Treatment of cancer and infectious diseases with killed intact bacteria

Abstract

The present disclosure relates generally to compositions, dosage forms, and methods for the prevention and treatment of cancer and infections. One example method entails administering to a patient an effective amount of a composition comprising 1 * 107 to 500 * 107 intact, stable and substantially inactive Escherichia coli cells that have been treated to result in an untreated Escherichia coli cell, when measured by a tachypleus amebocyte lysate (LAL) assay, as compared to an untreated wild-type Escherichia coli cell. Lipopolysaccharide (LPS)-related endotoxin activity is reduced by about 70% to 99%, and wherein the composition contains LPS of 124 to 62000 endotoxin units (EU).

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 426,245, filed on November 17, 2022, under 35 U.S.C. § 119(e), the entire content of which is incorporated herein by reference. Background of the Invention

[0002] Cancer is the second leading cause of death in the United States. In the past decade, advancements in cancer immunotherapy have transformed cancer treatment and led to many new treatment approaches. Inhibiting immune checkpoints with blocking antibodies against programmed cell death 1 (PD1) or programmed cell death ligand 1 (PD - L1) and cytotoxic T - lymphocyte antigen 4 (CTLA - 4) has resulted in long - term disease - free survival in several advanced malignancies. However, many patients do not respond to immunotherapy, and many patients who benefit from current immunotherapy ultimately experience disease progression.

[0003] Tumors evade immune surveillance by inhibiting innate and adaptive immune effectors, restricting neoantigen presentation, and impairing infiltrating immune effector cells. The failure of PD - 1 / PD - L1 inhibitors may be due to insufficient generation of anti - tumor T cells, exclusion of T cells from the tumor, insufficient function of tumor - specific T cells, and / or impaired formation of T - cell memory.

[0004] Effective and durable anti - tumor immune responses require systemic innate and adaptive immunity. Many steps involved in innate and adaptive anti - tumor immune responses, including the generation, mobilization, migration, activation, and antigen presentation of immune cells, occur outside the tumor microenvironment. These steps are largely controlled by pattern - recognition receptors (PRRs), which recognize a wide variety of endogenous and exogenous danger, pathogen, and exogenous - associated molecular patterns. Toll - like receptors (TLRs) represent the most prominent family of PRRs, including nine functional TLRs in mammals, and a subset of which are expressed on essentially all immune cells, including monocytes, macrophages, neutrophils, natural killer (NK) cells, γδ T cells, NKT cells, dendritic cells, CD4+ T cells, and CD8+ T cells.

[0005] Although TLR agonists (TLRa) can be released from dying normal or malignant human cells, most naturally occurring TLRa are present in bacteria, viruses, and other microorganisms, alerting the immune system to the presence of pathogens and activating appropriate defense responses. Activation of TLR signaling leads to the direct activation of immune cell functions and the indirect activation through the induction of the secretion of cytokines and chemokines, which act through autocrine and paracrine mechanisms.

[0006] Due to the role of TLRs in host-mediated anti-pathogen and anti-tumor immune responses, significant efforts have been made to produce TLR agonist adjuvants and therapeutic agents for use in infectious and anti-tumor immunotherapies. A variety of monospecific, purified or synthetic TLR agonists have been produced and tested in preclinical and clinical settings. TLR agonists are used as adjuvants in prophylactic vaccines. However, despite observing anti-pathogen and anti-tumor activity in the context of therapeutic vaccines, these efforts have faced significant challenges. Issues encountered include lack of potency and excessive toxicity, indicating a need for further improvement of TLR agonists for the prevention and treatment of existing infections and cancers.

[0007] Lipopolysaccharide (LPS) endotoxin accounts for approximately 75% of the outer cell membrane of Gram-negative bacteria and is a potent TLR4 agonist that triggers direct and indirect activation of innate and adaptive immune cells in a dose-dependent manner. LPS endotoxin is considered a major factor in the anti-tumor activity and intravenous toxicity of Gram-negative bacteria. SUMMARY OF THE INVENTION

[0008] The present disclosure demonstrates that attenuated, inactivated, intact, and stable bacteria produced from non-pathogenic Gram-negative bacterial cells, such as Escherichia coli, exhibit potent efficacy in inhibiting tumor growth and viral replication and activity. At the same time, these treated bacterial cells are well tolerated in in vivo studies, indicating a high therapeutic index and suitability for clinical development and use.

[0009] Accordingly, one embodiment of the present disclosure provides a method for treating or preventing cancer or an infectious disease in a patient in need thereof, comprising administering to the patient an effective amount of a composition comprising 1 × 10 7 to 500 × 10 7 intact, stable, and substantially non-viable Escherichia coli cells that have been treated to result in a reduction of lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% as measured by the Limulus amebocyte lysate (LAL) assay compared to untreated wild-type Escherichia coli cells, and wherein the composition contains 124 to 62,000 endotoxin units (EU) of LPS.

[0010] According to one embodiment of the present disclosure, there is provided a method for treating or preventing cancer or an infectious disease in a patient in need thereof, comprising administering to the patient an effective amount of a composition comprising 1 × 10 7 to 500 × 10 7A complete and substantially non-viable Escherichia coli cell that has been treated to result in a reduction of lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% as measured by the Limulus amoebocyte lysate (LAL) assay, compared to untreated wild-type Escherichia coli cells, and wherein the composition contains 124 to 62,000 endotoxin units (EU) of LPS.

[0011] In some embodiments, the composition comprises 2 × 10 7 to 200 × 10 7 complete and substantially non-viable Escherichia coli cells. In some embodiments, the composition comprises 3 × 10 7 to 100 × 10 7 complete and substantially non-viable Escherichia coli cells. In some embodiments, wherein the composition comprises 5 × 10 7 to 50 × 10 7 complete and substantially non-viable Escherichia coli cells. In some embodiments, the composition comprises 3 × 10 7 、7 × 10 7 、10× 10 7 、20 × 10 7 or 70 × 10 7 complete and substantially non-viable Escherichia coli cells.

[0012] In some embodiments, the composition contains 372 EU to 24,800 EU of LPS. In some embodiments, the composition contains 372 EU to 8,680 EU of LPS. In some embodiments, the composition contains 868 EU to 2,480 EU of LPS.

[0013] In some embodiments, the complete and substantially non-viable Escherichia coli cells have been treated to result in a reduction of LPS-associated endotoxin by about 85% to 98%. In some embodiments, the complete and substantially non-viable Escherichia coli cells have been treated to result in a reduction of LPS-associated endotoxin by about 90% to 98%.

[0014] In some embodiments, the administration is once daily, every other day, every 3 days, every 5 days, every 6 days, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, once every 9 months, or once a year.

[0015] In some embodiments, the treatment of the E. coli cells is carried out using polymyxin, preferably polymyxin B or polymyxin E. In some embodiments, the treatment of the E. coli cells is carried out at a temperature of about 2°C to about 10°C, preferably at about 4°C. In some embodiments, the treatment of the E. coli cells is carried out using polymyxin and glutaraldehyde. In some embodiments, the treatment is carried out using polymyxin B in a dose range of about 3 mg / mL to about 1,000 mg / mL and glutaraldehyde in a dose range of about 0.1% to about 1.0%.

[0016] In some embodiments, the composition further comprises phosphate buffer, Mg 2+ and trehalose. In some embodiments, the composition comprises 0.3 × 10 9 / mL to 5 × 10 9 / mL of intact and substantially non-viable E. coli cells, 0.5 mg / mL to 2 mg / mL of disodium phosphate dihydrate, 0.1 mg / mL to 0.4 mg / mL of potassium dihydrogen phosphate, 3 mg / mL to 12 mg / mL of sodium chloride, 0.05 mg / mL to 0.3 mg / mL of potassium chloride, 0.15 mg / mL to 0.6 mg / mL of magnesium chloride hexahydrate and 50 mg / mL to 200 mg / mL of trehalose dihydrate, with a pH value of 7.0 to 7.7.

[0017] In some embodiments, the administration is intravenous, intratumoral, subcutaneous, intramuscular, intrahepatic, intravesical, intranasal or intraperitoneal.

[0018] In some embodiments, the patient has a solid tumor. In some embodiments, the solid tumor is a metastatic solid tumor. In some embodiments, the cancer is selected from bladder cancer, gastrointestinal cancers (esophageal cancer, gastric cancer, liver cancer, colorectal cancer, pancreatic cancer), cervical cancer, ovarian cancer, endometrial cancer, leukemia, lymphoma, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, kidney cancer, melanoma, prostate cancer and thyroid cancer.

[0019] In some embodiments, the method further comprises administering to the patient a second agent selected from cyclophosphamide, IL-2, non-steroidal anti-inflammatory drugs (NSAIDs), anti-PD-1 or anti-PD-L1 antibodies, anti-CTLA-4 antibodies and anti-CD20 antibodies.

[0020] In some embodiments, the patient has an infection. In some embodiments, the infection is caused by hepatitis B virus (HBV) or human immunodeficiency virus (HIV).

[0021] In one embodiment, a method for providing a therapeutically acceptable composition is also provided, comprising: lyophilizing a solution to prepare a lyophilized composition, the solution comprising at least 1 × 10 6 intact and substantially non-viable Escherichia coli cells that have been treated to result in a reduction in lipopolysaccharide (LPS)-associated endotoxin activity of about 70% to 99% compared to untreated wild-type Escherichia coli cells when measured by the Limulus amebocyte lysate (LAL) assay; and storing the lyophilized composition (a) at a temperature of 1°C to 10°C for at least 2 months or (b) at a temperature of -15°C or below for at least 2 years, thereby providing a therapeutically acceptable composition suitable for therapeutic use.

[0022] In some embodiments, the solution further comprises a phosphate buffer, Mg 2+ and trehalose. In some embodiments, the solution comprises from 0.3 × 10 9 / mL to 5 × 10 9 / mL of intact and substantially non-viable Escherichia coli cells, 0.5 mg / mL to 2 mg / mL of disodium phosphate dihydrate, 0.1 mg / mL to 0.4 mg / mL of potassium dihydrogen phosphate, 3 mg / mL to 12 mg / mL of sodium chloride, 0.05 mg / mL to 0.3 mg / mL of potassium chloride, 0.15 mg / mL to 0.6 mg / mL of magnesium chloride hexahydrate, and 50 mg / mL to 200 mg / mL of trehalose dihydrate, with a pH value of 7.0 to 7.7.

[0023] In one embodiment, a method for treating or preventing cancer or an infectious disease in a patient in need thereof is also provided, comprising administering to the patient (a) an effective amount of a composition comprising intact and substantially non-viable Escherichia coli cells that have been treated to result in a reduction in lipopolysaccharide (LPS)-associated endotoxin activity of about 70% to 99% compared to untreated wild-type Escherichia coli cells when measured by the Limulus amebocyte lysate (LAL) assay, and (b) an exogenous antigen associated with the cancer or infectious disease.

[0024] In some embodiments, the antigen is a tumor-associated antigen. In some embodiments, the antigen is a viral or bacterial antigen. In some embodiments, the composition comprises from 1 × 10 7 to 500 × 10 7 intact and substantially non-viable Escherichia coli cells and contains 124 to 62,000 endotoxin units (EU) of LPS. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is shown that the manufacturing process of decoy bacteria stabilizes bacterial cells.

[0026] Figure 2 It is shown that based on three 48-hour in vitro (full) dose responses, the levels of the same cytokines secreted by human PBMCs induced by decoy bacteria are approximately 3,500-fold higher than those of mouse PBMCs.

[0027] Figure 3 It is shown that decoy bacteria cooperate with human PBMCs to kill human MDA-MB-231 breast cancer cells in vitro.

