of its purI gene, and therefore no further modification is needed to make it auxotrophic to adenosine. Therefore, the implementation scheme of the immunostimulatory bacterial strains provided in this paper is auxotrophic to adenosine. These auxotrophic bacteria selectively replicate in the tumor microenvironment, further increasing the accumulation and replication of the applied bacteria in the tumor and reducing the levels of adenosine within and around the tumor, thereby reducing or eliminating immunosuppression caused by adenosine accumulation. An example of such bacteria provided in this paper is one containing purI... - / msbB - Mutations were made to provide modified strains of *Salmonella typhimurium* that provide adenosine auxotrophic nutrition. For other strains and bacteria, the purI gene can be disrupted as in VNP20009, or it can contain a complete or partial deletion of the purI gene, ensuring no reversion to the wild-type gene. As described elsewhere in this document, in strain VNP20009, purI... - The gene is inactivated by inversion. Similarly, the msbB gene in VNP20009 is not completely deleted. As exemplified in this paper, strains in which both purI and msbB genes have been completely deleted to eliminate any risk of inversion show excellent fitness in in vitro culture growth assessments.

[0553] Immunostimulating bacteria are modified to be auxotrophic to one or more essential nutrients such as purines (e.g., adenine), nucleosides (e.g., adenosine), amino acids (e.g., aromatic amino acids, arginine, and leucine), or adenosine triphosphate (ATP). Specifically, in embodiments of the immunostimulating bacteria, such as *Salmonella typhimurium* strains provided herein, the bacteria are made auxotrophic to adenosine and optionally to ATP, and preferentially accumulate in the tumor microenvironment (TME). Therefore, the immunostimulating bacterial strains described herein are attenuated because they require purines, adenosine, and / or ATP to grow, and they preferentially colonize TMEs rich in these metabolites, as described below. Since adenosine accumulation in the tumor microenvironment of certain tumors has immunosuppressive effects, adenosine auxotrophy eliminates the immunosuppressive effects of adenosine accumulation in the tumor microenvironment of certain cancers.

[0554] 3. Plasmid maintenance and delivery

[0555] a.asd missing

[0556] The *asd* gene in bacteria encodes aspartate-semialdehyde dehydrogenase. *Salmonella typhimurium* *asd*... - The mutant exhibits a specific requirement for diaminopimelic acid (DAP), which is essential for cell wall synthesis, and will lyse in a DAP-deprived environment. This DAP trophic deficiency can be used for plasmid selection and maintenance of in vivo plasmid stability when the asd gene is trans-complemented on a bacterial plasmid without the use of antibiotics. Antibiotic-free plasmid selection systems are advantageous and allow for 1) the use of antibiotics as a rapid clearance mechanism in the event of adverse symptoms, and 2) the use of antibiotics for large-scale production in situations where such use is generally avoided. The asd gene complementation system provides for this antibiotic-free plasmid selection (see, for example, Galán et al. (1990) Gene 94(1):29-35). The aim is to use the asd gene complementation system to maintain plasmids in the tumor microenvironment to increase the efficacy of Salmonella typhimurium engineered to deliver plasmids encoding genetic payloads / therapeutic products, such as immunostimulatory proteins (e.g., cytokines, chemokines, co-stimulatory molecules); cytoplasmic DNA / RNA sensors that induce type I IFN, such as STING and IRF3 and their gain-of-function / constitutive active mutants; antibodies and their fragments (e.g., checkpoint inhibitors, or anti-IL-6 or anti-VEGF antibodies); and bispecific T-cell connectors (branded...). (Sales); interfering RNA; and other therapeutic products discussed in other parts of this document and known in the art; and complementary combinations of all the aforementioned therapeutic products.

[0557] An alternative use of the asd mutant of *Salmonella typhimurium* is to generate autolytic (or suicide) strains that utilize the auxotrophic nature of DAP, enabling the delivery of therapeutic products / molecules to infected cells without the ability to persistently colonize host tumors. The deletion of the asd gene renders the bacteria auxotrophic to DAP during in vitro or in vivo growth. The examples described herein provide an asd-deficient strain that is auxotrophic to DAP and contains a plasmid suitable for delivering immunostimulatory proteins, which does not contain the asd complementary gene, resulting in a strain with in vivo replication defects. This strain multiplies and grows normally in vitro in the presence of DAP and is then administered as an immunotherapeutic agent to a mammalian host lacking DAP. The suicide strain is able to invade host cells but cannot replicate due to the absence of DAP in mammalian tissues, thus automatically lysing and delivering its cytoplasmic contents (e.g., plasmids or proteins).

[0558] The corresponding genes encoding homologs or orthologs of aspartate-halfaldehyde dehydrogenase (asd) in other bacterial species can also be deleted or disrupted to obtain similar results. These genes include, but are not limited to, asd, encoding aspartate-halfaldehyde dehydrogenase in *Escherichia coli*; asd(STY4271), encoding aspartate-halfaldehyde dehydrogenase in *Salmonella typhi*; asd(lmo1437), encoding aspartate-halfaldehyde dehydrogenase in *Listeria monocytogenes*; asd(BL0492), encoding aspartate-halfaldehyde dehydrogenase in *Bifidobacterium longum*; and NT01CX_RS04325(asd), encoding aspartate-halfaldehyde dehydrogenase in *Clostridium novyi*.

[0559] b.endA is missing / damaged

[0560] The endA gene (e.g., SEQ ID NO:250) encodes a nuclease (a DNA-specific nuclease, see, for example, SEQ ID NO:251) that mediates the degradation of double-stranded DNA (dsDNA) in the periplasm of Gram-negative bacteria. The most common strain of *E. coli* in the laboratory is endA-, as mutations in the endA gene allow for higher yields of plasmid DNA. This gene is conserved across species. To facilitate the delivery of intact plasmid DNA, the endA gene in engineered, immunostimulated bacteria is deleted or mutated to prevent its nuclease activity. Examples of device mutations are the E208K amino acid substitution (see, for example, Durfee et al. (2008) J. Bacteriol. 190(7):2597-2606) or the corresponding mutation in the species of interest. endA, including E208, is conserved in bacterial species, including *Salmonella*. Therefore, the E208K mutation can be used to eliminate nuclease activity in other species, including *Salmonella* species. Those skilled in the art can introduce other mutations or deletions to eliminate endA activity. In the immunostimulatory bacteria described herein, such as Salmonella, this mutation, deletion, or disruption of the gene to eliminate endA activity improves the efficiency of delivery of intact plasmid DNA, thereby increasing the expression of one or more immunomodulatory proteins / therapeutic products encoded on the plasmid and enhancing antitumor response and efficacy.

[0561] 4. Flagellin knockout strains

[0562] Flagella are organelles on the surface of bacteria that consist of filaments connected by hooks to a rotating motor that can rotate clockwise or counterclockwise to provide a means of translocation. Flagella, for example in Salmonella typhimurium, are important for chemotaxis and for establishing infection via the oral route due to their ability to mediate motility across the mucus layer of the gastrointestinal tract. While flagella have been shown to be essential for chemotaxis and in vitro colonization of tumor ellipsoids (see, for example, Kasinskas and Forbes (2007) Cancer Res. 67(7):3201-3209), and motility has been shown to be important for tumor penetration (see, for example, Toley and Forbes (2012) Integr. Biol. (Camb) 4(2):165-176), flagella are not essential for tumor colonization in animals when bacteria are administered intravenously (see, for example, Stritzker et al. (2010) International Journal of Medical Microbiology 300:449–456). Each flagellum filament is composed of tens of thousands of flagellin subunits. The Salmonella typhimurium chromosome contains two genes, fliC and fljB, which encode flagellin monomers with different antigens. When administered orally, mutants deficient in both fliC and fljB are immobile and non-toxic, but retain toxicity when administered parenterally.

[0563] Flagellin is a major pro-inflammatory determinant of Salmonella (see, for example, Zeng et al. (2003) J. Immunol. 171:3668-3674) and is directly recognized by TLR5 on the cell surface and NLCR4 in the cytosol (see, for example, Lightfield et al. (2008) Nat. Immunol. 9(10):1171–1178). Both pathways lead to a pro-inflammatory response, resulting in the secretion of cytokines, including IL-1β, IL-18, TNF-α, and IL-6. An attempt was made to enhance the pro-inflammatory response to flagellin by engineering bacteria to secrete Vibrio vulnificus flagellin B, thereby making Salmonella-based cancer immunotherapy more effective. Vibrio vulnificus flagellin B induces greater inflammation compared to flagellins encoded by fliC and fljB (see, for example, Zheng et al. (2017) Sci. Transl. Med. 9 (376): eaak9537).

[0564] In this study, the Salmonella spp. *Salmonella typhimurium* was engineered to lack the flagellated protein subunits *fliC* and *fljB* to reduce TLR5-mediated pro-inflammatory signaling. Other flagellated bacteria can also be engineered to eliminate the flagella. For example, as shown in this study, Salmonella strains lacking *msbB* and / or *pagP* (which lead to reduced TNF-α induction) were combined with *fliC* and *fljB* knockout. This resulted in Salmonella strains with combined reduced TNF-α induction and reduced TLR5 recognition. These bacteria were modified with *msbB*. - pagP - fliC - and fljB - These modifications can be combined with immunostimulatory plasmids, optionally containing CpG, which encode therapeutic products, such as immunomodulatory proteins, either alone or in combination. The resulting bacteria exhibit reduced pro-inflammatory signaling but potent antitumor activity. These genomic modifications can also be combined with other genomic modifications described herein.

[0565] For example, as illustrated and provided in this paper, a double mutant of fliC and fljB was constructed in the asd-deficient strain of Salmonella Typhimurium, VNP20009. VNP20009 attenuates virulence by disrupting purI / purM and contains a modification (partial deletion) of the msbB gene, resulting in the production of lipid A subunits with lower virulence than wild-type lipid A. This leads to reduced TNF-α production in a mouse model after intravenous administration compared to strains with wild-type lipid A. The resulting strain is an example of bacterial inflammation attenuation achieved by modifying lipid A to reduce TLR2 / 4 signaling and deleting flagellin subunit expression to reduce TLR5 recognition and inflammasome induction.

[0566] The pathogenesis of certain bacterial species, including Salmonella species such as *Salmonella typhimurium*, involves a group of genes known as Salmonella pathogenic islands (SPIs). Salmonella uses a type 3 secretion system (T3SS) encoded by Salmonella pathogenic island 1 (SPI-1) to invade non-phagocytic intestinal epithelial cells, forming needle-like structures that inject effector proteins directly into the host cell's cytoplasm. These effector proteins cause a rearrangement of the eukaryotic cytoskeleton to facilitate invasion of the intestinal epithelium and also induce pro-inflammatory cytokines. SPI-1 mediates the invasion of epithelial cells. SPI-1 genes include, but are not limited to: avrA, hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP. Deletion of one or more of these genes reduces or eliminates the ability of bacteria to infect epithelial cells, but does not affect their ability to infect or invade phagocytes (including phagocytes of immune cells). For example, it has been shown that simultaneous deletion of the fliC and fljB genes significantly reduces the expression of SPI-1 genes, such as hilA, hilD, invA, invF and sopB, thereby reducing their ability to invade non-phagocytic cells (see, for example, Elhadad et al. (2015) Infect. Immun. 83(9):3355-3368).

[0567] In bacteria such as Salmonella, flagellin, in addition to the SPI-1 type 3 secretion system (T3SS), is essential for initiating macrophage pyroptosis and can be detected by the macrophage NLRC4 inflammasome. Elimination of flagellin subunits reduces macrophage pyroptosis. For example, compared to wild-type strains, Salmonella Typhimurium with deletions of fliC and fljB resulted in significantly reduced IL-1β secretion, while bacterial uptake and intracellular replication were unaffected. This suggests that flagellin plays an important role in inflammasome activation. In addition, it was found that engineered Salmonella Typhimurium strains constitutively expressing fliC could induce macrophage pyroptosis (see, for example, Li et al. (2016) Scientific Reports 6:37447; Fink and Cookson (2007) Cellular Microbiology 9(11):2562-2570; and Winter et al. (2015) Infect. Immun. 83(4):1546-1555).