[0028] Figure 4 It is shown that single-agent decoy inhibits metastasis and prolongs the survival of mice with orthotopic mouse CT26 colorectal cancer.

[0029] Figure 5 It is shown that single-agent decoy bacteria inhibit the in vivo growth of mouse colorectal cancer in a dose-dependent manner without obvious toxicity.

[0030] Figure 6 It is shown that decoy cooperates with low-dose cyclophosphamide (LDC) to inhibit the growth of subcutaneous CT26 colorectal tumors.

[0031] Figure 7 It is shown that decoy cooperates with low-dose interleukin-2 (IL-2) and / or low-dose indomethacin to inhibit subcutaneous CT26 colorectal cancer: when the tumor is 75 mm 3 in size, treatment is started on day 10, with 5 mice in each group.

[0032] Figure 8 It is shown that decoy inhibits the growth of subcutaneous CT26 colorectal cancer in mice.

[0033] Figure 9 It is shown that single-agent decoy prolongs the survival of mice with metastatic mouse Pan02 pancreatic cancer.

[0034] Figure 10 It is shown that in a metastatic mouse Pan02 pancreatic cancer model, decoy has single-agent activity and cooperates with oral low-dose NSAID indomethacin.

[0035] Figure 11 It is shown that decoy has single-agent activity and cooperates with NSAID and anti-PD-1 to achieve a 10 / 12 regression of 194 mm 3 subcutaneous H22 hepatocellular carcinoma (HCC) tumors in mice.

[0036] Figure 12It is shown that cured mice re-challenged with fresh HCC tumor cells reject the tumors (immune memory).

[0037] Figure 13 It is shown that the decoy synergizes with NSAID to cause regression of 4 / 6 183 mm 3 subcutaneous H22 hepatocellular carcinoma (HCC) tumors in mice.

[0038] Figure 14 It is shown that the decoy (once a week), NSAID and anti-PD-1 synergize to cause 100% regression of 183 mm 3 subcutaneous H22 hepatocellular carcinoma (HCC) tumors in mice.

[0039] Figure 15 It is shown that the decoy synergizes with the orally administered low-dose NSAID indomethacin to cause regression of established subcutaneous H22 hepatocellular carcinoma (HCC) in mice.

[0040] Figure 16 It is shown that the decoy and NSAID synergize with anti-PD-1 to cause regression of 200 mm 3 H22 hepatocellular carcinoma in mice, with a therapeutic index > 33 (the decoy group also received anti-PD-1 + oral NSAID).

[0041] Figure 17 It is shown that cured mice re-challenged with fresh HCC tumor cells reject the tumors (immune memory).

[0042] Figure 18 It is shown that the results of NanoString gene expression analysis of tumor RNA reveal an increase in the treatment-related HCC tumor inflammation score (cold to hot tumors).

[0043] Figure 19 It is shown that the decoy bacteria synergize with low-dose cyclophosphamide (LDC) to induce 100% regression of established A20 non-Hodgkin lymphoma (NHL) in mice after only two weeks of treatment.

[0044] Figure 20 It is shown that the synergistic regression of A20 non-Hodgkin lymphoma (NHL) tumors by the decoy and low-dose cyclophosphamide (LDC) is persistent and induces immune memory.

[0045] Figure 21 It is shown that A20 non-Hodgkin lymphoma (NHL) tumors regenerated after sub-optimal decoy and low-dose cyclophosphamide (LDC) treatment for 1 or 2 weeks are sensitive to optimal retreatment, and decoy + LDC treatment can cause regression of very large tumors.

[0046] Figure 22It is shown that the decoy synergizes with low-dose cyclophosphamide (LDC) to eradicate 200 mm 3 subcutaneous murine A20 non-Hodgkin lymphoma (NHL), with immune memory, and is reproducible.

[0047] Figure 23 It is shown that high percentage eradication of subcutaneous A20 non-Hodgkin lymphoma (NHL) by the decoy and low-dose cyclophosphamide (LDC) requires CD4+ and CD8+ T cells (adaptive immunity) and NK cells (innate immunity) (but single depletion results in transient regression and some eradication).

[0048] Figure 24 It is shown that two different decoy strains produced similar anti-tumor activity against murine A20 non-Hodgkin lymphoma (NHL).

[0049] Figure 25 It is shown that the decoy and low-dose cyclophosphamide (LDC) synergize with rituximab to induce regression of subcutaneous human Ramos non-Hodgkin lymphoma (NHL) in SCID mice.

[0050] Figure 26 It is shown that the decoy and low-dose cyclophosphamide (LDC) can synergize with rituximab to induce immune memory through the innate immune system.

[0051] Figure 27 It is shown that the expression of exogenous antigen (e.g., HER2, below) significantly enhances the anti-tumor effect of the decoy alone (above). After treatment with the decoy alone, two out of five animals had complete remission (below).

[0052] Figure 28 It is shown that the decoy inhibited the replication of human hepatitis B virus (HBV) in a mouse model of chronic HBV infection (AAV-HBV).

[0053] Figure 29 It is shown that the decoy inhibited the HBeAg level of human hepatitis B virus (HBV) in a mouse model of chronic HBV infection (AAV-HBV).

[0054] Figure 30 It is shown that the decoy inhibited the HBsAg level of human hepatitis B virus (HBV) in a mouse model of chronic HBV infection (AAV-HBV).

[0055] Figure 31 It is shown that the decoy inhibited the expression of human hepatitis B virus (HBV) DNA in the liver of mice infected with HBV (AAV-HBV model).

[0056] Figure 32It was shown that the decoy inhibited the expression of human hepatitis B virus (HBV) HBeAg in the livers of mice infected with HBV (AAV-HBV model).

[0057] Figure 33 It was shown that the decoy and entecavir inhibited the expression of hepatitis B virus (HBV) cccDNA-like molecules in the livers of mice infected with HBV (AAV-HBV model).

[0058] Figure 34 It was shown that entecavir, the decoy, and their combination inhibited the replication of human hepatitis B virus (HBV) in a mouse model of chronic human HBV infection (AAV-HBV).

[0059] Figure 35 It was shown that the decoy inhibited the levels of human hepatitis B virus (HBV) HBeAg in a mouse model of chronic HBV infection.

[0060] Figure 36 It was shown that the decoy inhibited the levels of human hepatitis B virus (HBV) HBsAg in a mouse model of chronic HBV infection.

[0061] Figure 37 It was shown that the decoy reduced the levels of human HIV virus in a (humanized) mouse model of chronic human HIV infection.

[0062] Figure 38 It was shown that the decoy induced the expression of transient plasma cytokines, chemokines, and biomarkers in human subjects. The reference ranges (healthy volunteers, pg / mL or units / mL) are provided in the figure. Most baseline measurements reflected the lower limit of quantification of the assay.

[0063] Figure 39 It was shown that pharmacokinetic analysis confirmed the rapid clearance of the decoy following systemic administration in human subjects. Detailed description

[0064] The following description sets forth exemplary embodiments of the present technology. However, it should be recognized that such description is not intended to limit the scope of the present disclosure, but is provided as a description of exemplary embodiments.

[0065] As used in this specification, the following terms, phrases, and symbols generally have the following meanings, unless the context in which they are used indicates otherwise. Therapeutic use of treated bacteria

[0066] Experimental embodiments of the present disclosure show that attenuated, intact, stable, and inactivated Gram-negative bacterial cells (such as Escherichia coli) (bait bacteria or bait) that have been processed to significantly reduce LPS-related endotoxin activity contain agonists for all functional human TLR receptors and receptor heterodimers (TLR 2, 2 / 1, 2 / 6, 3, 4, 5, 7, 8, and 9), as well as agonists for NOD-like (NLR) and stimulators for the interferon (IFN) gene (STING) receptor. Surprisingly, compared to untreated parental bacteria, the bait bacteria reduced pyrogenicity and acute toxicity in vivo but increased the ability to induce the secretion of many cytokines and chemokines by immune cells. Thus, such processed bacterial cells are suitable for providing a safe and effective method to stimulate the immune response of a subject, which can be used to treat tumors and bacterial, fungal, parasitic, or viral infections.

[0067] These processed bacterial cells, also referred to as "bait bacteria" or simply "bait", are attenuated, 100% killed, stable, and intact bacteria produced from non-pathogenic Gram-negative bacterial cells (such as Escherichia coli), resulting in a reduction of LPS endotoxin activity and pyrogenicity by approximately 90%. A comprehensive non-clinical pharmacology program has been developed to support the first-in-human (FIH) study of the bait. The primary pharmacodynamic (PD) studies of the bait included in vitro evaluation of cytokine and chemokine secretion induction in murine and human peripheral blood mononuclear cells, and in vivo evaluation of the anti-tumor activity of intravenous injection on established subcutaneous (s.c.) murine colorectal cancer, metastatic murine pancreatic cancer, established subcutaneous murine hepatocellular carcinoma (HCC), and established subcutaneous murine and human non-Hodgkin lymphoma (NHL) models. The bait was also tested against established murine breast cancer tumors with and without exogenous antigen expression. The bait was tested as a single agent and in combination with certain other therapeutic drugs. Significant single-agent anti-tumor activity was observed in several models, including the regression of established murine breast cancer tumors expressing exogenous antigens. Synergistic effects were observed with low-dose cyclophosphamide (LDC), indomethacin, rituximab, and anti-PD-1 checkpoint therapy, but not with drugs such as anti-GITR antibody, INF-γ, phenformin, gemcitabine, or 5-FU. In addition, tumor regression therapy was associated with the induction of immune memory, which was demonstrated by the rejection of tumor re-challenge without additional treatment.

[0068] Similarly, in animal models, the bait, either as a single agent or in combination with other drugs such as entecavir (ETV), showed potent activity in inhibiting the replication and activity of hepatitis B virus (HBV) and human immunodeficiency virus (HIV).

[0069] The safety of the bait was established 1 hour after intravenous injection in single-dose, two-week repeat-dose range finding, and four-week repeat-dose toxicology studies in New Zealand white rabbits (NZW), a non-human laboratory species considered to be most similar to humans in terms of sensitivity to adverse reactions to LPS. Additional safety information was obtained using the bait in studies conducted in mice.

[0070] The maximum tolerated dose (MTD) of the single-dose bait in rabbits was 1.5 x 10 9 inactivated bacteria [KB] / kg, with 4 dose levels tested. Bait was administered twice weekly for two weeks, with four dose levels tested, yielding a no-observed-adverse-effect level (NOAEL) of 6 x 10 7 KB / kg / dose. In the key 4-week repeat-dose toxicology study in rabbits (4 dose levels), the NOAEL of the bait was determined to be 4 x 10 7 KB / kg / dose. The study also found that the bait was 97% less pyrogenic (rectal temperature testing) in rabbits than the parental (untreated) bacteria and 3-fold less toxic (acute LD 100 ) than the parental (untreated) bacteria.