[0568] The genomes of the immunostimulatory bacteria described in this article can be modified to delete or mutate the flagellin genes fliC and fljB in *Salmonella typhimurium*, thereby reducing cell death in tumor-resident immune cells (e.g., macrophages) and enhancing the antitumor immune response of the immunostimulatory bacteria. The combination of flagellin subunit deletion and LPS modification can improve host tolerance, limit uptake by phagocytes alone and reduce their pyroptosis, and direct the immunostimulatory response to the delivery of therapeutic products such as immunomodulatory proteins to the TME, particularly tumor-resident bone marrow cells. The resulting immunostimulatory bacteria induce antitumor responses and promote adaptive immune responses against tumors.

[0569] Genes encoding flagellated proteins in other bacterial species can also be deleted to obtain similar results. These genes include, but are not limited to, for example, in *Escherichia coli*, *fliC*, which encodes a flagellated filament protein, and *fliE*, which encodes a flagellated basal body protein; in *Salmonella typhi*, *fliC*, which encodes a flagellated protein, and *flgB*, which encodes a flagellated basal body rod protein; in *Listeria monocytogenes*, *flaA*, which encodes a flagellated protein, *fliE*, which encodes a flagellated hook basal body protein, and *flgB*, which encodes a flagellated basal body rod protein; and in *Clostridium nodosum*, NT... 01CX_RS04995, NT01CX_RS04990, NT01CX_RS05070 and NT01CX_RS05075 encode flagellin, NT01CX_RS05080(flgB) encodes flagellin basal rod protein FlgB, NT01CX_RS05085(flgC) encodes flagellin basal rod protein FlgC, and NT01CX_RS05215(flgG) encodes flagellin basal rod protein FlgG.

[0570] 5. Engineered bacteria to promote adaptive immunity and enhance T cell function

[0571] L-asparaginase II (ansB) deficiency / disruption

[0572] L-Asparaginase II is an enzyme that catalyzes the conversion of L-asparagine to ammonia and aspartic acid. Some bacterial strains, such as *Escherichia coli* and *Salmonella typhimurium*, utilize L-asparaginase to remove fructose-asparagine as a carbon and nitrogen source (see, for example, Sabag-Daigle et al. (2018) *Appl. Environ. Microbiol.* 84(5):e01957-17). Malignant T cells, such as those in acute lymphoblastic leukemia (ALL), require asparagine because these T cells lack the enzyme to synthesize it. Administration of L-asparaginase has been a first-line therapy for ALL since the early 1970s (see, for example, Batool et al. (2016) *Appl. Biochem. Biotechnol.* 178(5):900-923). L-asparaginase II production by *Salmonella typhimurium* is necessary and sufficient for T cell suppression because it directly induces downregulation of the T cell receptor (TCR), reduces the production of T cell cytokines, and inhibits tumor cell lysis (see, for example, Kullas et al. (2012) Cell Host Microbe. 12(6) 791-798; and van der Velden et al. (2005) Proc. Natl. Acad. Sci. USA 102(49): 17769-17774). Under conditions of rapid clonal expansion, such as those occurring during T cell activation in the tumor microenvironment, asparagine is essential, and its consumption by L-asparaginase II leads to T cell suppression. Therefore, L-asparaginase II has been used as an anticancer therapeutic agent in cancer, where T cell suppression is a therapeutic modality.

[0573] Contrary to existing uses of L-asparaginase as an anticancer therapeutic agent, this paper demonstrates that the elimination of L-asparaginase activity in immunostimulatory bacteria provided herein enhances T cell function in the tumor microenvironment. Elimination of L-asparaginase activity can be achieved by modifying the bacterial genome to eliminate the expression of the active enzyme. Modifications include nucleic acid insertion, deletion, inversion, and substitution, resulting in an inactive, unexpressed, or eliminated encoded enzyme. This paper demonstrates that the complete or partial deletion or disruption of the gene ansB encoding L-asparaginase II to eliminate the expression of the encoded enzyme in immunostimulatory bacteria enhances T cell function in the tumor microenvironment colonized by the bacteria. Inhibition of L-asparaginase II activity is achieved through the complete or partial deletion or interruption / disruption of the gene ansB in immunostimulatory bacteria, thereby preventing the production of L-asparaginase II. Therefore, immunostimulatory bacteria whose genomes are modified to prevent the production of L-asparaginase II are provided. The immunostimulatory bacteria described in this article are used to colonize tumor-resident immune cells to enhance anti-tumor immune responses; the genomic modifications include deletion, insertion, disruption and / or elimination of L-asparaginase II expression and other modifications.

[0574] As illustrated herein, the genomes of the immunostimulatory bacteria described herein can be modified to delete, disrupt, or otherwise modify ansB, resulting in the inactivation of the encoded L-asparaginase II, or the elimination of asparaginase, preventing T cell suppression and enhancing anti-tumor T cell function in vivo. This paper demonstrates that strains with intact ansB induce significant T cell immunosuppression in T cells infected with this strain. Strains with deleted ansB do not induce immunosuppression, thus addressing another problem in the art regarding the delivery of encoded therapeutic products to tumors using bacteria. Therefore, immunostimulatory bacteria combining the deletion or disruption of the ansB gene, thereby preventing the expression of a functionally encoded enzyme, with other modifications described herein that lead to increased accumulation in the tumor microenvironment and / or tumor-resident immune cells, provide superior therapeutic immunostimulatory bacteria.

[0575] In other bacterial species, the corresponding genes encoding homologs or orthologs of L-asparaginase II (ansB) can also be deleted or disrupted to obtain similar results. These genes include, but are not limited to, for example: ansB, which encodes L-asparaginase 2 in *Escherichia coli*; ansB(STY3259), which encodes L-asparaginase in *Salmonella typhi*; ansB(lmo1663), which encodes L-asparagine synthase in *Listeria monocytogenes*; and BL1142, which encodes an L-asparaginase precursor in *Bifidobacterium longum*.

[0576] 6. Salmonella gene deletion / destruction required for expression of curly pili

[0577] Bacteria and fungi can form multicellular structures called biofilms. Bacterial biofilms are encapsulated within a mixture of secreted, cell wall-associated polysaccharides, glycoproteins, glycolipids, and extracellular DNA, collectively known as extracellular polymers. These extracellular polymers protect bacteria from a variety of attacks, such as detergents, antibiotics, and antimicrobial peptides. Bacterial biofilms allow for surface colonization and are a cause of severe infection of prosthetics (e.g., injection sites and catheters). Biofilms can also form in tissues during infection, leading to prolonged bacterial persistence and shedding, and limiting the effectiveness of antibiotic treatment. Prolonged bacterial presence in biofilms has been associated with increased tumorigenesis, for example in Salmonella typhi infection of the gallbladder (see, for example, Di Domenico et al. (2017) Int. J. Mol. Sci. 18: 1887).

[0578] In Salmonella, such as *Salmonella typhimurium*, biofilm formation is regulated by csgD, which activates the csgBAC operon and leads to increased production of the curled pili subunits CsgA and CsgB (see, for example, Zakikhany et al. (2010) *Molecular Microbiology* 77(3):771–786). CsgA is recognized as a PAMP by TLR2 and induces the production of IL-8 in human macrophages (see, for example, Tukel et al. (2005) *Molecular Microbiology* 58(1):289–304). Furthermore, csgD indirectly increases cellulose production by activating the adrA gene, which encodes diguanylate cyclase. The small cyclic diguanylate monophosphate (c-di-GMP) produced by adrA is a ubiquitous secondary messenger found in almost all bacterial species. Increased c-di-GMP enhances the expression of the cellulose synthase gene bcsA, which in turn increases cellulose production by stimulating the bcsABZC and bcsEFG operons, leading to cellulose biofilm formation. Thus, bacteria, such as Salmonella typhimurium, can form biofilms in solid tumors to prevent phagocytosis by host immune cells. Mutants of bacteria that cannot form biofilms, such as Salmonella mutants, are more readily absorbed by host phagocytes and more easily cleared from infected tumors (see, for example, Crull et al. (2011) Cellular Microbiology 13(8):1223-1233). This increased intracellular localization within phagocytes can reduce the persistence of extracellular bacteria and, as shown in this paper, can enhance the effectiveness of plasmid delivery of therapeutic products such as immunomodulatory proteins and other anticancer agents described herein. The reduced ability of immune-stimulated bacteria, such as Salmonella typhimurium, to form biofilms can be achieved by the deletion or disruption of genes involved in biofilm formation, such as csgD, csgA, csgB, adrA, bcsA, bcsB, bcsZ, bcsE, bcsF, bcsG, dsbA, or dsbB (see, for example, Anwar et al. (2014) PLoS ONE 9(8):e106095).

[0579] This article demonstrates that engineering immunostimulatory bacteria to reduce biofilm formation can improve clearance from tumors / tissues, enhance treatment tolerability, and prevent prosthesis colonization in patients, thereby increasing the therapeutic benefit of these strains. Adenosine mimics are known to inhibit Salmonella typhimurium biofilm formation, suggesting that high adenosine concentrations in the tumor microenvironment contribute to tumor-associated biofilm formation (see, for example, Koopman et al. (2015) Antimicrob. Agents Chemother. 59:76-84). This article shows that strains lacking csgD exhibit improved antitumor efficacy due to increased bacterial uptake into tumor-resident bone marrow cells.

[0580] Genes encoding homologs and orthologs of csgD in other bacterial species, as well as other genes required for curly pili and biofilm formation, can also be deleted, disrupted, or otherwise modified to achieve similar results. These genes include, but are not limited to, for example: csgD, which encodes the DNA-binding transcription dual regulator CsgD in *Escherichia coli*; csgD(STY1179), which encodes the regulatory protein CsgD in *Salmonella typhi*; and lcp, which encodes a Listeria cellulose-binding protein involved in biofilm formation in *Listeria monocytogenes*.

[0581] Modifications to the bacterial genome, such as by deleting or disrupting genes to make bacteria csgD - This leads to the elimination of curly pili and inflammatory cyclic dinucleotides (CDNs), and the elimination of cellulose secretion. This eliminates inflammatory and immunosuppressive factors, prevents TLR4 recognition through altered LPS acylation, eliminates cellulose secretion and thus potential biofilm formation, thereby improving safety and efficacy.

[0582] As described herein, bacterial strains, such as *Salmonella typhimurium* strains, are engineered to be adenosine auxotrophs; and their ability to induce pro-inflammatory cytokines is reduced by modifying LPS and / or deleting flagellin; and / or they do not express L-asparaginase II to improve T cell function; and / or they contain deletions of genes required for biofilm formation; and / or they are further modified to maintain a significant plasmid copy number per cell, at least low to moderate or higher, in the absence of antibiotic selection; and they deliver gene expression cassettes encoding therapeutic products to promote a robust anti-tumor immune response. The plasmids include regulatory sequences to facilitate the secretion of the encoded therapeutic product into the tumor microenvironment.

[0583] 7. Improved resistance to complement

[0584] The complement system is the first line of defense against invading pathogens that directly activate the lectin pathway or alternative pathway (AP) cascade in the human host. The complement system involves more than 30 soluble and cell membrane-bound proteins that play a role in the innate immune response to recognize and kill pathogens such as bacteria, viruses-infected cells, and parasites, and in antibody-mediated immunity. Activation of the complement cascade leads to opsonization of foreign microorganisms, release of chemokines, and ultimately, disruption of the bacterial cell membrane. Three homologous glycoproteins in the complement system, C3, C4, and C5, play a central role in complement function and interact with other complement components. C3b and C4b, generated from C3 and C4 respectively, are important components of convertases that promote complement cascade activation. C5a is a cleavage fragment of C5 that induces phagocyte migration to the site of infection, and C5b initiates the formation of the membrane attack complex and bacterial lysis (see, for example, Ramu et al. (2007) FEBS Letters 581:1716-1720).

[0585] To survive, pathogens have developed strategies to prevent the harmful consequences of complement activation. For example, members of the Ail / Lom family of outer membrane proteins can protect many pathogens from complement-dependent killing. Members of the Ail / Lom family, including Ail (attachment site) of Yersinia, such as Yersinia enterocolitica and Yersinia pseudotuberculosis, and Rck (complement-killing resistance) and PagC of Salmonella, as well as OmpX of Escherichia coli, are outer membrane proteins with significant amino acid sequence similarity and homogeneity and similar membrane topology. Although members of this protein family exhibit different functions, some of them, including Ail from Yersinia enterocolitica and Yersinia pseudotuberculosis, and Rck from Salmonella enterica, at least partially protect bacteria from complement-mediated lysis (see, for example, Bartra et al. (2008) Infection and Immunity 76:612–622).

[0586] Another bacterial product that helps avoid or mitigate complement is surface protease, known in Salmonella as PgtE (outer membrane serine protease), and other members of the omptin family. The surface protease PgtE of *Salmonella enterica* belongs to the omptin family of outer membrane aspartic proteases. PgtE and other omptins require rough LPS for activation but are sterically inhibited by the O antigen. PgtE expression is upregulated during Salmonella growth within macrophages, and bacteria released from macrophages exhibit potent PgtE-mediated proteolytic activity. PgtE proteolytic activation of mammalian plasma plasminogen activator plasminogen activator inactivates α2-antiplasmin, the main physiological inhibitor of plasmin, and mediates bacterial adhesion to the extracellular matrix of human cells. In this way, PgtE mediates the degradation of extracellular matrix components and generates potent local proteolytic activity, which can promote the migration of Salmonella across the extracellular matrix. PgtE also degrades α-helical antimicrobial peptides, which may be important during the intracellular growth of Salmonella. Omptin Pla from Yersiniapestis is a close ortholog of PgtE and shares its function. Pla cleaves C3, and PgtE increases Salmonella serum resistance by cleaving complement components C3b, C4b, and C5. Genes of pgtE and their orthologs from other bacterial species can be included in the immunostimulatory bacteria discussed in this article to increase complement resistance.

[0587] This article demonstrates that the role of complement in human serum explains the failure of therapeutically immunostimulating bacteria, such as Salmonella strain VNP20009, which has been shown to effectively colonize tumors in rodent models. Systemic administration of VNP20009 resulted in tumor colonization in mice (see, for example, Clairmont et al. (2000) J. Infect. Dis. 181:1996-2002; and Bermudes et al. (2001) Biotechnol. Genet. Eng. Rev. 18:219-33); while systemic administration of VNP20009 in human patients resulted in very little colonization. In a phase 1 study of patients with advanced melanoma, very little VNP20009 was detected in human tumors after 30 minutes of intravenous infusion (see Toso et al. (2002) J. Clin. Oncol. 20:142-52). Patients enrolled in a follow-up study evaluating a longer 4-hour infusion of VNP20009 also showed a lack of detectable VNP20009 after tumor biopsy (see Heimann et al. (2003) J. Immunother. 26:179–180). Colonization of VNP20009 derivatives was detected after intratumoral administration (see Nemunaitis et al. (2003) Cancer Gene Ther. 10:737-744). Direct intratumoral administration of VNP20009 to human tumors resulted in higher tumor colonization, indicating that human tumors can colonize at high levels, and that the difference in tumor colonization between mice and humans only occurred after systemic administration.

[0588] This article demonstrates and describes how VNP20009, although previously unknown in wild-type Salmonella typhimurium, is inactivated by human complement, explaining the low tumor colonization observed in humans following systemic administration of VNP20009. The strain presented in this article exhibits complement resistance. It can be modified to express Rck and other proteins involved in mediating complement resistance or avoidance, such as Ail from Yersinia enterocolitica or PgtE from Salmonella typhimurium, or, if it naturally expresses such a protein, it can be modified to overexpress Rck and / or other such proteins. Rck can be introduced into bacteria lacking homologs, such as Escherichia coli.

[0589] Rck expression

[0590] Rck (complement-killing resistance) is a 17 kDa outer membrane protein encoded by a large virulence plasmid of Salmonella species such as *Salmonella enteritidis* and *Salmonella typhimurium*, which induces epithelial cell adhesion and invasion. Rck proteins have been shown to protect *Salmonella enteritidis* from complement destruction by inhibiting C9 polymerization and subsequent assembly of a functional membrane attack complex. Epithelial cell invasion is reduced 2–3 times in rck mutants compared to wild-type strains, while overexpression of rck in wild-type strains leads to increased invasion. Rck proteins induce cell entry through receptor-mediated processes, promoting local actin remodeling and membrane elongation with weak and tight adhesion. Therefore, *Salmonella* can enter cells via two distinct mechanisms: a triggering mechanism mediated by the T3SS-1 complex and a zipper mechanism induced by rck (see, for example, Manon et al. (2012), *Salmonella*, Chapter 17, eds. *Annous and Gurtler*, Rijeka, pp. 339–364). Expression of rck on Salmonella virulence plasmids confers high levels of resistance to human complement neutralization by preventing the formation of the membrane attack complex. When rck-containing Salmonella typhimurium virulence plasmids are expressed in serum-sensitive Escherichia coli strains, Rck restores complement resistance.

[0591] This document describes immunostimulatory bacteria that retain or provide Rck to confer resistance to human complement. It shows that immunostimulatory bacteria, such as *Escherichia coli*, can be modified to encode rck on a plasmid within the bacteria, thereby conferring resistance to complement. The immunostimulatory bacteria described herein endogenously encode rck, or can be modified to encode it to increase resistance to complement. Methods for conferring complement resistance are also provided. For example, therapeutic *E. coli* species described in U.S. Patent Application Publications 2018 / 0325963 and 2018 / 0273956 and U.S. Patents 9,889,164 and 9,688,967 can be modified to enhance or provide resistance to complement, for example, by introducing nucleic acids encoding a *Salmonella* rck gene on a plasmid. Complement-resistant bacteria can be administered systemically, and sufficient bacteria can survive to produce a therapeutic effect. Introducing nucleic acids encoding a *Salmonella* rck gene into bacteria, such as therapeutic *E. coli*, confers or increases complement resistance.

[0592] Other orthologs and homologs of rck from other bacterial species can also be expressed in immunostimulatory bacteria. For example, Ail, a rck homolog from Yersinia enterocolitica, enhances complement resistance upon heterologous expression. PgtE, a surface protease of Salmonella typhimurium, has also been shown to enhance complement resistance upon heterologous expression.

[0593] 8. Deletion of the gene required for lipoprotein expression in Salmonella and other Gram-negative bacteria.

[0594] LPS and Braun lipoprotein (Lpp) are major components of the outer membrane of Gram-negative intestinal bacteria and can act as potent stimulants of inflammatory and immune responses. Braun lipoprotein (Lpp) is one of the most abundant components of the outer membrane of Salmonella typhimurium and leads to TLR2-induced pro-inflammatory cytokines such as TNFα, IL-6, and IL-8 (in humans). Two functional copies of the lipoprotein gene located on the Salmonella bacterial chromosome (lppA (SEQ ID NO:387) and lppB (SEQ ID NO:388)) contribute to bacterial virulence. Deletion of lppA and lppB genes and elimination of lipoprotein expression reduce virulence and decrease the production of pro-inflammatory cytokines (see, for example, Sha et al. (2004) Infect. Immun. 72(7):3987-4003; Fadlet et al. (2005) Infect. Immun. 73(2):1081-1096). It is expected that the deletion of Lpp genes would reduce cell infection, thereby reducing plasmid delivery and the expression of encoded therapeutic products or proteins. However, as shown in Example 18 below, while the deletion of these genes does reduce tumor colonization, the amount of plasmids delivered to target cells, tumor-resident immune cells, and particularly macrophages is significantly increased. As illustrated herein, the deletion or disruption of these genes (lppA and lppB) thus results in reduced virulence due to their inability to survive in infected macrophages, but leads to enhanced plasmid delivery of immunostimulatory bacteria, thereby increasing the expression of encoded therapeutic genes on target cells, namely tumor-resident immune cells, particularly macrophages.

[0595] 9. Highly immunostimulatory bacteria whose genomes are modified to be optimized for anti-tumor therapy and encode therapeutic products, including a variety of therapeutic products.

[0596] As described herein, bacterial strains, such as *Salmonella typhimurium* strains, are engineered to be adenosine auxotrophs and are modified to reduce their ability to induce pro-inflammatory cytokines by modifying LPS and / or deleting flagellin, and / or modified to improve T cell function by deleting or eliminating L-asparaginase II expression, and / or modified by deleting or disrupting genes required for biofilm formation, and / or exhibit enhanced human serum viability due to increased rck expression. These bacterial strains are further modified to deliver therapeutic products, such as immunomodulatory proteins, and to promote potent antitumor immune responses.

[0597] The table below summarizes bacterial genotypes / modifications, their functions, and some of the effects / benefits achieved in this study.

[0598]

[0599] The strains provided in this article are ΔFLG, and / or ΔpagP, and / or ΔansB, and / or ΔcsgD. Furthermore, the strains are one or more of ΔpurI (ΔpurM), ΔmsbB, and Δasd (in the bacterial genome). Specifically, the strains are ΔpurI (ΔpurM), ΔmsbB, ΔpagP, ΔansB, and Δasd. The strains may also be lppA. - and / or lppB - Especially lppA - / lppB - The plasmid is modified to encode a therapeutic product under the control of a host-recognized promoter (e.g., a eukaryotic promoter, such as the RNA polymerase II promoter, including promoters from eukaryotic and animal viruses). The plasmid can encode asd to allow bacterial replication in vivo, and can encode nucleic acids with other beneficial functions (e.g., CpG), and can encode gene products as described elsewhere in this document.

[0600] The immunostimulatory bacteria described herein can be modified to eliminate the ability to infect epithelial cells, for example, by eliminating flagella. As described elsewhere herein, the ability to eliminate epithelial cell infection can also be achieved by inactivating SPI-1-dependent invasion, by inactivating or knocking out one or more genes involved in the SPI-1 pathway. These genes include, but are not limited to, one or more of the following: avrA, hilA, hilD, invA, invB, invC, invE, invF, invG, invH, invI, invJ, iacP, iagB, spaO, spaP, spaQ, spaR, spaS, orgA, orgB, orgC, prgH, prgI, prgJ, prgK, sicA, sicP, sipA, sipB, sipC, sipD, sirC, sopB, sopD, sopE, sopE2, sprB, and sptP. Alternatively, immunostimulatory bacteria may contain gene knockouts or deletions to inactivate products involved in SPI-1-independent infection / invasion, such as one or more of the genes fljB, fliC, rck, pagN, hlyE, pefI, srgD, srgA, srgB, and srgC, and / or immunostimulatory bacteria may contain gene products knockouts or deletions to inactivate cell death inducing tumor-resident immune cells, such as genes encoding proteins directly recognized by inflammasomes, including fljB, fliC, prgI (needle protein), and prgJ (bar protein). However, the rck gene is preferable because it is protected from complement inactivation. Bacteria that do not endogenously encode rck can be modified to encode a heterologous rck gene.

[0601] The immunostimulatory bacteria are derived from suitable bacterial strains. These strains may be attenuated strains, strains attenuated using standard methods, or strains attenuated through modifications provided herein, and their colonization ability is primarily limited to immune-exempt tissues and organs, particularly tumor-resident immune cells, tumor mesenteric enzymes (TMEs), and tumor cells, including solid tumors. Bacteria include, but are not limited to, strains such as Salmonella, Shigella, Listeria, Escherichia coli, and Bifidobacteriae. For example, bacterial species include: Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Listeria monocytogenes, Salmonella typhi, Salmonella typhimurium, Salmonella gallinarum, and Salmonella enteritidis. Other suitable bacterial species include: Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus, and Erysipelothrix.Examples include Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, and Citrobacter freundii. freundii), Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, or Agrobacterium tumerfacium.