[0071] Based on these non-clinical findings, human clinical trials have been designed and conducted, and a formulation suitable for clinical use has been developed. Table 4 shows an example formulation. Example doses are shown in Table 5. The starting dose for the first part of the clinical study was 7 x 10 7 KB / patient, approximately 1 / 10 of the human equivalent dose (HED) determined based on the no-observed-adverse-effect level (NOAEL) observed in the 4-week rabbit toxicology study with twice-weekly dosing (4 x 10 7 KB / kg dose). Based on the 4-week Good Laboratory Practice (GLP) study data, including a 3.1-fold allometric scaling factor (dose reduction) for HED plus a 10-fold dose reduction safety adjustment, the starting human dose was 1.29 x 10 6 KB / kg or approximately 16 bait-related endotoxin units (EU) / kg, i.e., 7.74 x 10 7 KB per 60 kg subject; equivalent to approximately 960 EU per 60 kg subject (not the conventional 70 kg to account for lower patient weights). The starting dose in the study will be slightly lower, at 7.0 x 10 7KB: Equivalent to 868 EU or 1.8 ng / kg LPS per 60 kg subject. Based on published results of the systemic clearance of live and inactivated bacteria in mice, rabbits, and humans, rapid clearance of the bait by the liver and spleen is expected (within minutes to approximately 1 hour), and thus dose adjustment based on body weight is not considered necessary. The starting dose of LPS in the study was lower than the well-tolerated maximum dose (4 ng / kg) determined after intravenous injection of purified LPS in over 1000 healthy human volunteers. In the later stages of the clinical study, dosing will be repeated once weekly. If there are no safety concerns, subjects will take the bait continuously for up to 2 years.

[0072] As shown by the preliminary results of the Phase I clinical trial (Example 4), in all four cancer patients, including three patients with tumor progression prior to treatment, a one-hour intravenous infusion of 7 x 10 7 Inactivated bait bacteria led to disease stabilization. Equally importantly, within 30 - 120 minutes after the end of the infusion, the bait bacteria were cleared from the blood and a transient induction of over 50 cytokines, chemokines, and biomarkers was produced in the plasma, many of which are known to directly participate in stimulating innate and / or adaptive immune responses, including anti-tumor responses. The transient induction of cytokines and chemokines is an important and novel feature of the response to the bait bacteria, contributing to reducing the likelihood of systemic toxicity known to result from continuous or long-term systemic exposure to these potent immune-activating molecules.

[0073] Accordingly, in one embodiment of the present disclosure, there is provided a method for treating or preventing cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of a composition of treated bacteria.

[0074] In another embodiment, there is provided a method for stimulating an immune response in a subject in need thereof. In another embodiment, there is provided a method for preventing or treating an infection in a patient in need thereof. In another embodiment, there is provided a method for treating an immunodeficiency in a patient in need thereof. In another embodiment, there is provided a method for vaccinating a subject at risk of infection or cancer.

[0075] In some embodiments, the treated bacterial cells are intact, stable, substantially non-viable Gram-negative bacterial cells that have been treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity and / or pyrogenicity. In some embodiments, the intact and substantially non-viable Gram-negative bacterial cells have been treated to result in a reduction of approximately 70% to 99% in LPS-associated endotoxin activity as measured by the Limulus amebocyte lysate (LAL) assay, compared to untreated wild-type Gram-negative bacteria.

[0076] Candidate bacterial organisms useful in the methods herein are Gram-negative bacteria, including wild-type organisms having LPS-related endotoxin activity. The term "Gram-negative bacteria" refers to bacteria that do not retain the initial basic dye stain (such as crystal violet), which is part of the Gram staining procedure. In an exemplary Gram stain, cells are first fixed to a slide by heating and stained with a basic dye (such as crystal violet), which is taken up by both Gram-negative and Gram-positive bacteria. The slide is then treated with a mordant (such as Gram's iodine), which binds to the basic dye (e.g., crystal violet) and traps it within the cell. The cells are then washed with acetone or alcohol and then counterstained with a second dye of a different color (such as safranin). Gram-positive bacteria retain the initial purple stain, while Gram-negative bacteria are decolorized by the organic washing solvent and thus show the counterstain. Exemplary Gram-negative bacteria include, but are not limited to, Escherichia spp, Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp., and Vibrio spp.

[0077] Among Gram-negative bacteria, there is the Enterobacteriaceae, a large family that includes many well-known pathogens in addition to many harmless symbiotic bacteria, such as Salmonella, E. coli, Yersinia pestis, Klebsiella and Shigella, Proteus, Enterobacter, Serratia, and Citrobacter. Members of the Enterobacteriaceae are called enterobacteria because some members live in the intestines of animals.

[0078] In one embodiment, E. coli is selected as the organism. A particular strain considered is the E. coli strain 2617-143-312, (Migula) Castellani and Chalmers (ATCC ® 13070™). Another E. coli strain that can be used includes MG1655 (ATCC® 47076).

[0079] The term "lipopolysaccharide" (LPS) refers to a macromolecule composed of covalently linked lipid and polysaccharide (glycolipid). LPS consists of three parts: 1) O antigen; 2) core oligosaccharide; and 3) lipid A. The O antigen is a repeating glycan polymer attached to the core oligosaccharide and contains the outermost domain of the LPS molecule. The core oligosaccharide is directly attached to lipid A and usually contains sugars such as heptose and 3-deoxy-D-manno-octulosonic acid (also known as KDO, keto-deoxyoctulosonate). Lipid A is a phosphorylated glucosamine disaccharide linked to multiple fatty acids. The fatty acids anchor the LPS in the bacterial outer membrane, and the rest of the LPS extends from the cell surface.

[0080] Endotoxin activity resides in the lipid A domain portion of LPS and is thus also referred to as "LPS-related endotoxin activity or LPS endotoxin activity". Gram-negative bacteria contain additional TLR agonists, including agonists of TLR2 / 1, 2 / 6, 2, 3, 5, 7, 8, and 9, as well as other immune-stimulatory molecules such as STING (stimulator of interferon genes) and NOD (nucleotide-binding oligomerization domain-containing protein) agonists. Intact bacteria that enter the circulatory system are rapidly phagocytosed by immune cells in the liver and spleen, leading to direct and indirect immune cell / pathway activation through the induction of cytokine and chemokine secretion. The rapid clearance of circulating bacterial cells by immune cells in the liver and spleen helps to localize immune activation to key immune organs. Large amounts of LPS endotoxin are released or shed by live proliferating bacteria. If live bacterial cells proliferate, invade normal tissues and cells, and / or break down in the circulation, large amounts of immune activators can be released systemically. This can produce an inappropriate and excessive inflammatory response throughout the body, leading to potentially fatal shock (referred to as endotoxin or septic shock). Thus, the present invention and the use of killed and stabilized bacteria with reduced LPS endotoxin activity should allow for transient or short-lived immune activation in the liver and spleen after systemic administration, while significantly reducing the likelihood of inappropriate systemic inflammation produced by live bacteria that are capable of invading normal cells / tissues and proliferating, proliferating and breaking down in the systemic circulation, and shedding or releasing many different types of immune activators throughout the body. The most potent bacteria-related immune stimulant is LPS endotoxin, which can promote anti-tumor and anti-viral efficacy as well as systemic toxicity. LPS-related endotoxin activity can be measured by methods well known in the art, including, for example, the Limulus amebocyte lysate (LAL) assay, which utilizes the blood of the horseshoe crab and can detect very low levels of LPS. The presence of endotoxin activity will cause the horseshoe crab blood lysate to clot due to enzyme cascade amplification. Gel clotting, turbidimetric, and chromogenic forms of the LAL assay are available on the market.

[0081] Endotoxin activity assays based on enzyme-linked immunosorbent assay (ELISA) are also known, such as EndoLISA® from Hyglos in the Munich region of Germany. This assay uses an LPS-specific bacteriophage protein attached to a solid phase to capture LPS, and after a washing step, the presence of LPS is determined by adding recombinant factor C, which cleaves a compound and then emits fluorescence when activated by LPS. Factor C present in Limulus amebocyte lysate is normally present in a zymogen form and is the primer for the coagulation cascade that occurs in the LAL assay.

[0082] Pyrogenicity refers to the ability of a reagent to cause fever in a subject. Pyrogenicity can be measured by the elevation of rectal temperature in a rabbit following intravenous injection of a TLR agonist, an organism, or its derivative.

[0083] There are various methods for reducing the endotoxin activity and / or pyrogenicity of Gram-negative organisms. These methods include treating the organisms with reagents that bind LPS or disrupt its formation.

[0084] In one embodiment, reduction of endotoxin activity or pyrogenicity is achieved by treating bacterial organisms with antibiotics that inactivate endotoxin. Suitable such antibiotics are the polymyxins, including polymyxin B or polymyxin E. Determining the amount of antibiotic and the treatment conditions are within the skill of those in the art. In one embodiment, the polymyxin, whether polymyxin B or E, can be used at a concentration of about 3 micrograms to 5000 micrograms per milliliter. In another embodiment, the concentration of the polymyxin can be about 200 micrograms to 5000 micrograms per milliliter. In one embodiment, the antibiotic is administered to the bacteria for 10 minutes to 4 hours or about 30 minutes to about 3 hours.

[0085] In one embodiment, the bacteria are grown in the presence of magnesium (Mg) in the form of MgCl2. In one embodiment, the bacteria are treated with polymyxin in the presence of MgCl2 and at a temperature suitable for maintaining bacterial integrity. In one embodiment, the concentration of MgCl2 in the growth medium is about 0.5 mM to about 5.0 mM, or about 2 mM, and the concentration of MgCl2 in the treatment medium is about 5.0 mM to about 30 mM, or about 20 mM. In one embodiment, the temperature of the treatment medium is about 2°C to about 10°C, or about 4°C. Bacterial integrity is determined by the efficiency of recovery in well-defined pellets after centrifugation at 3,000 x g for 10 minutes, and by electron microscopy or light microscopy with Gram staining. In a preferred embodiment, the recovery rate of bacteria after treatment and washing is greater than about 80%, and the bacteria appear intact by light or electron microscopy.

[0086] In another embodiment, reduction of endotoxin activity is achieved by treating bacterial organisms with antibiotics known to disrupt KDO2-lipid IV A biosynthesis. For example, Goldman et al., J Bacteriol. 170(5):2185-91, 1988 describes antibacterial agents, including antibacterial agent III, which specifically inhibits CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity and can be used to block the incorporation of 3-deoxy-D-manno-octulosonic acid (KDO) into the LPS of Gram-negative bacteria. As LPS synthesis ceases, bacterial growth also stops. In Salmonella typhimurium and Escherichia coli, addition of the LPS precursor species lipid IV AThe addition of KDO in [substance] is the main pathway for the formation of lipid A-KDO. In one embodiment, the antibiotic is antimycin III, and Gram-negative bacteria are treated with a suitable amount, such as 5 micrograms to 500 micrograms per milliliter, for a suitable time, such as 2 to 8 hours.

[0087] Similarly, the known compound α-C-(1,5-anhydro-7-amino-2,7-dideoxy-D-mannoheptopyranosyl)-carboxylate inhibits 3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase), a cytoplasmic enzyme that activates 3-deoxy-D-manno-octulosonate (KDO) for incorporation into LPS (Nature. 1987 Oct 16;329(6135):162-4). Thus, treating an organism with this compound can also reduce LPS-related endotoxin activity.

[0088] In another embodiment, the reduction of endotoxin activity is achieved by treating the organism with an LPS inhibitor. For example, a bacterial cyclic lipopeptide, surfactin, has been shown to bind to lipid A and inhibit its activity (J Antibiot 2006 59(1):35-43).

[0089] In addition to LPS-related endotoxins, various other components of Gram-negative bacteria can also induce or cause pyrogenicity and septic shock, including outer membrane proteins, pili, fimbriae, lipopeptides, and lipoproteins (reviewed in Jones, M., Int. J. Pharm.Compd., 5(4):259-263, 2001). Pyrogenicity can be measured by the rabbit method well known in the art, which involves assessing rectal temperature after intravenous injection of a putative pyrogen.

[0090] It has been found that treating Gram-negative bacteria with a combination of polymyxin B and glutaraldehyde reduces pyrogenicity measured in rabbits by 30-fold. In one embodiment, 1000 micrograms per milliliter (µg / mL) of polymyxin B and 1% glutaraldehyde are used to reduce pyrogenicity measured in rabbits by 30-fold. The reaction of polymyxin B with LPS and the reaction of glutaraldehyde with LPS and other bacterial components act together to reduce pyrogenicity. The bifunctional chemical cross-linking activity of glutaraldehyde also contributes to killing and stabilizing bacterial cells, thus playing a triple role in this context (reducing pyrogenic activity, killing cells, and stabilizing cells).

[0091] Accordingly, in one embodiment, there is provided a method of reducing endotoxin activity and pyrogenicity and killing and stabilizing Gram-negative bacteria by treating the Gram-negative bacteria with a combination of 1000 µg / mL polymyxin B and 1% glutaraldehyde. In another embodiment, the Gram-negative bacteria are treated with a combination of polymyxin B in a dose range of from about 3 µg / mL to about 1000 µg / mL and glutaraldehyde in a dose range of from about 0.1% to about 1.0%. In another embodiment, the dose range of polymyxin B is from about 100 µg / mL to about 1000 µg / mL and the dose range of glutaraldehyde is from about 0.25% to about 1.0%. Additionally, the Gram-negative bacteria can be treated, for example, with polymyxin B in a dose range of from about 1000 µg / mL to about 3000 µg / mL and glutaraldehyde in a dose range of from about 0.25% to about 1.0%. In another aspect, the Gram-negative bacteria can be treated, for example, with polymyxin B in a dose range of from about 3000 µg / mL to about 5000 µg / mL and glutaraldehyde in a dose range of from about 0.25% to about 2.0%.

[0092] In some embodiments, the LPS-associated endotoxin activity (e.g., measured by the LAL assay) of intact and substantially non-viable Gram-negative bacterial cells is reduced by at least about 70% compared to untreated wild-type bacteria. In some embodiments, the reduction is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or 99.98%. In some embodiments, the reduction is not greater than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction is from about 70% to about 99.99%, from about 80% to about 99.9%, from about 90% to about 99.5% or 99%, from about 91% to about 99%, from about 92% to about 98%, from about 93% to about 97%, from about 94% to about 96%, from about 94.5% to about 95.5%, from about 94% to about 97%, from about 95% to about 98%, from about 96% to about 99%, from about 97% to about 99.5%, or from about 98% to about 99.9%, but is not limited thereto.

[0093] In some embodiments, certain residual levels of active LPS are preferred. For example, in some embodiments, in the compositions of the present disclosure, there are about 1 to 200 ng of active LPS per 1 x 10 8 cells. In some embodiments, per 1 x 10 8Each cell has about 2 to 200 ng, about 5 to 150 ng, about 5 to 120 ng, about 10 to 120 ng, about 20 to 100 ng, about 20 to 50 ng, or about 10 to 50 ng of active LPS.

[0094] In some embodiments, compared to untreated wild-type bacteria, the pyrogenicity (e.g., measured by an in vivo rabbit test) of intact and substantially non-viable Gram-negative bacterial cells is reduced by at least about 70%. In some embodiments, the reduction is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or 99.98%. In some embodiments, the reduction is no greater than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction is about 70% to about 99.99%, about 80% to about 99.9%, about 90% to about 99.5% or 99%, about 91% to about 99%, about 92% to about 98%, about 93% to about 97%, about 94% to about 96%, about 94.5% to about 95.5%, about 94% to about 97%, about 95% to about 98%, about 96% to about 99%, about 97% to about 99.5%, or about 98% to about 99.9%, but is not limited thereto.

[0095] As described above, in addition to LPS-related endotoxins, various other components of Gram-negative bacteria can also induce or contribute to pyrogenicity, such as outer membrane proteins, fimbriae, pili, lipopeptides, and lipoproteins. In some embodiments, intact and substantially non-viable Gram-negative bacterial cells are treated in such a way that the reduction in pyrogenicity is achieved by reducing the activity of LPS-related endotoxins and reducing non-LPS-related pyrogenicity, the latter such as the inactivation, removal, or blocking of outer membrane proteins, fimbriae, pili, lipopeptides, or lipoproteins. In some embodiments, the reduction in non-LPS-related pyrogenicity is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or 99.98%. In some embodiments, the reduction is no greater than about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%.

[0096] The bacteria administered according to the methods of the present disclosure become inactivated or substantially inactivated either before or after administration. "Inactivated" means that the organism is killed by an exogenous agent and / or contains mutations that render the organism unable to survive in a mammalian host. Substantially inactivated bacteria are strains whose survival rate has been reduced by at least 80%, 85%, 90%, 95%, 99% or more.

[0097] Bacteria can be inactivated by treatment with compounds such as polymyxin. Polymyxin binds to LPS and interferes with membrane integrity during bacterial division, resulting in a reduced survival rate due to decreased permeability of the cell envelope. If the survival rate is reduced by this method, measures need to be taken to prevent cell lysis and maintain cell integrity. Another method is to culture bacterial strains with conditional mutations in the LPS biosynthetic pathway, which are suppressed during growth and then transferred to non-permissive conditions to activate the mutations and disrupt LPS biosynthesis. In each case, the methods applied are to render the bacteria non-viable by determining the optimal treatment time or compound dose in each environment, thereby substantially eliminating the viability of the bacteria while retaining significant bacterial cell integrity. In cases where the non-survival rate is less than 100%, bacteria containing mutations that prevent further proliferation of the surviving bacteria in the mammalian host can be used (e.g., the diaminopimelic acid auxotrophs described in Bukhari and Taylor, J. Bacteriol. 105(3):844 - 854, 1971 and Curtiss et al., Immunol. Invest. 18(1 - 4):583 - 596, 1989). Dose and Administration Regimen

[0098] Suitable effective amounts (doses) and administration regimens have also been determined for therapeutic and prophylactic uses. In this context, the preferred Gram-negative bacterial species is Escherichia coli. In some embodiments, the effective amount of treated E. coli cells comprises from 1 × 10 7 to 500 × 10 7 intact and substantially inactivated E. coli cells.

[0099] As demonstrated in the appended examples, the bait products have strong efficacy against tumors and viral infections, particularly when used in the presence of immune cells or in combination with certain other therapeutic agents such as cyclophosphamide, IL-2, non-steroidal anti-inflammatory drugs (NSAIDs) (such as indomethacin), anti-PD-1 or anti-PD-L1 antibodies, anti-CTLA-4 antibodies and anti-CD20 antibodies (such as rituximab). Thus, the effective amount can be as low as (or greater than) 1 × 10 7 treated cells, or as low as (or greater than) 2 × 10 7 、3 × 107 , 4 × 10 7 , 5 × 10 7 , 7 × 10 7 , 7.74 × 10 7 , 10 × 10 7 , 15 × 10 7 , 20 × 10 7 , 30 × 10 7 , 40 × 10 7 , 50 × 10 7 , 60 × 10 7 , 70 × 10 7 , 80 × 10 7 , 90 × 10 7 , 100 × 10 7 , 150 × 10 7 , 200 × 10 7 , 250 × 10 7 , 300 × 10 7 or 400 × 10 7 treated cells.

[0100] As demonstrated, the bait product is safe enough to be used at high doses clinically. Thus, the effective amount can be as high as (or lower than) 500 × 10 7 treated cells, or as high as (or lower than) 2 × 10 7 , 3 × 10 7 , 4 × 10 7 , 5 × 10 7 , 7 × 10 7 , 7.74 × 10 7 , 10 × 10 7 , 15 × 10 7 , 20 × 10 7 , 30 × 10 7 , 40 × 10 7 , 50 × 10 7 , 60 × 10 7 , 70 × 10 7 , 80 × 10 7 , 90 × 10 7 , 100 × 10 7 , 150 × 10 7 , 200 × 10 7 , 250 × 10 7 , 300 × 10 7 or 400 × 107 One treated cell.

[0101] In some embodiments, the effective amount is 1 × 10 7 to 500 × 10 7 treated cells. In some embodiments, the effective amount is 1 × 10 7 to 400 × 10 7 、1 × 10 7 to 300 × 10 7 、1 × 10 7 to 200 ×10 7 、1 × 10 7 to 150 × 10 7 、1 × 10 7 to 100 × 10 7 、1 × 10 7 to 70 × 10 7 、1 × 10 7 to 50 × 10 7 、1 × 10 7 to 20 × 10 7 or 1 × 10 7 to 10 × 10 7 treated cells. In some embodiments, the effective amount is 3 × 10 7 to 400 × 10 7 、3 × 10 7 to 300 × 10 7 、3 × 10 7 to 200 × 10 7 、3 × 10 7 to 150 × 10 7 、3 × 10 7 to 100 × 10 7 、3 × 10 7 to 70 × 10 7 、3 × 10 7 to 50× 10 7 、3 × 10 7 to 20 × 10 7 or 3 × 10 7 to 10 × 10 7 treated cells. In some embodiments, the effective amount is 7 × 10 7 to 400 × 10 7 、7 × 10 7 to 300 × 107 , 7 × 10 7 to 200 × 10 7 , 7× 10 7 to 150 × 10 7 , 7 × 10 7 to 100 × 10 7 , 7 × 10 7 to 70 × 10 7 , 7 × 10 7 to 50 ×10 7 , 7 × 10 7 to 20 × 10 7 or 7 × 10 7 to 10 × 10 7 treated cells. In some embodiments, the effective amount is 7.74 × 10 7 to 400 × 10 7 , 7.74 × 10 7 to 300 × 10 7 , 7.74 × 10 7 to 200 ×10 7 , 7.74 × 10 7 to 150 × 10 7 , 7.74 × 10 7 to 100 × 10 7 , 7.74 × 10 7 to 70 × 10 7 , 7.74 × 10 7 to 50 × 10 7 , 7.74 × 10 7 to 20 × 10 7 or 7.74 × 10 7 to 10 × 10 7 treated cells.

[0102] In some embodiments, the effective amount is 10 × 10 7 to 400 × 10 7 , 10 × 10 7 to 300 ×10 7 , 10 × 10 7 to 200 × 10 7 , 10 × 10 7 to 150 × 10 7 , 10 × 10 7 to 100 × 10 7, 10 × 10 7 to 70 × 10 7 , 10 × 10 7 to 50 × 10 7 or 10 × 10 7 to 20 × 10 7 processed cells. In some embodiments, the effective amount is 15 × 10 7 to 400 × 10 7 , 15 × 10 7 to 300 × 10 7 , 15 × 10 7 to 200 × 10 7 , 15 × 10 7 to 150 × 10 7 , 15 × 10 7 to 100 × 10 7 , 15 × 10 7 to 70 × 10 7 , 15 × 10 7 to 50 × 10 7 or 15 × 10 7 to 20 × 10 7 processed cells. In some embodiments, the effective amount is 20 × 10 7 to 400 × 10 7 , 20 × 10 7 to 300 × 10 7 , 20 × 10 7 to 200 × 10 7 , 20× 10 7 to 150 × 10 7 , 20 × 10 7 to 100 × 10 7 , 20 × 10 7 to 70 × 10 7 or 20 × 10 7 to 50 × 10 7 processed cells.