[0602] Examples of immunostimulatory bacteria provided in this article are Salmonella species. Examples of modified bacteria as described herein are wild-type strains of Salmonella, such as strains possessing all the distinguishing characteristics of the strain deposited at the American Center for Type Culture Collection (ATCC) accession number #14028. Engineered Salmonella Typhimurium strains, such as strain YS1646 (ATCC catalog #202165, also known as VNP20009; see also International PCT application publication number WO99 / 13053), are engineered with plasmids to supplement asd gene knockout and allow for antibiotic-free plasmid maintenance. The strains are then modified to delete the flagellin gene and / or pagP. The combination of flagellation knockout and pagP deletion renders the strain highly resistant to human serum complement. These strains are also conferred purine auxotrophs, particularly adenosine, and are asd... - and msbB - For example, strains with complete deletions of purI and msbB are more suited (grow faster) than strain VNP20009, in which these genes are not deleted but modified to eliminate expression. The asd gene can be provided on the plasmid for replication in the eukaryotic host. The strain also modifies the ansB gene, such as by deletion, disruption, or other modifications, to prevent the production of immunosuppressive L-asparaginase II and improve tumor T cell function. The strain is also modified to eliminate biofilm formation, for example by csgD deletion, which prevents the production of curly pili, cellulose, and c-di-GMP, thereby reducing unwanted inflammatory responses and preventing biofilm formation.

[0603] These genomic deletions and plasmids are described and illustrated elsewhere in this document. Any nucleic acid encoding therapeutic products such as immunostimulatory proteins and other products, as described elsewhere in this document and / or known to those skilled in the art, may be included on plasmids. As described elsewhere in this document, plasmids are typically present at low to moderate copy numbers. Therapeutic products include gain-of-function mutants of cytoplasmic DNA / RNA sensors that can constitutively induce / induce type I IFN expression, as well as other immunostimulatory proteins, such as cytokines, chemokines, and co-stimulatory molecules, that promote antitumor immune responses in the tumor microenvironment, and other such products as described herein. Plasmids may also encode antibodies and fragments thereof, such as single-chain antibodies, that target immune checkpoints and other cancer targets, such as VEGF, IL-6, and TGF-β, and other molecules, such as bispecific T-cell connectors, or The plasmid can also encode IL-6-binding decoy receptors, TGF-β-binding decoy receptors, and TGF-β peptide antagonists. As described below, the plasmid can encode one or more therapeutic products / genetic payloads (i.e., multi-complexes) for delivering anticancer therapeutic products to the tumor / tumor microenvironment. The products can be operatively linked to transport signals, such as secretion signals. The products can also be engineered for expression on the cell surface, for example, in tumor-resident bone marrow cells.

[0604] 10. M2 phenotype macrophages transform into M1 and M1-like phenotype macrophages.

[0605] As described in this article, the immunostimulatory bacteria presented accumulate in and / or target macrophages. Macrophages are phagocytic immune cells; they play a role in clearing senescent and apoptotic cells, phagocytizing immune-related complexes and pathogens, and maintaining homeostasis. The phenotype and function of macrophages can be polarized by the microenvironment. There are two types: M1 type (classical activated macrophages) and M2 type (alternative activated macrophages).

[0606] M1 macrophages function by secreting pro-inflammatory cytokines and chemokines, and presenting antigens, thereby participating in positive immune responses and acting as immune surveillance mechanisms. M1 macrophages produce pro-inflammatory cytokines, including IL-6, IL-12, and TNF-α. M2 macrophages secrete arginase 1, IL-10, TGF-β, and other anti-inflammatory cytokines, possessing functions such as reducing inflammation, promoting tumor growth, and immunosuppression. Therefore, the M1 or M1-like phenotype is advantageous for the treatment of cancer and other related diseases and conditions.

[0607] M2 macrophages can transform into M1 macrophages or macrophages with an M1-like phenotype. The immunostimulatory bacterial infection of macrophages described in this article can transform M2 macrophages into M1 or M1-like phenotypes. Phenotypic markers for M1 macrophages include CD80 (also known as B7, B7.1, or BB1), CD86 (also known as B7.2), CD64 (also known as high-affinity immunoglobulin γ-Fc receptor I), CD16, and CD32 (also known as low-affinity immunoglobulin γ-Fc receptor IIb). The expression of nitric oxide synthase (iNOS) in M1 macrophages can also serve as a phenotypic marker. CD163 and CD206 are markers for identifying M2 macrophages. Arginase 1 (Arg1) and DECTIN-1 are also ideal phenotypic indicators for identifying M2 macrophages. Therefore, the expression of these markers can be used to monitor or assess transformation.

[0608] Tumor-associated macrophages (TAMs) are associated with an immunosuppressive M2 phenotype. The immunostimulatory bacteria described herein can convert these macrophages into an M1 or M1-like phenotype. This conversion is achieved by immunostimulatory bacteria, which encode a therapeutic product leading to type I interferon (IFN) expression. This is a unique property of the immunostimulatory bacteria described herein and utilizes the bacteria's ability, including genomic modifications leading to macrophage infection. The encoded therapeutic product includes those that are part of a cytoplasmic DNA / RNA sensor pathway, such as STING variants (described in detail herein). Following infection of tumor-resident macrophages and expression of the therapeutic product, the immunostimulatory bacteria can achieve conversion to an M1 phenotype (or M1-like phenotype). This ability to convert macrophage phenotypes is demonstrated and illustrated in Example 12 below. The M1 macrophage phenotype is converted to an M2 macrophage phenotype by expressing a modified STING protein through the immunostimulatory bacteria described herein that infect macrophages.

[0609] The immunostimulatory bacteria described herein, including genomic modifications such as flagellar ablation and LPS modification, transform infected M2 macrophages into those macrophages that induce an M1 macrophage cytokine profile. Immunostimulatory bacteria expressing STING protein variants lead to constitutive type I IFN expression in primary human M2 macrophages, transforming these cells into M1-like (with typical M1 macrophage phenotypic markers and / or expression profiles) type I IFN-producing cells.

[0610] Examples demonstrate this change from M2 to M1-like or M1-like phenotypes. Comparison of the cytokine profile in uninfected M2 macrophages with that inducible cytokines in M2 macrophages infected with Salmonella strains (Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD M2) induced higher levels of IFNγ, CXCL10, and CXCL11 secretion. Infection with the same strains transformed with plasmids encoding the huSTINGtazCTT N154S+R284G variant, WT huIL-12, and huSTINGtazCTT N154S+R284G variant or WT huIL-15 induced higher CXCL10 and CXCL11 secretion than untransformed strains without the plasmid. Characteristic cytokine profiles of M1 or M1-like phenotypes were induced in the strains with various different payloads. Example results are detailed in the examples.

[0611] D. Immunostimulatory bacteria with an enhanced therapeutic index that encode genetic payloads that stimulate immune responses in the tumor microenvironment.

[0612] The immunostimulatory bacteria described herein are modified to accumulate in the tumor microenvironment and tumor-resident bone marrow cells, whereby the therapeutic products are expressed under the control of eukaryotic promoters. The bacteria encode therapeutic products, particularly anticancer products, including those that stimulate the immune system and / or reverse or alleviate tumor immunosuppression. As described herein, the bacteria can encode multiple products, each expressed under the control of a separate promoter, or under the control of a single promoter, and may include sequences leading to discrete product expression, and, where appropriate, regulatory sequences to ensure the secretion of the encoded products into the tumor microenvironment. The immunostimulatory bacteria express the encoded therapeutic products on plasmids. As discussed, plasmids can encode one or more products. Each product can be expressed under the control of a different eukaryotic promoter, or multiple encoded products can be expressed under the control of a single promoter, for example by including 2A self-cleaving peptides between the coding portions, such as T2A (SEQ ID NO:327), P2A (SEQ ID NO:328), E2A (SEQ ID NO:329), and F2A (SEQ ID NO:330). The encoded products include those described herein, and they can be complementary anticancer immunostimulatory products. The immunostimulatory bacteria provided herein allow for the combined administration of multiple immunomodulatory products or payloads (multiple payloads) that would otherwise be too toxic for systemic administration. Examples of multiple payloads include one or more cytokines, immunostimulatory proteins that stimulate or induce type I IFN expression, such as STING or variants thereof with increased or constitutive activity, and co-stimulatory molecules, such as engineered 4-1BBL co-stimulatory molecules. This document provides modified 4-1BBL peptides and encoding nucleic acids that exhibit improved expression and activity when encoded on plasmids in the immunostimulatory bacteria provided herein, which deliver the plasmids to bone marrow cells for expression under the control of host transcriptional and translational mechanisms.

[0613] The immunostimulatory bacteria described herein possess potent antitumor activity, including providing cure, for example, after intravenous administration with multiple or single-drug payloads. Upon systemic administration, these immunostimulatory bacteria infiltrate and enrich in solid tumors, tumor-terminal myeloid lesions (TMEs), and tumor-resident myeloid cells, subsequently expressing the therapeutic product encoded therein, and then locally delivering it to the tumor microenvironment. Following consumption (phagocytosis) by tumor-resident myeloid cells, the bacteria deliver a plasmid encoding the genetic payload, which allows for ectopic, single, or multiple payload expression in a tumor-specific manner.

[0614] 1. Immune-stimulating proteins

[0615] The immunostimulatory bacteria described herein can be modified to encode immunostimulatory proteins that promote, induce, or enhance antitumor responses. As illustrated and described in the examples, the order in which the encoded nucleic acids are arranged on the plasmid can improve overall expression, and modifications to the plasmid can improve the fitness of bacteria containing plasmids encoding proteins.

[0616] Immunostimulatory proteins can be encoded on plasmids in bacteria under the control of eukaryotic promoters, such as those recognized by RNA polymerase II, for expression in eukaryotic subjects, particularly in subjects receiving immunostimulated bacteria (such as humans). In addition to eukaryotic promoters, nucleic acids encoding immunostimulatory proteins may also include other regulatory signals for expression or transport within the cell (e.g., for secretion or expression on the cell surface).

[0617] Immunostimulatory proteins are those proteins that can promote, participate in, or enhance the antitumor response in subjects administered immunostimulatory bacteria in appropriate environments, such as the tumor microenvironment (TME). Immunostimulatory proteins include, but are not limited to, cytokines, chemokines, and co-stimulatory molecules. These include cytokines such as, but not limited to, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-12p70 (IL-12p40+IL-12p35), the IL-15 / IL-15Rα chain complex, IL-36γ, GM-CSF, IFNα, IFNβ, IL-2 with attenuated binding to IL-2Ra, and IL-2 modified to not bind to IL-2Ra; chemokines such as, but not limited to, CCL3, CCL4, ... CCL5, CXCL9, CXCL10, and CXCL11; and / or co-stimulatory molecules, such as, but not limited to, CD40, CD40L, OX40, OX40L, 4-1BB, 4-1BBL, 4-1BBL with truncated or missing cytoplasmic domains (4-1BBLΔcyt), TNF / TNFR superfamily members (e.g., CD27 and CD27L), and B7-CD28 family members (e.g., CD80, CD86, ICOS, and ICOS ligand (B7RP1)).

[0618] Other such immunostimulatory proteins known to those skilled in the art for treating tumors or for promoting, enhancing, or otherwise increasing or eliciting antitumor responses are intended for encoding in the immunostimulatory bacteria provided herein. For example, said immunostimulatory bacteria may deliver a genetic payload encoding truncated costimulatory molecules (e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, and OX40L) with complete or partial deletion of cytoplasmic domains expressed in APCs, wherein the truncated gene product is capable of constitutive immunostimulatory signaling to T cells via costimulatory receptors and is unable to send counterregulatory signals to APCs due to the deletion or truncation of the cytoplasmic domains. As described herein, for example, modified truncated cytoplasmic domains of 4-1BBL contain specific residues to ensure the correct orientation of the protein domain, thereby increasing protein expression. As described herein, the deletion (complete or partial) and modification of the cytoplasmic domains of costimulatory molecules enhance the activation of the costimulatory molecules without immunosuppressive reverse signal transduction. As described in the examples and below using 4-1BBL as an example; the same modifications, including replacing residues in truncated cytoplasmic domains to ensure correct orientation in the membrane, can be applied to any co-stimulatory molecule, as well as other transmembrane peptides.