[0103] In some embodiments, the effective amount is 30 × 10 7 to 400 × 10 7 , 30 × 10 7 to 300 ×10 7 , 30 × 10 7 to 200 × 10 7, 30 × 10 7 to 150 × 10 7 , 30 × 10 7 to 100 × 10 7 , 30 × 10 7 to 70 × 10 7 or 30 × 10 7 to 50 × 10 7 or 30 × 10 7 to 400 × 10 7 , 40 × 10 7 to 300 × 10 7 , 40 × 10 7 to 200 × 10 7 , 40 × 10 7 to 150 × 10 7 , 40 × 10 7 to 100 × 10 7 , 40 × 10 7 to 70 × 10 7 or 40 × 10 7 to 50 × 10 7 or 40 × 10 7 to 400 × 10 7 , 50 × 10 7 to 300 × 10 7 , 50 × 10 7 to 200 × 10 7 , 50 × 10 7 to 150 × 10 7 , 50 × 10 7 to 100 × 10 7 or 50 × 10 7 to 70 × 10 7 or 70 × 10 7 to 400 × 10 7 , 70 × 10 7 to 300 × 10 7 , 70 × 10 7 to 200 × 10 7 , 70 × 10 7 to 150 × 10 7 or 70 × 10 7to 100 × 10 7 treated cells. In some embodiments, the effective amount is 100 × 10 7 to 400 × 10 7 、100 × 10 7 to 300 × 10 7 、100 × 10 7 to 200 × 10 7 or 100 × 10 7 to 150 × 10 7 treated cells.

[0104] In some embodiments, the effective amount is about 1 (or 0.5 - 1.5) × 10 7 、2 (or 1.5 - 2.5) × 10 7 、3 (or 2 - 4) × 10 7 、4 (or 3 - 5) × 10 7 、5 (or 4 - 6) × 10 7 、7 (or 6 - 8) × 10 7 、7.74 × 10 7 、10 (or 8 - 12) × 10 7 、15 (or 13 - 17) × 10 7 、20 (or 15 - 25) × 10 7 、30 (or 25 - 35) × 10 7 、40 (or 30 - 50) × 10 7 、50 (or 40 - 60) × 10 7 、60 (or 50 - 70) × 10 7 、70 (or 60 - 80) × 10 7 、80 (or 70 - 90) × 10 7 、90 (or 80 - 100) × 10 7 、100 (or 80 - 120) × 10 7 、150 (or 130 - 170) × 10 7 、200 (or 150 - 250) × 10 7 、250 (or 200 - 300) × 10 7 、300 (or 200 - 400) × 10 7 or 400 (or 300 - 500) × 10 7 treated cells.

[0105] In some embodiments, for each kilogram of body weight of the patient, the effective amount is at least 0.02 × 10 7A treated cell. In some embodiments, for a patient per kilogram of body weight, the effective amount is at least 0.05 × 10 7 、0.12 × 10 7 、0.13 × 10 7 、0.17 × 10 7 、0.33 × 10 7 、0.83 × 10 7 、1.17 × 10 7 、1.67 × 10 7 、2.50 × 10 7 、3.33 × 10 7 、5.00 × 10 7 or 6.67 × 10 7 treated cells. In some embodiments, for a patient per kilogram of body weight, the effective amount is not more than 0.05 × 10 7 、0.12 × 10 7 、0.13 × 10 7 、0.17 × 10 7 、0.33 × 10 7 、0.83 × 10 7 、1.17 × 10 7 、1.67 × 10 7 、2.50 × 10 7 、3.33 × 10 7 、5.00 × 10 7 、6.67 × 10 7 or 8.33 × 10 7 treated cells.

[0106] In some embodiments, the effective amount of the treated cell comprises a predetermined amount of active LPS, which can be measured in endotoxin units (EU). In some embodiments, the effective amount comprises 124 to 62000 endotoxin units (EU) of LPS. In some embodiments, the effective amount comprises at least 124, 372, 868, 960, 1240, 2480, 6200, 8680, 12400, 18600, 24800, 37200 or 49600 EU of LPS. In some embodiments, the effective amount comprises not more than 62000 endotoxin units (EU) of LPS. In some embodiments, the effective amount comprises not more than 372, 868, 960, 1240, 2480, 6200, 8680, 12400, 18600, 24800, 37200 or 49600 EU of LPS.

[0107] In some embodiments, the effective amount comprises from 124 to 62,000 endotoxin units (EU) of LPS. In some embodiments, the effective amount comprises from 372 to 62,000 EU of LPS, or from 868 to 62,000 EU, from 960 to 62,000 EU, from 1240 to 62,000 EU, from 2480 to 62,000 EU, from 6200 to 62,000 EU, from 8680 to 62,000 EU, from 12400 to 62,000 EU, from 18600 to 62,000 EU, from 24800 to 62,000 EU, from 37200 to 62,000 EU or from 49600 to 62,000 EU of LPS. In some embodiments, the effective amount comprises from 372 to 49,600 EU, from 868 to 49,600 EU, from 960 to 49,600 EU, from 1240 to 49,600 EU, from 2480 to 49,600 EU, from 6200 to 49,600 EU, from 8680 to 49,600 EU, from 12400 to 49,600 EU, from 18600 to 49,600 EU, from 24800 to 49,600 EU or from 37200 to 49,600 EU of LPS. In some embodiments, the effective amount comprises from 372 to 37,200 EU, from 868 to 37,200 EU, from 960 to 37,200 EU, from 1240 to 37,200 EU, from 2480 to 37,200 EU, from 6200 to 37,200 EU, from 8680 to 37,200 EU, from 12400 to 37,200 EU, from 18600 to 37,200 EU or from 24800 to 37,200 EU of LPS.

[0108] In some embodiments, the effective amount comprises from 372 to 24800 EU, from 868 to 24800 EU, from 960 to 24800 EU, from 1240 to 24800 EU, from 2480 to 24800 EU, from 6200 to 24800 EU, from 8680 to 24800 EU, from 12400 to 24800 EU, or from 18600 to 24800 EU of LPS. In some embodiments, the effective amount comprises from 372 to 18600 EU, from 868 to 18600 EU, from 960 to 18600 EU, from 1240 to 18600 EU, from 2480 to 18600 EU, from 6200 to 18600 EU, from 8680 to 18600 EU, or from 12400 to 18600 EU of LPS. In some embodiments, the effective amount comprises from 372 to 12400 EU, from 868 to 12400 EU, from 960 to 12400 EU, from 1240 to 12400 EU, from 2480 to 12400 EU, from 6200 to 12400 EU, or from 8680 to 12400 EU of LPS. In some embodiments, the effective amount comprises from 372 to 8680 EU, from 868 to 8680 EU, from 960 to 8680 EU, from 1240 to 8680 EU, from 2480 to 8680 EU, or from 6200 to 8680 EU. In some embodiments, the effective amount comprises from 372 to 6200 EU, from 868 to 6200 EU, from 960 to 6200 EU, from 1240 to 6200 EU, or from 2480 to 6200 EU of LPS. In some embodiments, the effective amount comprises from 372 to 6200 EU, from 868 to 2480 EU, from 960 to 2480 EU, or from 1240 to 2480 EU of LPS. In some embodiments, the effective amount comprises from 372 to 1240 EU, from 868 to 1240 EU, or from 960 to 1240 EU of LPS. In some embodiments, the effective amount comprises from 372 to 960 EU, or from 868 to 960 EU of LPS. In some embodiments, the effective amount comprises from 372 to 868 EU.

[0109] In some embodiments, for a patient per kilogram of body weight, the effective amount comprises at least 2.07 endotoxin units (EU) of LPS. In some embodiments, for a patient per kilogram of body weight, the effective amount comprises at least 6.20, 14.47, 16.00, 20.67, 41.33, 103.33, 144.67, 206.67, 310.00, 413.33, 620.00, or 826.67 endotoxin units (EU) of LPS. In some embodiments, for a patient per kilogram of body weight, the effective amount comprises no more than 6.20, 14.47, 16.00, 20.67, 41.33, 103.33, 144.67, 206.67, 310.00, 413.33, 620.00, 826.67, or 1033.33 endotoxin units (EU) of LPS.

[0110] In some embodiments, the effective amount of the treated cells comprises a predetermined amount of active LPS, which can be measured by the amount of active LPS. In some embodiments, the effective amount comprises 15 ng to 7714 ng of active LPS. In some embodiments, the effective amount comprises at least 15 ng of active LPS, or at least 46, 108, 119, 154, 309, 771, 1080, 1543, 2314, 3086, 4629, or 6171 ng of active LPS. In some embodiments, the effective amount comprises no more than 46, 108, 119, 154, 309, 771, 1080, 1543, 2314, 3086, 4629, 6171, or 7714 ng of active LPS.

[0111] The term "active LPS" refers to LPS in the composition that is capable of exhibiting LPS-related endotoxin activity, e.g., as measured by the LAL assay, wherein based on a standard LPS preparation, 5 - 9 endotoxin units (EU) are considered equivalent to 1 ng of active LPS. The amount of active LPS in the composition can be described as the weight of un-inhibited LPS that is capable of exhibiting the same level of LPS-related endotoxin activity as the composition.

[0112] In some embodiments, the effective amount comprises from 15 ng to 7714 ng of active LPS. In some embodiments, the effective amount comprises from 46 ng to 7714 ng, from 108 ng to 7714 ng, from 119 ng to 7714 ng, from 154 ng to 7714 ng, from 309 ng to 7714 ng, from 771 ng to 7714 ng, from 1080 ng to 7714 ng, from 1543 ng to 7714 ng, from 2314 ng to 7714 ng, from 3086 ng to 7714 ng, from 4629 ng to 7714 ng, or from 6171 ng to 7714 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 6171 ng, from 46 ng to 6171 ng, from 108 ng to 6171 ng, from 119 ng to 6171 ng, from 154 ng to 6171 ng, from 309 ng to 6171 ng, from 771 ng to 6171 ng, from 1080 ng to 6171 ng, from 1543 ng to 6171 ng, from 2314 ng to 6171 ng, from 3086 ng to 6171 ng, or from 4629 ng to 6171 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 4629 ng, from 46 ng to 4629 ng, from 108 ng to 4629 ng, from 119 ng to 4629 ng, from 154 ng to 4629 ng, from 309 ng to 4629 ng, from 771 ng to 4629 ng, from 1080 ng to 4629 ng, from 1543 ng to 4629 ng, from 2314 ng to 4629 ng, or from 3086 ng to 4629 ng of active LPS.

[0113] In some embodiments, the effective amount comprises from 15 ng to 3086 ng, from 46 ng to 3086 ng, from 108 ng to 3086 ng, from 119 ng to 3086 ng, from 154 ng to 3086 ng, from 309 ng to 3086 ng, from 771 ng to 3086 ng, from 1080 ng to 3086 ng, from 1543 ng to 3086 ng, or from 2314 ng to 3086 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 2314 ng, from 46 ng to 2314 ng, from 108 ng to 2314 ng, from 119 ng to 2314 ng, from 154 ng to 2314 ng, from 309 ng to 2314 ng, from 771 ng to 2314 ng, from 1080 ng to 2314 ng, or from 1543 ng to 2314 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 1543 ng, from 46 ng to 1543 ng, from 108 ng to 1543 ng, from 119 ng to 1543 ng, from 154 ng to 1543 ng, from 309 ng to 1543 ng, from 771 ng to 1543 ng, or from 1080 ng to 1543 ng of active LPS.

[0114] In some embodiments, the effective amount comprises from 15 ng to 1080 ng, from 46 ng to 1080 ng, from 108 ng to 1080 ng, from 119 ng to 1080 ng, from 154 ng to 1080 ng, from 309 ng to 1080 ng, or from 771 ng to 1080 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 771 ng, from 46 ng to 771 ng, from 108 ng to 771 ng, from 119 ng to 771 ng, from 154 ng to 771 ng, or from 309 ng to 771 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 309 ng, from 46 ng to 309 ng, from 108 ng to 309 ng, from 119 ng to 309 ng, or from 154 ng to 309 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 154 ng, from 46 ng to 154 ng, from 108 ng to 154 ng, or from 119 ng to 154 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 119 ng, from 46 ng to 119 ng, or from 108 ng to 119 ng of active LPS. In some embodiments, the effective amount comprises from 15 ng to 46 ng of active LPS.