[0619] The full-length sequence of human 4-1BBL (SEQ ID NO:389, Uniprot P41273) is:

[0620]

[0621]

[0622] The cytoplasmic domain corresponds to amino acids 1-28 (italicized), the transmembrane domain corresponds to amino acids 29-49 (bold), and the extracellular domain corresponds to amino acids 50-254 (underlined). The human 4-1BBLΔcyt sequence (see SEQ ID NO:390) is:

[0623]

[0624] It is identical to the full-length protein, but lacks the cytoplasmic domain. Therefore, the transmembrane domain corresponds to amino acid residues 2-22 (bold), and the extracellular domain corresponds to amino acid residues 23-227 (underlined).

[0625] An example of human 4-1BBL with a truncated cytoplasmic domain is shown below (see SEQ ID NO:391):

[0626]

[0627] The truncated cytoplasmic domains correspond to residues RVLP (italicized), starting from M; the transmembrane domains correspond to residues 6-26 (bold); and the extracellular domains correspond to residues 27-231 (underlined).

[0628] For full-length 4-1BBLs, positively charged amino acids, such as R and K, tend to be located in the cytoplasm (internal / cytoplasmic domain), which orients the transmembrane domain so that the N-terminus is internal. However, when the cytoplasmic domain is truncated, this alters the charge balance, so that only positive charges are present on the exterior (in the extracellular domain). This favors a conformation where the N-terminus of the protein is external rather than cytoplasmic, resulting in an "inside-out conformation." If this is observed, for example, through significantly lower activity or expression or other parameters, 4-1BBL variants with truncated cytoplasmic domains can be modified to include positive residues to ensure the correct orientation of protein expression in the cell membrane. Examples of possible modifications to 4-1BBLs include those where residues are replaced by positively charged residues or include c-myc tags. Other similar substitutions / additions can be envisioned by those skilled in the art to achieve the same result.

[0629] Examples of modified human 4-1BBL variants with truncated cytoplasmic domains include the following variants, which include additional positive residues (arginine (R), lysine (K), italicized) in the cytoplasmic domain region, as shown below, so that the resulting protein is correctly oriented (with the correct conformation, rather than an "inside-out" conformation) when expressed in the cell.

[0630] See SEQ ID NO:391 and 392 respectively:

[0631] Truncated cytoplasmic domains:

[0632]

[0633] This involves adding a positive charge back to the N-terminus (R), which facilitates the proper orientation of the N-terminus within the cytoplasm. In another example, a MYC tag is added.

[0634] Truncated cytoplasmic domains with MYC tags:

[0635]

[0636]

[0637] For the full-length mouse 4-1BBL sequence, and example sequences of mu4-1BBLΔcyt (mouse 4-1BBL with cytoplasmic domain deletion), namely mu4-1BBL with truncated cytoplasmic domain and mu4-1BBL with truncated cytoplasmic domain and MYC tag, see Example 19 below; also see SEQ ID NO:393-396 respectively.

[0638] Co-stimulatory molecules may include additional or alternative amino acid substitutions to ensure the correct orientation of proteins expressed in the membrane. Other similar modifications can be readily prepared by those skilled in the art to ensure the correct orientation of transmembrane proteins with truncated cytoplasmic domains.

[0639] Besides deletion or truncation of the cytoplasmic domain, co-stimulatory molecules used for expression on APCs (e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, and OX40L) can also be modified by introducing amino acid modifications, such as insertion, deletion, and / or substitution of the cytoplasmic domain, so that the modified gene product can participate in constitutive immunostimulatory signaling to T cells via co-stimulatory receptors and cannot emit counter-regulatory signals to APCs due to the modification of the cytoplasmic domain. For example, the immunosuppressive reverse (intracellular) signaling can be eliminated by modifying the phosphorylation site of the cytoplasmic domain, for example by replacing one or more Ser residues at one or more appropriate loci with residues that reduce or eliminate reverse signaling. For example, for human 4-1BBL, immunosuppressive reverse (intracellular) signaling can be eliminated by modifying the phosphorylation site of the cytoplasmic domain, said site including Ser5 and Ser8, referring to the sequence of full-length human 4-1BBL (SEQ ID NO:389). Serine residues in the cytoplasmic domain can be replaced by any other residues that reduce or eliminate reverse signal transduction.

[0640] Additional or alternative amino acid substitutions may be included in the co-stimulatory molecule to eliminate immunosuppressive intracellular (reverse) signaling. Those skilled in the art can readily prepare other similar modifications to eliminate immunosuppressive reverse signaling while still retaining the co-stimulatory molecule's ability to participate in the transmission of constitutive immunostimulatory signals to T cells via co-stimulatory receptors.

[0641] a. Cytokines and chemokines

[0642] In some embodiments, the immunostimulatory bacteria described herein are engineered to express cytokines that stimulate the immune system, including but not limited to IL-2, IL-7, IL-12, IL-12p70 (IL-12p40+IL-12p35), IL-15 (and the IL-15:IL-15Rα chain complex), IL-18, IL-21, IL-23, IL-36γ, IL-2 with attenuated binding to IL-2Ra, IL-2 modified to not bind to IL-2Ra, IFN-α, and IFN-β. These cytokines stimulate immune effector cells and stromal cells at tumor sites and enhance the recognition of tumor cells by cytotoxic cells. In some embodiments, the immunostimulatory bacteria may be engineered to express chemokines such as CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11.

[0643] IL-2

[0644] Interleukin-2 (IL-2) was the first cytokine approved for cancer treatment. It participates in the activation of the immune system through several mechanisms, including activating and promoting the growth of cytotoxic T lymphocytes (CTLs), producing lymphokine-activated killer (LAK) cells, promoting the growth and proliferation of Treg cells, stimulating tumor-infiltrating lymphocytes (TILs), and promoting the proliferation and differentiation of T cells, B cells, and NK cells. Recombinant IL-2 (rIL-2) has been approved by the FDA for the treatment of metastatic renal cell carcinoma (RCC) and metastatic melanoma (see, for example, Sheikhi et al. (2016) Iran J. Immunol. 13(3):148-166).

[0645] IL-7

[0646] IL-7, a member of the IL-2 superfamily, is involved in T cell survival, proliferation, and homeostasis. Mutations in the IL-7 receptor have been shown to lead to T cell loss and the development of severe combined immunodeficiency (SCID), highlighting the crucial role of IL-7 in T cell development. IL-7 is a homeostatic cytokine that provides continuous signaling to resting naive and memory T cells, and accumulates during lymphopenia, leading to T cell proliferation and increased T cell repertoire diversity. Compared to IL-2, IL-7 selectively expands CD8+. + T cells instead of CD4 + FOXP3 +Regulatory T cells. Recombinant IL-7 has been shown to enhance antigen-specific T cell responses following vaccination and adoptive cell therapy in mice. IL-7 also plays a role in promoting T cell recovery after hematopoietic stem cell transplantation chemotherapy. Early clinical trials in patients with advanced malignancies have demonstrated that recombinant IL-7 is well-tolerated and has limited toxicity (i.e., circulating CD4+) at bioactive doses. + and CD8 + The number of T cells increased 3 to 4 times (see, for example, Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893). IL-7 has been shown to have antitumor effects against tumors such as glioma, melanoma, lymphoma, leukemia, prostate cancer, and glioblastoma, and in vivo administration of IL-7 in mouse models resulted in reduced cancer cell growth. IL-7 has also been shown to enhance the antitumor effect of IFN-γ in rat glioma tumors and induce monocytes to produce IL-1α, IL-1β, and TNF-α, thereby inhibiting melanoma growth. Furthermore, administration of recombinant IL-7 after treatment of pediatric sarcoma led to a promotion of immune recovery (see, for example, Lin et al. (2017) Anticancer Research 37:963-968).

[0647] IL-12(IL-12p70(IL-12p40+IL-12p35))

[0648] The bioactive IL-12 (IL-12p70) that promotes cell-mediated immunity is a heterodimer composed of p35 and p40 subunits, while IL-12p40 monomers and homodimers act as IL-12 antagonists. IL-12 is secreted by antigen-presenting cells, promotes the secretion of IFN-γ by NK cells and T cells, inhibits tumor angiogenesis, and leads to increased activity of NK cells and CD8+. + T cells and CD4 + T cell activation and proliferation, enhancing natural CD4+ + T cells differentiate into Th1 cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells. IL-12 has shown antitumor effects in mouse models of melanoma, colon cancer, breast cancer, and sarcoma (see, for example, Kalinski et al. (2001) Blood 97:3466-3469; Sheikhi et al. (2016) Iran J. Immunol. 13(3):148-166; and Lee, S. and Margolin, K. (2011) Cancer 3:3856-3893).

[0649] IL-15 and IL-15:IL-15Rα

[0650] IL-15 is structurally similar to IL-2. While both IL-2 and IL-15 provide early stimulation for T cell proliferation and activation, IL-15 blocks IL-2-induced apoptosis, a process that leads to the elimination of stimulated T cells, induces T cell tolerance, limits memory T cell responses, and potentially limits the therapeutic efficacy of IL-2 alone. IL-15 also supports memory CD8+. + T cell persistence is crucial for maintaining long-term anti-tumor immunity, and this is achieved through direct activation of CD8 cells in an antigen-independent manner. + Effector T cells showed significant antitumor activity in preclinical mouse models. (Except for CD8) + In addition to T cells, IL-15 is responsible for the development, proliferation and activation of effector natural killer (NK) cells (see, for example, Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893; and Han et al. (2011) Cytokine 56(3):804-810).

[0651] IL-15 and its receptor α (IL-15Rα) are co-expressed by antigen-presenting cells (e.g., monocytes and dendritic cells), and IL-15 is trans-presented via IL-15Rα to cells on CD8. + The IL-15βγc receptor complex is expressed on the surface of T cells and NK cells. Soluble IL-15:IL15-Rα complexes have been shown to modulate immune responses via the IL-15βγc complex, and administration of IL-15 as a pre-formed IL-15 and soluble IL-15Rα complex has been shown to increase IL-15 bioactivity by 50-fold and have a longer half-life compared to IL-15 alone. This significant enhancement of IL-15 therapeutic efficacy through pre-association with IL-15Rα has been demonstrated in mouse tumor models (see, for example, Han et al. (2011) Cytokine 56(3):804-810).

[0652] IL-18

[0653] IL-18 via NK and CD8 + T cells induce IFN-γ secretion, enhancing its cytotoxicity. IL-18 also activates macrophages and stimulates Th1 helper CD4 cells. + T cell development. IL-18 has shown promising antitumor activity in some preclinical mouse models. For example, administration of recombinant IL-18 (rIL-18) activates CD4+. +T-cell and / or NK-cell mediated responses led to regression of melanoma or sarcoma in homogeneous mice. Other studies have shown that the antitumor effect of IL-18 is mediated by IFN-γ and involves an anti-angiogenic mechanism. Combination of IL-18 with other cytokines (such as IL-12) or co-stimulatory molecules such as CD80 enhances the IL-18-mediated antitumor effect. Phase I clinical trials in patients with advanced solid tumors and lymphomas have shown that administration of IL-18 is safe and leads to increased immunomodulatory activity and serum IFN-γ and GM-CSF levels and moderate clinical responses in patients. Clinical trials have shown that IL-18 can be combined with other anticancer agents, such as monoclonal antibodies, cytotoxic drugs, or vaccines (see, for example, Fabbi et al. (2015) J. Leukoc. Biol. 97:665-675; and Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893).

[0654] Attenuated strains of Salmonella Typhimurium engineered to express IL-18 have been found to inhibit the growth of subcutaneous (SC) tumors or lung metastases in homogeneous mice after systemic administration without any toxicity. Treatment with this engineered bacterium induces the accumulation of T cells, NK cells, and granulocytes in tumors and leads to intratumoral production of cytokines (see, for example, Fabbi et al. (2015) J. Leukoc. Biol. 97:665-675).

[0655] Chemokines

[0656] Chemokines are a family of small cytokines that mediate leukocyte migration to damaged or inflammatory areas and participate in mediating immune and inflammatory responses. Based on the position of cysteine ​​residues in their sequence, chemokines are divided into four subfamilies: XC-, CC-, CXC-, and CX3C- chemokine ligands, or XCL, CCL, CXCL, and CX3CL. Chemokine ligands bind to their homologous receptors and regulate the circulation, homing, and retention of immune cells, with each chemokine ligand-receptor pair selectively regulating a particular type of immune cell. Different chemokines attract different leukocyte populations and create concentration gradients in vivo, with attracted immune cells moving towards areas with higher chemokine concentrations (see, for example, Argyle D. and Kitamura, T. (2018) Front. Immunol. 9:2629; and Dubinett et al. (2010) Cancer J. 16(4):325-335). Chemokines can improve antitumor immune responses by increasing the infiltration of immune cells into tumors, promoting the migration of antigen-presenting cells (APCs) to tumor-draining lymph nodes, and eliciting innate T and B cells (see, for example, Lechner et al. (2011) Immunotherapy 3(11):1317-1340). The immunostimulatory bacteria described in this article can be engineered to encode chemokines, including but not limited to CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11.

[0657] CCL3, CCL4, CCL5

[0658] CCL3, CCL4, and CCL5 share high homology and are associated with several cell types in humans and mice, including immature DCs and T cells, including CCR5 (CCL3, CCL4, and CCL5) and CCR1 (CCL3 and CCL5). Therapeutic T cells have been shown to induce innate immune cell chemotaxis to tumor sites via tumor-specific secretion of CCL3, CCL4, and CCL5 (see, for example, Dubinett et al. (2010) Cancer J. 16(4):325-335).

[0659] Inducing a type 1 (Th1) T helper cell response releases CCL3. In vivo and in vitro studies in mice have shown that CCL3 exhibits chemotaxis towards both neutrophils and monocytes; specifically, CCL3 can mediate the mobilization of bone marrow progenitor cells (MPCs) from the bone marrow and has both MPC-regulating and stimulatory effects. Human ovarian cancer cells transfected with CCL3 showed enhanced intratumoral T cell infiltration and macrophage activity, leading to a modified antitumor response, and demonstrated that CCL3-mediated neutrophil chemotaxis inhibits tumor growth. DCs recruited by CCL3 and transfected with the tumor antigen human melanoma-associated gene (MAGE)-1 exhibited superior antitumor activity in a mouse model of melanoma, including increased lymphocyte proliferation, cell lysis, viability, and reduced tumor growth. The combined use of CCL3 with the MAGE-1 antigen-specific platform has also been used to treat gastric cancer. CT26 is a highly immunogenic mouse colon tumor that produces CCL3 that slows tumor growth in vivo; this process has been shown to be driven by CCL3-dependent accumulation of natural killer (NK) cells, and thus IFNγ leads to the production of CXCL9 and CXLC10 (see, for example, Allen et al. (2017) Oncoimmunology 7(3):e1393598; and Schaller et al. (2017) Expert Rev. Clin. Immunol. 13(11):1049-1060).

[0660] CCL3 has been used as an adjuvant in cancer treatment. Administration of the active CCL3 variant ECI301 after radiofrequency ablation of hepatocellular carcinoma in mice increased tumor-specific responses and further demonstrated that this mechanism depends on the expression of CCR1. CCL3 has also been shown to be successful as an adjuvant in systemic cancers, with mice inoculated with CCL3 and IL-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF) in leukemia / lymphoma models showing increased survival (see, for example, Schaller et al. (2017) Expert Rev. Clin. Immunol. 13(11):1049-1060).

[0661] CCL3 and CCL4 guide CD8 + T cells infiltrate the primary tumor sites of melanoma and colon cancer to exert their effects. Tumorigenesis of CCL4 leads to CD103. + DC accumulation; inhibition of CCL4 via the WNT / β-catenin-dependent pathway, preventing CD103 in melanoma tumors. + DC invasion (see, for example, Spranger et al. (2015) Nature 523(7559):231-235). In a mouse model of colon cancer, CCL3 also showed enhancement of CD4.+ and CD8 + T cell infiltration into the primary tumor site (see, for example, Allen et al. (2017) Oncoimmunology 7(3):e1393598).

[0662] The binding of CCL3 or CCL5 to their receptors (CCR1 and CCR5, respectively) translocates immature dendritic cells (DCs), monocytes, memory cells, and T effector cells from circulation to sites of inflammation or infection. For example, CCL5 expression in colorectal tumors contributes to T lymphocyte chemotaxis and survival. CCL3 and CCL5 have been used alone or in combination therapy in several preclinical models to induce tumor regression and immunity. For example, studies have shown that subcutaneous injection of genetically modified Chinese hamster ovarian cells expressing CCL3 resulted in tumor suppression and neutrophil infiltration. In another study, recombinant oncolytic adenovirus expressing CCL5 (Ad-RANTES-E1A) led to primary tumor regression and prevented metastasis in a mouse model of breast cancer (see, for example, Lechner et al. (2011) Immunotherapy 3(11):1317-1340).

[0663] In translational studies of colorectal cancer, CCL5 induces an “antiviral response pattern” in macrophages. CCL5 is generated as a result of CXCR3-mediated lymphocyte migration at the invasive margin of colorectal cancer liver metastases. Blocking CCR5 (CCL5 receptor) leads to tumor death, driven by IFN-producing macrophages and reactive oxygen species. Although macrophages are present in the tumor microenvironment, CCR5 inhibition induces a phenotypic shift from M2 to M1. CCR5 blockade also leads to clinical responses in colorectal cancer patients (see, for example, Halama et al. (2016) Cancer Cell 29(4):587-601).

[0664] CCL3, CCL4, and CCL5 can be used to treat conditions including lymphoma, bladder cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, ovarian cancer, cervical cancer, or liver cancer (see, for example, US Patent Publication No. US 2015 / 0232880; International Patent Publications Nos. WO 2015 / 059303, WO 2017 / 043815, WO 2017 / 156349, and WO 2018 / 191654).

[0665] CXCL9, CXCL10, CXCL11

[0666] CXCL9 (MIG), CXCL10 (IP10), and CXCL11 (ITAC) are induced by the production of IFN-γ. These chemokines bind to CXCR3, are preferentially expressed on activated T cells, and play roles in angiogenesis inhibition and leukocyte recruitment and activation. The prognosis of colorectal cancer is closely related to tumor-infiltrating T cells, especially Th1 and CD8. + Effector T cells; high intratumoral expression of CXCL9, CXCL10, and CXCL11 indicates a favorable prognosis. For example, in a sample of 163 colorectal cancer patients, those with high levels of CXCL9 or CXCL11 showed increased postoperative survival, while those with high CXC expression showed increased CD3... + T cells, CD4 + T helper cells and CD8 + The number of cytotoxic T cells was significantly increased. In liver metastases of colorectal cancer patients, CXCL9 and CXCL10 levels increased at the invasive margin ...

Claims

1. An immunostimulatory bacterium comprising a plasmid encoding one or more therapeutic products under the control of one or more eukaryotic promoters, wherein the genome of the immunostimulatory bacterium is modified by deletion or disruption of one or more genes encoding L-asparaginase II, thereby preventing the bacterium from activating the synthesis of secreted asparaginase or from expressing L-asparaginase II, thus being ansB - .

2. The immunostimulatory bacteria of claim 1, wherein the genome of said bacteria is modified by deletion or disruption of one or more genes encoding L-asparaginase II and by deletion or disruption of gene csgD, thereby said bacteria is ansB. - It does not express active L-asparaginase II and is csgD - It does not activate the synthesis of curly pili.

3. The immunostimulatory bacteria of claim 1, wherein the therapeutic product contributes to an antitumor immune response in the tumor microenvironment.

4. The immunostimulatory bacteria of claim 1, wherein the therapeutic product comprises a modified cytoplasmic DNA / RNA sensor protein, which is a modified interferon gene stimulating factor (STING) protein, wherein the STING protein comprises one or more modifications, thereby constitutively inducing type I interferon (IFN) by the modified STING protein.

5. The immunostimulatory bacteria of claim 4, wherein the modified STING protein has lower NF-κB signaling activity compared to human STING.

6. The immunostimulatory bacteria of claim 1, comprising a nucleic acid encoding a variety of anticancer products as a polycistronic sequence under the control of a single promoter, wherein: The encoded product comprises at least two proteins; one protein is part of a cytoplasmic DNA / RNA sensor pathway that leads to the expression of type I interferon (IFN) and contains one or more modifications, wherein the constitutive activity of the protein induces the expression of type I interferon (IFN); the second protein is different from the first and is one or more proteins that confer or contribute to an anti-tumor immune response in the tumor microenvironment; and the polycistronic sequence is contained in a 2A peptide between each open reading frame (ORF) encoding each product.

7. The immunostimulatory bacteria of claim 6, wherein the 2A peptide is one or more of T2A, P2A, E2A or F2A.

8. The immunostimulatory bacteria of claim 6, wherein, The proteins that are part of the cytoplasmic DNA / RNA sensor pathway are interferon gene stimulating factor (STING), RIG-I, MDA-5, IRF-3, or IRF-7.

9. The immunostimulatory bacteria of claim 6, comprising a nucleic acid encoding a variety of anticancer products as a polycistronic sequence under the control of a single promoter, wherein: One product is a modified STING protein; and the modified STING protein comprises one or more modifications, thereby the modified STING protein having constitutive activity inducing type I interferon expression and lower NF-κB signaling activity compared with unmodified human STING.

10. The immunostimulatory bacteria of claim 1, wherein the plasmid-encoded therapeutic product is a modified STING protein selected from: a) a modified non-human STING protein, wherein: The non-human STING protein has lower NF-κB signaling activity compared to wild-type (WT) human STING protein; and the non-human STING protein contains a mutation, wherein the STING protein constitutively induces type I interferon (IFN); b) a modified chimeric STING protein, wherein the C-terminal tail (CTT) region of the STING protein is replaced by the CTT of a STING protein from another species that has lower NF-κB signaling activity than human STING, and the modified chimeric STING protein further contains a mutation, wherein the chimeric STING protein constitutively induces type I IFN; c) a STING protein modified to constitutively induce type I IFN and containing a mutation that eliminates phosphorylation sites to reduce NF-κB signaling activity, optionally wherein the sequence is aligned with and compared to any of the human STING sequences shown in SEQ ID NO: 305-309, the phosphorylation sites being located at residues corresponding to 324-326; and d) the modified STING protein of a), b), or c), wherein the TRAF6 binding site in the CTT of the STING protein is missing.

11. The immunostimulatory bacteria of claim 10, wherein the unmodified human STING protein has the sequence shown in any one of SEQ ID NO: 305-309.

12. The immunostimulatory bacteria of claim 10, wherein the non-human STING protein is selected from Tasmanian devil, marmoset, cattle, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, and ghost shark STING proteins.

13. The immunostimulatory bacteria of claim 6, wherein the second protein encoded in the polycistronic sequence is a cytokine.

14. The immunostimulatory bacteria of claim 6, wherein the second protein encoded in the polycistronic sequence is selected from: (a) IL-2, IL-7, IL-12p70 (IL-12p40 + IL-12p35), IL-15, IL-2 with weakened IL-2Ra binding, IL-15 / IL-15R. α-chain complex, 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, proteins involved in or enabling or enhancing T cell recruitment and / or persistence, CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), proteins with cytoplasmic domain deletion or truncation to eliminate immunosuppression. One or more of the following: 4-1BBL, members of the B7-CD28 family, CD47 antagonists, anti-IL6 antibodies or IL-6-binding decoy receptors, TGF-β peptide antagonists, bispecific T-cell adaptor antibodies, and members of the tumor necrosis factor receptor (TNFR) superfamily; and / or (b) 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. The α-chain complex, IL-36γ, IL-2 with weakened IL-2Ra binding, IL-2 modified to not bind to IL-2Ra, CXCL9, CXCL10 (IP-10), CXCL11, CCL3, CCL4, CCL5, molecules involved in potential T cell recruitment and / or persistence, CD40, CD40 ligand (CD40L), OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), 4-1BBL with a missing cytoplasmic domain (4-1BBLΔcyt) or with a partially missing cytoplasmic domain where the cytoplasmic domain is missing or truncated to eliminate immunosuppressive reverse signaling, members of the B7-CD28 family, and members of the tumor necrosis factor receptor (TNFR) superfamily.