[0115] In some embodiments, for a patient per kilogram (kg) of body weight, the effective amount comprises at least 0.26 ng of active LPS. In some embodiments, for a patient per kilogram (kg) of body weight, the effective amount comprises at least 0.77, 1.80, 1.99, 2.57, 5.14, 12.86, 18.00, 25.71, 38.57, 51.43, 77.14 or 102.86 ng of active LPS. In some embodiments, for a patient per kilogram (kg) of body weight, the effective amount comprises no more than 0.77, 1.80, 1.99, 2.57, 5.14, 12.86, 18.00, 25.71, 38.57, 51.43, 77.14, 102.86 or 128.57 ng of active LPS.

[0116] In some embodiments, the treated cells are administered once, daily, for two consecutive days per week, for three consecutive days per week, for four consecutive days per week, for five consecutive days per week, for six consecutive days per week, or once every day, every other day, every three days, every five days, every week, every two weeks, monthly, every two months, every three months, every four months, every six months or annually. In a preferred embodiment, the administration is once a week. Diseases and disorders

[0117] The intact and substantially non-viable Gram-negative bacterial cells disclosed herein can be used to treat or prevent various types of cancer as well as infectious diseases and disorders.

[0118] "Treatment" is a method of obtaining a beneficial or desired result, including a clinical result. Beneficial or desired clinical results can include one or more of the following: a) inhibiting a disease or disorder (e.g., alleviating one or more symptoms caused by the disease or disorder, and / or reducing the degree of the disease or symptom); b) slowing or arresting the development of one or more clinical symptoms associated with the disease or disorder (e.g., stabilizing the disease or disorder, preventing or delaying the worsening or progression of the disease or disorder, and / or preventing or delaying its spread (e.g., metastasis)); and / or c) remitting the disease, i.e., causing the disappearance of clinical symptoms (e.g., improving the disease state, providing partial or complete remission of the disease or disorder, enhancing the effect of another drug, delaying the progression of the disease, improving the quality of life, and / or prolonging the survival period).

[0119] "Prevention" or "prevention of" refers to any treatment of a disease or disorder that results in the non-development of clinical symptoms of the disease or disorder. In some embodiments, the bacterial cells can be administered to a subject (including a human) at risk or with a family history of the disease or disorder.

[0120] "Subject" refers to an animal that has been or will be the subject of treatment, observation, or experiment, such as a mammal (including humans). The methods described herein can be used for human therapy and / or veterinary applications. In some embodiments, the subject is a mammal, such as a human, dog, cat, cow, sheep, etc. In one embodiment, the subject is a human.

[0121] In some embodiments, the cancer is a solid tumor, including metastatic solid tumors and advanced metastatic solid tumors. In some embodiments, the cancer is leukemia or lymphoma. Non-limiting examples of cancers include bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, hepatocellular or liver cancer, pancreatic cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, and small cell lung cancer.

[0122] Other cancerous diseases or conditions include, but are not limited to, the progression and / or metastasis of malignancies and related diseases, such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblast, promyelocyte, granulomonocyte, monocyte, and erythroleukemia))) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0123] In some embodiments, for the treatment of cancer, a second agent can be used in combination with the treated cells. The combination can be used simultaneously (e.g., at the same time, on the same day, or in the same dosage form) or sequentially. Exemplary second agents include, but are not limited to, cyclophosphamide, IL-2, non-steroidal anti-inflammatory drugs (NSAIDs) (such as indomethacin), anti-PD-1 or anti-PD-L1 antibodies, and anti-CD20 antibodies (such as rituximab). Each of these drugs has been shown to be able to treat cancer synergistically with the treated cells.

[0124] In one example, the cancer is colorectal cancer and the second agent is cyclophosphamide and / or an anti-CTLA-4 antibody. In another example, the cancer is pancreatic cancer and the second agent is cyclophosphamide, an NSAID (such as indomethacin), an anti-PD-1 or anti-PD-L1 antibody, or a combination thereof. In another example, the cancer is liver cancer and the second agent is an NSAID (such as indomethacin), an anti-PD-1 or anti-PD-L1 antibody, or a combination thereof. In another example, the cancer is non-Hodgkin lymphoma and the second agent is cyclophosphamide or an anti-CD20 antibody (such as rituximab).

[0125] The intact, stable, and substantially non-viable Gram-negative bacterial cells disclosed herein can also be used to enhance the immune system of a subject and thus can be used to prevent or treat diseases and disorders by improving the immune response. The intact and substantially non-viable Gram-negative bacterial cells disclosed herein can also be used as a vaccine or an immune adjuvant for a subject at risk of developing such a disease or disorder.

[0126] In some embodiments, the disease or disorder to be treated is an infectious disease. In some embodiments, the infection is caused by bacteria, fungi, parasites, or viruses. In particular, the bacterial cells of the present disclosure can be uniquely suitable for treating viral infections, optionally in combination with a second anti-infective agent.

[0127] In some embodiments, the disease being treated is HBV infection. In some embodiments, the disease being treated is HIV infection.

[0128] Administration can also be initiated before actual infection or before diagnosis of infection as a prophylactic vaccine or preventive measure.

[0129] In some embodiments, one or more additional therapeutic agents can be inhibitors of cyclooxygenase (COX) enzymes, such as non-steroidal anti-inflammatory drugs, including 6MNA, aspirin, carprofen, diclofenac, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, naproxen, flunixin, piroxicam, sulindac sulfide, suprofen, tenidap, tolmetin, tomoxiprol, zomepirac, celecoxib, etodolac, meloxicam, nimesulide, isopropylphosphorofluoridate, L745,337, NS398, rofecoxib, SC58125, S-aminosalicylic acid, ampyrone, diflunisal, nabumetone, acetaminophen, resveratrol, salicin, salicylaldehyde, sodium salicylate, sulfasalazine, sulindac, tamoxifen, ticlopidine, and pentasodium valproate.

[0130] In some embodiments, one or more additional therapeutic agents can be agonists of stimulatory immune checkpoints, such as CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS, or antagonists of inhibitory immune checkpoint molecules, such as A2AR, B7-H3, B7-H4, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, TIM-3, and VISTA.

[0131] Non-limiting examples of the one or more additional therapeutic agents also include abacavir, acyclovir, adefovir, amantadine, amprenavir, amprinavir, arbidol, atazanavir, atripol, balavir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, ecoliever, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonoacetic acid, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, lobivudine, maraviroc, moroxydine hydrochloride, metisazone, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogs, novir, oseltamivir (Tamiflu ®), peginterferon alpha-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil fumarate, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine. In one embodiment, the additional therapeutic agent is interferon alpha.

[0132] In some embodiments, the second agent is an exogenous antigen. As demonstrated in Example 2, when test animals are transfected with an exogenous antigen (HER2), the decoy significantly enhances the regression of established murine breast cancer tumors. The decoy is expected to act as a "super adjuvant" to facilitate antigen presentation. However, tumor cells often develop the ability to hide antigens to evade the immune response. When an exogenous antigen is provided, the decoy can promote antigen presentation to the immune system, resulting in a significant immune response.

[0133] Accordingly, another embodiment of the present disclosure provides a method for treating or preventing cancer in a patient in need thereof, which comprises administering to the patient (a) an effective amount of a composition comprising intact, stable, and substantially non-viable Escherichia coli cells that have been treated to result in a reduction of lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% as measured by the Limulus amebocyte lysate (LAL) assay compared to untreated wild-type Escherichia coli cells, and (b) an exogenous antigen associated with cancer.

[0134] In some embodiments, the antigen is selected from EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mesothelin, mucin, NY-ESO, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, Tenascin, and Claudin 18.2.

[0135] Another embodiment of the present disclosure provides a method for treating or preventing an infectious disease in a patient in need thereof, the method comprising administering to the patient (a) an effective amount of a composition comprising intact, stable, and substantially non-viable Escherichia coli cells that have been treated to result in a reduction of lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% as measured by the Limulus amebocyte lysate (LAL) assay compared to untreated wild-type Escherichia coli cells, and (b) an exogenous antigen associated with the infectious disease. In some embodiments, the antigen is a viral or bacterial antigen.

[0136] In some embodiments, the exogenous antigen can be encapsulated within the Escherichia coli cells, conjugated to the Escherichia coli cells, or expressed by the Escherichia coli cells. For example, if the antigen is a protein, a vector encoding the antigen can be introduced into the Escherichia coli cells to enable expression and secretion of the antigen. In some embodiments, the exogenous antigen is separated from the Escherichia coli cells. In some embodiments, they are mixed prior to administration to effect co-administration. In some embodiments, they can be administered simultaneously or sequentially separately. If administered separately, the dosing schedule and frequency can be determined as needed.

[0137] In some embodiments, the second agent is PBMC or engineered immune cells. Engineered immune cells are in clinical use, such as immune cells transduced to express a recombinant chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the immune cells are T cells, macrophages, monocytes, NK cells, or myeloid cells. In some embodiments, the CAR or TCR recognizes a tumor-associated antigen. Formulations, Dosage Forms, and Modes of Administration

[0138] Formulations and dosage forms of the treated bacterial cells are also provided. In one embodiment, an aqueous formulation is provided that comprises the treated cells as well as phosphate buffer, Mg 2+ and trehalose.

[0139] In some embodiments, the phosphate buffer comprises or can be prepared by mixing disodium hydrogen phosphate dihydrate with potassium dihydrogen phosphate. In some examples, the magnesium ions can be provided by magnesium chloride hexahydrate.

[0140] In some embodiments, the trehalose is trehalose dihydrate. In some embodiments, the concentration of trehalose or trehalose dihydrate is 2% to 30% or 5% to 20%, or 8% to 16%, 10% to 14% or 11% to 13% (w / v). In some embodiments, the concentration of trehalose or trehalose dihydrate is 20 mg / mL to 300 mg / mL, 50 mg / mL to 200 mg / mL, 80 mg / mL to 160 mg / mL, 100 mg / mL to 140 mg / mL or 110 mg / mL to 130 mg / mL.

[0141] Example formulations include disodium phosphate dihydrate at 0.5 mg / mL to 2 mg / mL, potassium dihydrogen phosphate at 0.1 mg / mL to 0.4 mg / mL, sodium chloride at 3 mg / mL to 12 mg / mL, potassium chloride at 0.05 mg / mL to 0.3 mg / mL, magnesium chloride hexahydrate at 0.15 mg / mL to 0.6 mg / mL, and trehalose dihydrate at 50 mg / mL to 200 mg / mL, with a pH of 7.3 to 7.7.

[0142] In another embodiment, the formulation includes disodium phosphate dihydrate at 0.8 mg / mL to 1.3 mg / mL, potassium dihydrogen phosphate at 0.14 mg / mL to 0.25 mg / mL, sodium chloride at 4 mg / mL to 8 mg / mL, potassium chloride at 0.1 mg / mL to 0.2 mg / mL, magnesium chloride hexahydrate at 0.2 mg / mL to 0.4 mg / mL, and trehalose dihydrate at 80 mg / mL to 160 mg / mL, with a pH of 7.3 to 7.7.

[0143] In some embodiments, the formulation contains 0.1 × 10 9 / mL to 20 × 10 9 / mL of treated cells. In some embodiments, the formulation contains 0.2 × 10 9 / mL to 10 × 10 9 / mL of treated cells. In some embodiments, the formulation contains 0.5 × 10 9 / mL to 5 × 10 9 / mL of treated cells.