15. The immunostimulatory bacteria of claim 1, comprising: (a) a nucleic acid encoding a 4-1BBL having a missing or partially missing cytoplasmic domain and optionally including an amino acid-modified 4-1BBL, wherein the resulting 4-1BBL is expressed in the correct orientation in the cell; and / or (b) a nucleic acid encoding a 4-1BBL variant having a missing or partially missing cytoplasmic domain or a modified 4-1BBL having a truncated and modified cytoplasmic domain, wherein the sequence of said 4-1BBL is as shown in SEQ ID NO:390, SEQ ID NO:391 and SEQ ID NO:

392.

16. The immunostimulatory bacteria of claim 1, wherein the plasmid encodes a modified STING and IL-15 or IL-15 / IL-15R α chain complex, the modified STING comprising one or more modifications, thereby constitutively inducing type I interferon (IFN) by the modified STING protein.

17. The immunostimulatory bacteria of claim 6, wherein the encoded polycistronic product comprises a constitutively active modified STING and IL-15 or an IL-15 / IL-15Rα chain complex.

18. The immunostimulatory bacteria of claim 6, wherein: The first protein is a modified STING; the modified STING contains one or more mutations, thereby making the STING protein constitutively active; and the TRAF6 binding site in the CTT of the STING protein is optionally missing.

19. The immunostimulatory bacteria of claim 6, wherein the encoded polycistronic product comprises modified STING and IL-12p70, wherein the modified STING comprises one or more modifications, thereby constitutively inducing type I interferon (IFN) by the modified STING protein.

20. The immunostimulatory bacteria of claim 1, wherein the encoded therapeutic product is: a modified STING comprising one or more modified STING proteins, wherein the modified STING protein constitutively induces type I interferon (IFN); said modified STING is a modified chimeric protein comprising STING from a first species and a CTT from a second species having lower NF-κB signaling activity than human STING, replacing the CTT of the first species; the first species is human; and the CTT is a STING protein from Tasmanian devil, marmoset, cattle, cat, ostrich, wild boar, bat, manatee, crested ibis, coelacanth, or ghost shark.

21. The immunostimulatory bacteria of claim 1, wherein the encoded therapeutic product: comprises one or more modified STINGs, whereby the modified STING protein constitutively induces type I interferon (IFN); said modified STING is a modified chimeric STING comprising CTT from a species having lower NF-κB signaling activity compared to human STING to replace the native CTT; and said replacement CTT is selected from the following species and has the following sequences: Tasmanian devil RQEEFAIGPKRAMTVTTSSTLSQEPQLLISGMEQPLSLRTDGF SEQ ID NO:371, marmoset EEEEVTVGSLKTSEVPSTSTMSQEPELLISGMEKPLPLRSDLF SEQ ID NO:372, bovine EREVTMGSTETSVMPGSSVLSQEPELLISGLEKPLPLRSDVF SEQ ID NO:373, feline EREVTVGSVGTSMVRNPSVLSQEPNLLISGMEQPLPLRTDVF SEQ ID NO:

373. 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, Crested Ibis CHEEYTVYEGNQPHNPSTTLHSTELNLQISESDLPQPLRSDCF SEQ ID NO:379, Coelacanth (Variant 1) QKEEYFMSEQTQPNSSSTSCLSTEPQLMISDTDAPHTLKRQVC SEQ ID NO:380, Coelacanth (Variant 2) QKEEYFMSEQTQPNSSSTSCLSTEPQLMISDTDAPHTLKSGF SEQ ID NO:381, and ghost sharkLTEYPVAEPSNANETDCMSSEPHLMISDDPKPLRSYCP SEQ ID NO:

383.

22. The immunostimulatory bacteria of claim 1, wherein the encoded therapeutic product is a modified STING comprising modifications selected from those conferring constitutive STING activity in inducing type I interferon (IFN): a) corresponding to one or more amino acid substitutions, with reference to an alignment with any of the human STING sequences shown in SEQ ID NO: 305-309: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273 A. S358A / E360A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A and S324A / S326A; b) Reference and SEQ Alignment with any of the human STING sequences shown in SEQ ID NO:305-309, corresponding to the permutation of C206Y or R284G; or c) reference to the alignment with any of the human STING sequences shown in SEQ ID NO:305-309, corresponding to the permutation of N154S / R284G.

23. The immunostimulatory bacteria of claim 6, wherein: The protein described as part of the cytoplasmic DNA / RNA sensor pathway is STING; the STING is chimeric STING, which contains a human STING polypeptide with CTT from Tasmanian devil and an amino acid substitution that leads to constitutive expression of type I interferon.

24. The immunostimulatory bacteria of claim 1, comprising a nucleic acid encoding a plurality of anticancer products as a polycistronic sequence under the control of a single promoter, wherein the construct encodes one of the following product combinations: IL-12, or IL-15, or IL12p70, or IL-15 / IL-15R. One or more of the α-chain complex; IL-2 and IL-12p70; IL-2 and IL-21; cytokine and interferon gene-stimulating factor (STING) pathway agonists; cytokines, STING pathway agonists, and co-stimulatory receptor ligands or immune checkpoint inhibitors; cytokines, STING pathway agonists, and TGF-β peptide antagonists; cytokines, STING pathway agonists, TGF-β peptide antagonists, and co-stimulatory receptor ligands or immune checkpoint inhibitors; anti-CTLA-4 antibodies, IL-15, and TGF-β receptor decoys or peptide antagonists; 4-1BBL and IL-15; 4-1BBL, IL-15, and TGF-β receptor decoys or peptide antagonists; anti-CTLA-4 antibodies and IL-12; anti- CTLA-4 antibody, IL-12 and TGF-β receptor decoys or peptide antagonists; 4-1BBL and IL-12; 4-1BBL, IL-12 and TGF-β receptor decoys or peptide antagonists; anti-CTLA-4 antibody and TGF-β receptor decoys or peptide antagonists; 4-1BBL and TGF-β receptor decoys or peptide antagonists; IL-15 and TGF-β receptor decoys or peptide antagonists; IL-12 and TGF-β receptor decoys or peptide antagonists; IL-12, IL-15 and TGF-β receptor decoys or peptide antagonists; and IL-15, IL-21 and TGF-β receptor decoys or peptide antagonists, wherein STING pathway agonists are products that increase type I interferon expression by activating the STING pathway.

25. The immunostimulatory bacteria of claim 1, comprising a nucleic acid encoding one of the following combinations of products: anti-CTLA-4 antibody and STING peptide, IL-15 and STING peptide, 4-1BBL and STING peptide, TGF-β receptor decoy or antagonist peptide, and STING peptide, IL-12 and STING peptide, anti-CTLA-4 antibody, IL-15 and STING peptide, 4-1BBL, IL-15 and STING peptide, TGF-β receptor decoy or antagonist peptide, IL-15 and STING peptide, anti-CTLA-4 antibody, IL-12 and STING peptide, 4-1BBL, IL-12 and STING peptide, TGF-β receptor decoy or multiple Peptide antagonists, IL-12 and STING peptides, anti-CTLA-4 antibodies, IL-15, TGF-β receptor decoys or peptide antagonists and STING peptides, 4-1BBL, IL-15, TGF-β receptor decoys or peptide antagonists and STING peptides, anti-CTLA-4 antibodies, IL-12, TGF-β receptor decoys or peptide antagonists and STING peptides, 4-1BBL, IL-12, TGF-β receptor decoys or peptide antagonists and STING peptides, anti-CTLA-4 antibodies, IL-12, IL-15 and STING peptides, 4-1BBL, IL-12, IL-15 and STING peptides, TGF-β receptor decoys or peptide antagonists, IL -12, IL-15 and STING peptide, TGF-β receptor decoy or peptide antagonist, IL-12, IL-15 and STING peptide, anti-CTLA-4 antibody, IL-12, IL-15, TGF-β receptor decoy or peptide antagonist and STING peptide, 4-1BBL, IL-12, IL-21, TGF-β receptor decoy or peptide antagonist and STING peptide, anti-CTLA-4 antibody, IL-12, IL-15 and TGF-β receptor decoy or peptide antagonist, 4-1BBL, IL-12, IL-21 and TGF-β receptor decoy or peptide antagonist, IL-12, IL-15 and STING peptide, IL-15, IL-21 and STING peptide, IL-12, IL-21 and STING peptide, anti-CTLA-4 antibody, IL-15, IL-21 and STING peptide, anti-CTLA-4 antibody, IL-12, IL-21 and STING peptide, 4-1BBL, IL-15, IL-21 and STING peptide, 4-1BBL, IL-12, IL-21 and STING peptide, anti-CTLA-4 antibody and IL-15, anti-CTLA-4 antibody, IL-15 and TGF-β receptor decoy or peptide antagonist, 4-1BBL and IL-15, 4-1BBL, IL-15 and TGF-β receptor decoy or peptide antagonist, anti-CTLA-4 antibody and IL-12,Anti-CTLA-4 antibody, IL-12 and TGF-β receptor decoys or peptide antagonists, 4-1BBL and IL-12, 4-1BBL, IL-12 and TGF-β receptor decoys or peptide antagonists, anti-CTLA-4 antibody and TGF-β receptor decoys or peptide antagonists, 4-1BBL and TGF-β receptor decoys or peptide antagonists, IL-15 and TGF-β receptor decoys or peptide antagonists, IL-12 and TGF-β receptor decoys or peptide antagonists, IL-12, IL-15 and TGF-β receptor decoys or peptide antagonists, and IL-15, IL-21 and TGF-β receptor decoys or peptide antagonists, wherein: 4-1BBL is a 4-1BBL with a missing cytoplasmic domain, a 4-1BBL with a modified cytoplasmic domain, a 4-1BBL with a truncated cytoplasmic domain, or a 4-1BBL with both truncated and modified cytoplasmic domains; the anti-CTLA-4 antibody is scFv or scFv-Fc; and the STING peptide is a variant STING peptide or a chimeric STING peptide or a chimeric STING peptide with amino acid substitutions; and the constitutively active STING peptide contains one or more modifications, thereby constitutively inducing type I interferon (IFN).

26. The immunostimulatory bacteria of claim 1, encoding a combination of therapeutic products selected from the following: IL-2, IL-12p70, and STING gain-of-function (GOF) variants; IL-2, IL-21, and STING GOF variants; IL-2, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt), wherein Δcyt is a missing cytoplasmic domain; IL-2, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα and STING GOF variants; IL-15 / IL-15Rα, STING... GOF variants 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 variants; IL-15 / IL-15Rα, IL-21 and STING GOF variants; IL-15 / IL-15Rα, IL-12p70, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); IL-15 / IL-15Rα, IL-21, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-21; IL-12p70, IL-21 and STING GOF variants; IL-12p70, IL-21, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and STING GOF variants; IL-12p70, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); IL-12p70 and IL-18; IL-12p70, IL-18 and STING GOF variants; IL-12p70, IL-18, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptors or TGF-β peptide antagonists, IL-2 and IL-12p70; TGF-β decoy receptors or TGF-β peptide antagonists, IL-2 and IL-21; TGF-β decoy receptors or TGF-β peptide antagonists, IL-2, IL-12p70 and STING GOF variants; TGF-β decoy receptors or TGF-β peptide antagonists, IL-2, IL-21 and STING GOF variants; TGF-β decoy receptors or TGF-β peptide antagonists, IL-2, IL-12p70, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt);TGF-β decoy receptors or TGF-β peptide antagonists, IL-2, IL-21, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptors or TGF-β peptide antagonists, IL-15 / IL-15Rα and STING GOF variants; TGF-β decoy receptors or TGF-β peptide antagonists, IL-15 / IL-15Rα, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); TGF-β bait receptors or TGF-β peptide antagonists, IL-15 / IL-15Rα and IL-12p70; TGF-β bait receptors or TGF-β peptide antagonists, IL-15 / IL-15Rα and IL-21; TGF-β bait receptors or TGF-β peptide antagonists, IL-15 / IL-15Rα, IL-12p70 and STING GOF variants; TGF-β bait receptors or TGF-β peptide antagonists, IL-15 / IL-15Rα, IL-21 and STING GOF variants; TGF-β bait receptors or TGF-β peptide antagonists, IL-15 / IL-15Rα, IL-12p70, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptors or TGF-β peptide antagonists, IL-15 / IL-15Rα, IL-21, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptors or TGF-β peptide antagonists, IL-12p70 and IL-21; TGF-β decoy receptors or TGF-β peptide antagonists, IL-12p70, IL-21 and STING GOF variants; TGF-β decoy receptors or TGF-β peptide antagonists, IL-12p70, IL-21, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); TGF-β bait receptors or TGF-β peptide antagonists, and IL-12p70; TGF-β bait receptors or TGF-β peptide antagonists, IL-12p70 and STING GOF variants; TGF-β bait receptors or TGF-β peptide antagonists, IL-12p70, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); TGF-β bait receptors or TGF-β peptide antagonists, IL-12p70 and IL-18; TGF-β bait receptors or TGF-β peptide antagonists, IL-12p70, IL-18 and STING GOF variants; TGF-β bait receptors or TGF-β peptide antagonists, IL-12p70, IL-18, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); TGF-β decoy receptors or TGF-β peptide antagonists, and STING GOF variants;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 variant; anti-CTLA-4 antibody, IL-2, IL-21, and STING GOF variant; anti-CTLA-4 antibody, IL-2, IL-12p70, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-2, IL-21, STING GOF variant, and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, and STING GOF variant; anti-CTLA-4 antibody, IL-15 / IL-15Rα, and STING GOF variant. GOF variants 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 variants; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21 and STING GOF variants; anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-12p70, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody, IL-15 / IL-15Rα, IL-21, STING GOF variants 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 variants; anti-CTLA-4 antibody, IL-12p70, IL-21, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody and IL-12p70; anti-CTLA-4 antibody, IL-12p70 and STING GOF variants; anti-CTLA-4 antibody, IL-12p70, STING GOF variants 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 variants; anti-CTLA-4 antibody, IL-12p70, IL-18, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); anti-CTLA-4 antibody and STING GOF variants; CD40 agonist, IL-2 and IL-12p70;CD40 agonists, IL-2, and IL-21; CD40 agonists, IL-2, IL-12p70, and STING GOF variants; CD40 agonists, IL-2, IL-21, and STING GOF variants; CD40 agonists, IL-2, IL-12p70, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonists, IL-2, IL-21, STING GOF variants, and 4-1BBL (including 4-1BBLΔcyt); CD40 agonists, IL-15 / IL-15Rα, and STING GOF variants; CD40 agonists, IL-15 / IL-15Rα, and STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); CD40 agonists, IL-15 / IL-15Rα and IL-12p70; CD40 agonists, IL-15 / IL-15Rα and IL-21; CD40 agonists, IL-15 / IL-15Rα, IL-12p70 and STING GOF variants; CD40 agonists, IL-15 / IL-15Rα, IL-21 and STING GOF variants; CD40 agonists, IL-15 / IL-15Rα, IL-12p70, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); CD40 agonists, IL-15 / IL-15Rα, IL-21, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); CD40 agonists, IL-12p70 and IL-21; CD40 agonists, IL-12p70, IL-21 and STING GOF variants; CD40 agonists, IL-12p70, IL-21, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); CD40 agonists and IL-12p70; CD40 agonists, IL-12p70 and STING GOF variants; CD40 agonists, IL-12p70, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); CD40 agonists, IL-12p70 and IL-18; CD40 agonists, IL-12p70, IL-18 and STING GOF variants; CD40 agonists, IL-12p70, IL-18, STING GOF variants and 4-1BBL (including 4-1BBLΔcyt); and CD40 agonists and STING GOF variants, wherein: 4-1BBL is 4-1BBL with a missing cytoplasmic domain (4-1BBLΔcyt), 4-1BBL with a modified cytoplasmic domain, 4-1BBL with a truncated cytoplasmic domain, or 4-1BBL with both truncated and modified cytoplasmic domains; the STING GOF variant is a variant of STING containing one or more modifications, thereby constitutively inducing type I interferon (IFN); and the anti-CTLA-4 antibody is scFv or scFv-Fc.

27. The immunostimulatory bacteria of claim 1, comprising: (a) a nucleic acid encoding IL-36γ; and / or (b) a nucleic acid encoding an immune checkpoint inhibitor antibody or its antigen-binding portion; and / or (c) a nucleic acid encoding a product, said product being an antibody against scFv or an scFv-Fc double-stranded polypeptide.

28. The immunostimulatory bacteria of claim 1, comprising nucleic acid encoding an immune checkpoint inhibitor antibody or its antigen-binding portion, wherein the immune checkpoint is CTLA-4, PD-1, or PD-L1.

29. The immunostimulatory bacteria of claim 1, wherein: The encoded product is a variant protein that is part of a cytoplasmic DNA / RNA sensor pathway leading to type I interferon (IFN) expression; said variant protein is a variant of STING, MDA5, RIG-1, or IRF-3; and said variant protein contains a mutation selected from those that confer constitutive activity in inducing type I interferon (IFN): a) refer to SEQ ID For the comparison of NO:305-309, the STING is selected from one or more of the following: S102P, V147L, V147M, N154S, V155M, G166E, C206Y, G207E, S102P / F279L, F279L, R281Q, R284G, R284S, R284M, R284K, R284T, R197A, D205A, R310A, R293A, T294A, E296A, R197A / D205A, S272A / Q273A, R310A / E316A, E316A, E316N, E316Q, S272A, R293A / T294A / E296A, D231A, R232A, K236A, Q273A, S358A / E36 0A / S366A, D231A / R232A / K236A / R238A, S358A, E360A, S366A, R238A, R375A, N154S / R284G and S324A / S326A; b) Reference and SEQ The comparison with SEQ ID NO:310, in MDA5, is selected from one or more of the following: T331R, A489T, R822Q, G821S, A946T, R337G, D393V, G495R, R720Q, R779H, R779C, L372F, and A452T; c) the comparison with SEQ ID NO:311, in RIG-I, is selected from one or both of E373A and C268F; and d) the comparison with SEQ ID NO:312, in IRF-3, is selected from the mutation S396D.

30. The immunostimulatory bacteria of claim 1, wherein said bacteria lacks flagella and is msbB - pagP - ansB - and csgD - Wild-type bacteria have flagella.

31. The immunostimulatory bacteria of claim 1, wherein the genome of said bacteria is modified such that: said bacteria lack flagella, wherein wild-type bacteria have flagella; or said bacteria contains a genome modification such that said bacteria is msbB. - / pagP - ; or the bacteria lack flagella and are pagP - ansB - and csgD - The wild-type bacteria have flagella; and / or the bacteria are asd - csgD - purI - msbB - and pagP - And lacking flagella, where wild-type bacteria possess flagella; or the bacteria are csgD - purI - msbB - and pagP - Furthermore, it lacks flagella, unlike wild-type bacteria which do possess flagella.

32. The immunostimulatory bacteria of claim 1, wherein the bacteria comprises a genomic modification, thereby the bacteria lacking flagella and being msbB - / pagP - .

33. The immunostimulatory bacteria of claim 1, wherein the genome of said bacteria is modified to asd - The plasmid encodes aspartate-semialdehyde dehydrogenase (asd).

34. The immunostimulatory bacteria of claim 1, wherein the bacteria is an adenine auxotroph.

35. The immunostimulatory bacteria of claim 1, wherein the immunostimulatory bacteria have a genome modification such that the genome confers a bacterial phenotype of Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD or Δasd / ΔFLG / ΔpagP / ΔansB / ΔcsgD / ΔmsbB / ΔpurI, wherein the plasmid optionally encodes aspartate semialdehyde dehydrogenase (asd).

36. The immunostimulatory bacteria of claim 1, wherein: The bacteria are Gram-negative bacteria; or the bacteria are *Salmonella*, *Shigella*, *Escherichia*, *Bifidobacteriae*, *Rickettsia*, *Vibrio*, *Listeria*, *Klebsiella*, *Bordetella*, *Neisseria*, *Aeromonas*, *Francisella*, *Cholera*, or *Corynebacterium*. Strains of *Haemophilus*, *Citrobacter*, *Chlamydia*, *Haemophilus*, *Brucella*, *Mycobacterium*, *Mycoplasma*, *Legionella*, *Rhodococcus*, *Pseudomonas*, *Helicobacter*, *Bacillus*, or *Erysipelothrix*, or any of the aforementioned bacterial strains, their attenuated strains or modified strains.Or the bacteria mentioned are Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, or Chlamydia pneumoniae. *Haemophilus somnus*, *Brucella abortus*, *Mycobacterium intracellulare*, *Legionella pneumophila*, *Rhodococcus equi*, *Pseudomonas aeruginosa*, *Helicobacter mustelae*, *Vibrio cholerae*, *Bacillus subtilis*, *Erysipelothrix rhusiopathiae*, *Yersinia enterocolitica*, *Rochalimaea quintana*, or *Agrobacterium tumerfacium*, or attenuated strains or modified strains thereof.

37. The immunostimulatory bacteria of claim 1, wherein the bacteria is a strain of Salmonella, or an attenuated strain thereof, or a modified strain thereof.

38. The immunostimulatory bacteria of claim 37, wherein the bacteria is a strain of Salmonella typhimurium, or an attenuated strain thereof, or a modified strain thereof.

39. The immunostimulatory bacteria of claim 38, wherein the bacteria are derived from strain YS1646, or strain ATCC 14028, or a strain having all the identifying characteristics of strain ATCC 14028.

40. Use of the immunostimulatory bacteria of any one of claims 1-39 in the preparation of a pharmaceutical composition for treating cancer.

41. The use of claim 40, wherein the cancer is selected from leukemia; lymphoma; gastric cancer; and cancers of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, rectum, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, cervix and liver.

42. The immunostimulatory bacteria of claim 37, wherein the bacteria comprises a genome modification, thereby the bacteria lacking flagella and being msbB - / pagP - .

43. The immunostimulatory bacteria of claim 6, wherein the cytoplasmic DNA sensor protein is a chimeric STING having the amino acid sequence shown in SEQ ID NO: 354 or 397.

44. A combination comprising: a composition comprising the immunostimulatory bacteria of any one of claims 1-39; and another anticancer or immunotherapeutic agent for immunotherapy, wherein the composition and agent are in the same or separate compositions.

45. The combination of claims 44, wherein the immunotherapy is selected from cell therapy, CRISPR therapy, bispecific T-cell adaptor proteins, PARP (poly(ADP-ribose) polymerase) inhibitors, histone deacetylase (HDAC) inhibitors, chemotherapeutic compounds, surgery, radiotherapy, cancer vaccines, oncolytic viruses, anti-PD-1 antibodies, anti-CTLA-4 antibodies, anti-PD-L1 antibodies, anti-IL-6 antibodies, anti-VEGF antibodies, anti-VEGFR antibodies, anti-VEGFR2 antibodies, anti-CD38 antibodies, anti-EGFR antibodies, anti-Her2 antibodies, anti-mesothelin antibodies, anti-BCMA antibodies, and antibody fragments thereof.

46. ​​The combination of claim 44, wherein the immunotherapy is selected from CAR-T therapy.

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