[0144] In some embodiments, the LPS-related endotoxin activity (e.g., measured by the LAL assay) of intact and substantially non-viable Gram-negative bacterial cells in the dosage form is reduced by at least about 70% compared to untreated wild-type bacteria. In some embodiments, the reduction is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or 99.98%. In some embodiments, the reduction is no greater than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction is from about 70% to about 99.99%, from about 80% to about 99.9%, from about 90% to about 99.5% or 99%, from about 91% to about 99%, from about 92% to about 98%, from about 93% to about 97%, from about 94% to about 96%, from about 94.5% to about 95.5%, from about 94% to about 97%, from about 95% to about 98%, from about 96% to about 99%, from about 97% to about 99.5%, or from about 98% to about 99.9%, but is not limited thereto.

[0145] In some embodiments, the pyrogenicity (e.g., measured by an in vivo rabbit test) of intact and substantially non-viable Gram-negative bacterial cells in the dosage form is reduced by at least about 70% compared to untreated wild-type bacteria. In some embodiments, the reduction is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or 99.98%. In some embodiments, the reduction is no greater than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction is from about 70% to about 99.99%, from about 80% to about 99.9%, from about 90% to about 99.5% or 99%, from about 91% to about 99%, from about 92% to about 98%, from about 93% to about 97%, from about 94% to about 96%, from about 94.5% to about 95.5%, from about 94% to about 97%, from about 95% to about 98%, from about 96% to about 99%, from about 97% to about 99.5%, or from about 98% to about 99.9%, but is not limited thereto.

[0146] In some embodiments, the reduction in non-LPS-related pyrogenicity is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or 99.98%. In some embodiments, the reduction is no greater than about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the viability of the substantially non-viable bacteria is reduced by at least 80%, 85%, 90%, 95%, 99%, or more.

[0147] As demonstrated by the experimental examples, the manufactured treated bacterial cells are unexpectedly stable. The stability is reflected in strong resistance to disruption by sonication and long-term maintenance of integrity during storage. Accordingly, one embodiment of the present disclosure provides a method for providing a therapeutically acceptable composition. In some embodiments, the method entails lyophilizing a plurality of the treated bacterial cells disclosed herein to form a lyophilized composition and storing the lyophilized composition (a) at a temperature of 1°C to 10°C for at least 2 months or (b) at a temperature of -15°C or below for at least 2 years, thereby providing a therapeutically acceptable composition suitable for therapeutic use.

[0148] In some embodiments, the storage is at a temperature of 1°C to 10°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or for at least 1 day, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or at least 1 week, 2, 3, 4, 5, 6, 7, 8, or 9 weeks. In some embodiments, the storage is at a temperature of -15°C or below for at least 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 3, 4, 5, 6, 7, or 8 years.

[0149] In some embodiments, a plurality of the treated bacterial cells are provided in a solution further comprising phosphate buffer, Mg 2+ and trehalose, as any of the formulated compositions disclosed herein. In one embodiment, the formulated composition comprises 0.3 × 10 9 / mL to 5 × 10 9Complete and substantially inactivated Escherichia coli cells at / mL, disodium phosphate dihydrate at 0.5 mg / mL to 2 mg / mL, potassium dihydrogen phosphate at 0.1 mg / mL to 0.4 mg / mL, sodium chloride at 3 mg / mL to 12 mg / mL, potassium chloride at 0.05 mg / mL to 0.3 mg / mL, magnesium chloride hexahydrate at 0.15 mg / mL to 0.6 mg / mL, and trehalose dihydrate at 50 mg / mL to 200 mg / mL, with a pH value of 7.0 to 7.7.

[0150] The composition is formulated for pharmaceutical administration to mammals, preferably humans. This pharmaceutical composition of the invention can be administered in various ways, including intravenously, intratumorally, subcutaneously, intradermally, intramuscularly, intravesically, intranasally, or intraperitoneally.

[0151] In one embodiment, the treated cells or composition (as disclosed herein, before or after storage) are administered parenterally. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intravesical, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0152] The sterile injectable form of the composition can be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, a sterile fixed oil is commonly used as a solvent or suspending medium. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, can be used to prepare injectables, as can natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersing agents, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers, which are commonly used in manufacturing pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used in the formulation. The composition can be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion.

[0153] The pharmaceutical composition can be administered once or multiple times. The pharmaceutical composition can be administered by various methods, including, for example, rectal, oral, intranasal, and transdermal routes. In certain embodiments, the pharmaceutical composition can be administered by intra-arterial injection, intravenous, intravesical, intraperitoneal, parenteral, intramuscular, or subcutaneous injection.

[0154] One mode of administration is parenteral, for example by injection. Forms in which the pharmaceutical compositions described herein can be administered by injection include, for example, aqueous or oily suspensions or emulsions containing sesame oil, corn oil, cottonseed oil or peanut oil, as well as elixirs, mannitol, glucose or sterile aqueous solutions, and similar pharmaceutical carriers.

[0155] Some examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, trehalose, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup and methylcellulose. The formulations can also include lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl hydroxybenzoate and propyl hydroxybenzoate; sweetening agents; and flavoring agents. Examples

[0156] The following examples are included to demonstrate specific embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that function well in the practice of the present disclosure and can thus be considered to constitute specific modes of its practice. However, according to the present disclosure, those skilled in the art should understand that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain similar or analogous results. Example 1. Characterization of Bait Treated Bacterial Products

[0157] This example summarizes some of the non-clinical study results of the "bait" product of treated bacteria. The bait consists of attenuated, 100% killed, stable and intact bacteria produced by non-pathogenic Escherichia coli, reducing LPS endotoxin activity and pyrogenicity by approximately 90% ("treated bacteria", also referred to as "bait bacteria" or simply "bait"). Specific human immune receptor (TLR and NOD) activation or agonist activity was evaluated using a panel of human embryonic kidney reporter cell lines (HEK293), each transfected with a different immune receptor. STING agonist activity was evaluated using a human monocyte cell line carrying a luciferase reporter gene responsive to type 1 interferon induction. Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate and in vivo rabbit tests. Bacterial integrity was evaluated by electron microscopy and light microscopy. Stability was evaluated by sensitivity to disruption caused by probe sonication. Escherichia coli is auxotrophic and requires diaminopimelic acid (DAP) for growth. Mammals do not produce DAP, resulting in the inability of live strains to proliferate in mammals. This provides a fail-safe mechanism that enhances the safety of the product in addition to 100% killing.

[0158] Under various incubation conditions and times, the viability and endotoxin activity of wild-type Escherichia coli before and after various treatments were evaluated using optical density measurements at 600 nm, plating efficiency, and the Endosafe Endochrome K Kinetic LAL assay. Treatments included polymyxin B (an antibiotic that lyses Gram-negative bacteria but is also known to neutralize endotoxins), and drugs with the potential to kill (phenol) or kill and stabilize bacteria (formaldehyde, glutaraldehyde). Since the antibiotic action of polymyxin B results in cell lysis but is not related to the neutralization of endotoxin activity, the goal of the exploratory study was to develop a process with relevant conditions that could significantly reduce endotoxin activity and kill 100% of the cells while still keeping the cells intact.

[0159] It was determined that incubating Escherichia coli cells with polymyxin B at low temperature in the presence of fermentation medium and magnesium chloride could reduce endotoxin activity without lysing the cells. Subsequent incubation with glutaraldehyde in phosphate-buffered saline (PBS) resulted in 100% killing of the cells. The concentrations and exposure times of polymyxin B and glutaraldehyde were determined through dose-response and time-course experiments. The integrity of the product was confirmed by optical microscopy (after Gram staining) and electron microscopy. Routine assessments of DAP-dependence and viability were performed on all bacterial cultures used to produce the treated bait products before, during, and after the treatment process. Trehalose (final concentration of 12%) was added to the product as a cryoprotectant and then the product was frozen and stored at -70 °C.

[0160] The stability of the treated or bait bacteria relative to untreated parental bacteria was demonstrated by showing that the untreated parental bacteria were easily disrupted by sonication. However, as shown by optical microscopy (after Gram staining), the treated or bait bacteria were resistant to disruption by sonication ( Figure 1 ). Sonication was performed for 5 - 10 minutes at setting #3 using a Fisher Scientific Sonic Dismembrator with a microprobe fixed. Sonication was carried out in round-bottom tubes with the probe held just below the surface to enable effective sonication of the material.

[0161] After long-term storage, the stability of the bait bacteria was also tested. When frozen and stored at -70 °C, the bait bacteria were stable for at least 2 years. Under storage conditions of 4 °C, the bait bacteria were stable for at least 6 months with no detectable degradation.

[0162] In a dose-range finding study using New Zealand white rabbits (NZW) (4 rabbits per group), the pyrogenicity of untreated bacteria (DB100) and the bait was evaluated. Untreated bacteria (3x10 4 bacteria per milliliter) and bait doses (3x10 per milliliter5 bacteria and 9x10 per milliliter 5 bacteria) were slowly injected intravenously into New Zealand white rabbits (10 milliliters per dose) via the marginal ear vein. At a dose of 3x10 per milliliter 4 bacteria, the untreated bacteria increased the rectal temperature by 0.7°C. At a dose of 3x10 per milliliter 5 bacteria, the bait increased the rectal temperature by 0.1°C, while at a dose of 9x10 per milliliter 5 bacteria, it increased by 0.8°C to 1.0°C, indicating a reduction in pyrogenicity by approximately 30-fold or 97% (Table 1). Table 1. Treatment of live Escherichia coli cells with polymyxin B and glutaraldehyde (GA) kills bacteria and significantly reduces LPS endotoxin activity and in vivo pyrogenicity LAL = Limulus amebocyte lysate; LPS = lipopolysaccharide. Acute LD of bait bacteria in mice 100 (3x10 10 ) was 3 times higher than that of the untreated bacteria (1x10 10 ).

[0163] Untreated (DB100) Escherichia coli and bait were administered as a single intravenous injection dose to a group of 3 BALB / c mice, 1x10 per mouse 8 to 3x10 10 bacteria, and observed for up to 15 days. A dose of 1x10 10 DB100 was 100% lethal (death or dying was found), while the same dose of bait did not cause death. A 3-fold dose of 3x10 10 produced lethality. In this and subsequent pharmacological studies, the clinical observations of mice during bait treatment were mainly transient weight loss and transient fur ruffling. Although in the combination setting, the bait bacteria dose that did not produce clinical symptoms (e.g., 3x10 per animal 7 bacteria) showed antitumor activity. A panel of human embryonic kidney reporter cell lines (HEK293) was used to evaluate the activation or agonist activity of specific human immune receptors (TLRs and NODs), with each cell line transfected with a different immune receptor. Activation of the receptor stimulates NF-kB activity, inducing the expression of a reporter gene that produces quantifiable secreted embryonic alkaline phosphatase (SEAP). STING agonist activity was evaluated using a human monocytic cell line (THP1) carrying a luciferase reporter gene responsive to type I interferon induction. Control cell lines without transfected immune receptors but containing a reporter gene system were used to determine the specificity of the response. Activity was confirmed if the test compound produced ≥2-fold induction of the reporter gene. The activity of the test compound was also compared to the activity of commercially available positive control receptor activators. The treated bait bacteria contained agonists or activators of all functional TLR receptors and heterodimers, as well as NOD2 and STING (Table 2).

[0164] The pharmacodynamic effects of the bait on cytokine and chemokine secretion were evaluated using human peripheral blood mononuclear cells (PBMC). TLR ligands such as Escherichia coli LPS, double-stranded Escherichia coli DNA, Escherichia coli peptidoglycan (PGN), Flagellin, R848, polyinosinic acid (poly I:C), and CpG oligonucleotides (ODN) were also evaluated in these studies. Initial experiments were conducted with incubations of 24, 48, and 72 hours, and with and without activation of immune cells with anti-CD3. Activation of immune cells with anti-CD3 was not required for the bait bacteria to induce cytokine and chemokine secretion from PBMC. In addition, the maximum secretion induction of most cytokines and chemokines occurred after 48 hours. The treated bait bacteria induced higher levels of most cytokines and chemokines compared to the same dose of untreated bait bacteria (Table 2). Essentially, all cytokines and chemokines are involved in the activation of innate and / or adaptive (including anti-tumor and anti-viral) immune pathways, but can also cause severe toxicity if expressed in the wrong place, at the wrong time, for too long, or at inappropriate high levels. Table 2. Bait treatment does not reduce (most) cytokine secretion by human PBMC GM-CSF = granulocyte-macrophage colony-stimulating factor; IL = interleukin; PBMC = peripheral blood mononuclear cell; TNF = tumor necrosis factor.

[0165] In addition to the outer membrane Toll-like receptor 4 (TLR4) agonist endotoxin or LPS, Gram-negative bacteria such as Escherichia coli also contain agonists for a variety of other TLRs, including TLR2, TLR2 / 1, TLR2 / 6, TLR5, and TLR9. There is evidence that E. coli contains TLR3, TLR7, and TLR8 agonist RNA. E. coli also contains agonists for the NOD-like receptors (NLRs) NOD1 and NOD2, as well as the stimulator of interferon (IFN) genes (STING). Since decoy fabrication alters the surface but preserves the integrity of the bacteria, it is likely that a variety of other TLR agonists and immune-activating danger signals are retained in the product. This was verified by screening decoy bacteria against a panel of human embryonic kidney (HEK) cell lines, each individually transfected with a different human or mouse immune receptor or receptor heterodimer, including TLR2, 2 / 1, 2 / 6, 3, 4, 5, 7, 8, 9, NOD1, NOD2, or STING, each coupled to a reporter gene readout. Decoy bacteria showed agonist activity or activation against all human TLRs, most mouse TLRs, as well as NOD2 and STING (Table 3). Table 3. Activation of human immune receptors by decoy bacteria or positive controls

[0166] There is substantial evidence that TLR / TLRa signaling can enhance or be required for innate and adaptive anti-tumor immune responses. Several single, pure, or synthetic TLR agonists have shown anti-tumor activity in preclinical studies and have been tested in cancer clinical trials. This example compares the effects of decoy and various single TLR agonists on cytokine and chemokine secretion by PBMCs. Table 4 shows that decoy bacteria induced higher levels of cytokines and chemokines than any of the four individual TLR agonists, including purified E. coli LPS. These results indicate that decoy bacteria are more potent than many single pure TLR agonists in activating immune cell cytokine and chemokine secretion and support the finding that decoy bacteria contain multiple TLR agonists and other immune stimulants in addition to LPS. Table 4. Decoy bacteria induce higher levels and a distinct pattern of cytokines in human PBMCs compared to treatment with pure (single) TLR agonists (Study AB64785) GM-CSF = granulocyte-macrophage colony-stimulating factor; IFN = interferon; IL = interleukin; LPS = lipopolysaccharide; peripheral blood mononuclear cells; TLR = toll-like receptor; TNF = tumor necrosis factor.

[0167] On a ng / kg basis, humans are approximately 500 to 1000 times more sensitive to the toxicity mediated by intravenous injection of purified LPS than mice, and this difference in sensitivity has been found to be related to the difference in the ability of LPS to induce cytokine secretion in human and mouse immune cells. In addition, the difference in sensitivity was found to be mediated by factors in blood or serum. Additional in vitro pharmacological studies were conducted, and the results showed that, compared to mouse PBMC (in mouse serum), the same amount of bait bacteria induced 10 to 3500 times more cytokine secretion in the same amount of human PBMC (in human serum) ( Figure 2 ).

[0168] Since cytokines mediate anti-tumor activity and toxicity, this finding indicates that human immune cells are more sensitive to bait bacteria-induced cytokines (including anti-tumor cytokines) than mouse immune cells, suggesting that if there is an acceptable anti-tumor treatment index for mice, then there may also be one for humans. Example 2. Non-clinical studies of the bait

[0169] This example summarizes some of the non-clinical study results of the bait product of the treated bacteria. In vitro activity against human breast cancer cells

[0170] The growth inhibitory effect and cytotoxicity of the bait were evaluated in a co-culture model of human peripheral blood mononuclear cells and MDA-MB-231 breast tumor cell line. PBMC (1x10 4 ), alone or in combination with MDA-MB-231 Nuclight Red stable cells (2x10 4 ), were seeded in 96-well plates at concentrations of 1x10 5 cells / well, 1x10 6 cells / well, and 1x10 6 cells / well, and treated with the bait (3x10 5 bacteria) for 96 and 168 hours. At concentrations of 1x10 6 cells / well and 1x10 4 cells / well, PBMC alone inhibited the growth of MDA-MB-232 tumor cells, but the bait alone had minimal inhibitory effect on tumor cell growth. The combination of the bait and non-inhibitory concentration of PMBC (1x10 Figure 3 ) produced a significant synergistic effect of 90% growth inhibition and cytotoxicity of tumor cells after 7 days of incubation. In vivo activity in a mouse colorectal cancer model

[0171] In an orthotopic model of human colorectal cancer, the anti-tumor activity of the bait alone was investigated using female BALB / c mice (7 mice per group) carrying green fluorescent protein-labeled CT26 tumor fragments derived from subcutaneous tumors. Five days after cecal implantation (surgery), the mice were injected intravenously with the bait vector or the bait (2 x 10 8 bacteria / animal, intravenous injection, twice a week [QDx2] for 3 weeks). The results are as Figure 4 shown. Tumor growth was measured by in vivo palpation and postmortem tumor weight. Metastasis was measured by green fluorescent protein imaging in postmortem "open" animals. In this experiment, the bait alone could prolong survival and inhibit metastasis. Compared with the start of treatment, the maximum instantaneous weight loss in the first week of bait treatment was 1.3%, and there was no weight loss in the following weeks of treatment.

[0172] Using a subcutaneous murine syngeneic CT26.WT colorectal cancer model, the anti-tumor efficacy of the bait was evaluated in female BALB / c mice (8 animals per group). Exploratory trials were also conducted to orally administer cyclophosphamide and anti-CTLA-4 antibody in drinking water, alone or in combination with the bait. Starting from the 3rd day after tumor cell implantation, the single-agent bait was injected intravenously at a dose of 5 x 10 7 or 1 x 10 9 bacteria per mouse, twice a week (Q4D) for 3 weeks. Compared with the vehicle control, both doses produced statistically significant tumor growth inhibition, which was determined by unpaired T-tests on days 24 and 28 (low dose) and days 21, 24, and 28 (high dose) ( Figure 5 ). Compared with the vehicle control, the high dose produced a statistically significant inhibitory effect, measured by the time to reach 1000 mm 3 . No weight loss was recorded for the low-dose bait, and only one measurement of instantaneous weight loss (0.56%) was recorded for the high-dose bait.

[0173] In a subcutaneous model of colorectal cancer, the anti-tumor activity of the combination of the bait and low-dose intraperitoneal (i.p.) cyclophosphamide (LDC) was investigated using female BALB / c mice (8 mice per group) carrying established CT26 tumors (identical to CT26.WT). Eleven days after implantation, when the average tumor size was 202 mm 3 , the mice received no treatment, or the bait alone (1.5, 3.0, or 6.0 x 10 8 bacteria / animal, intravenous injection, twice a week [QDx2] for 3 weeks), LDC (20 mg / kg, intravenous injection, QDx4, starting one day before bait treatment for 3 weeks), or a combination of 3.0 x 10 8 bait and LDC. These results are asFigure 6 as shown

[0174] In this study, the use of the bait alone did not produce a statistically significant anti-tumor effect at 1.5 or 3.0 x 10 8 bacteria per mouse, but did produce a statistically significant tumor growth delay and a 25% increase in life span (%ILS) at 6.0 x 10 8 bacteria per mouse. LDC alone produced a 57% ILS, which was statistically significant. However, the combination of 3 x 10 8 bait and LDC produced a statistically significant 89% ILS and led to a complete and durable tumor regression with long-term survival until termination on day 81. During treatment with weekly doses of 1.5, 3.0, and 6.0 x 10 8 QDx2 bait, the transient maximum weekly weight loss in the first week was 8.9% to 11.2%, in the second week was 2.2% to 5.4%, and in the third week was 0% to 1.9%, which is consistent with the expected LPS tolerance and has been reported in mammals (including humans) upon intravenous injection of LPS. During the three-week treatment, the bait + LDC treatment transiently decreased the body weight of each animal by 12 - 16% without any animal loss.

[0175] Using a syngeneic subcutaneous mouse CT26.WT colorectal cancer model, the anti-tumor efficacy of the bait as a single agent or in combination with i.p. IL-2 + p.o. indomethacin was evaluated in female BALB / c mice (5 animals per group). CT26.WT cells were subcutaneously injected into the right flank of BALB / c mice. Starting from day 10, when the average tumor size was 75 mm 3 , the mice in each group received no treatment, or were administered the bait vehicle, the bait (1 x 10 9 bacteria / mouse, intravenous injection, Q4Dx2 x 2 weeks; IL-2 (2500 U / animal, intravenous injection, QDx28) + indomethacin (14 µg / mL, oral, ad libitum, QDx28) or the bait + IL-2 + indomethacin. Both the single-agent bait and IL-2 + indomethacin produced a statistically significant inhibition of tumor growth (median tumor growth delays were 7.0 days and 7.1 days, respectively). The combination of the bait + IL-2 + indomethacin produced a statistically significant median tumor growth delay of 18.9 days and led to a tumor regression or complete remission until termination on day 60 ( Figure 7 ). The maximum transient weight loss with the bait alone was 5% in the first week of treatment and 0% in the second week. The maximum transient weight loss with the bait + IL-2 + indomethacin was 7% in the first week of treatment and 3% in the second week.

[0176] This study also tested several other compounds, including anti-GITR antibody, INFγ (accidentally used at 10-fold the expected dose, resulting in toxicity), and phenformin, alone and in combination with each other and / or with the decoy. Some of the above groups received these compounds after day 31. These drugs or combinations did not show significant additional anti-tumor activity and / or synergistic effects.

[0177] In an in vivo pharmacodynamic study, using a subcutaneous mouse CT26.WT colorectal cancer model, the anti-cancer activity of the decoy (DB104) as a single agent or in combination with cyclophosphamide (CTX) was evaluated in female BALB / c mice (5 animals per group). Mouse CT26.WT colorectal cancer cells were implanted (injected) into the right flank of BALB / c mice. The animals were randomly grouped and treatment was initiated (starting day = SD), with an average group tumor weight of 75 to 79 mg. Mice were administered the decoy (3x10 8 bacteria / animal, intravenous injection, Q4Dx4 [SD+1]; Q8Dx4 [SD+1]), the decoy (1x10 8 , 3x10 8 , 1x10 9 , 3x10 9 bacteria / animal, intravenous injection, Q4Dx4 [SD+1]), and CTX (50 mg / kg, intravenous injection, Q4Dx2 [SD]; Q8Dx2 [SD]). A group of untreated animals was used as a control. The 3x10 9 bacteria / animal dose of the decoy was not tolerated, resulting in 4 deaths, but all other treatments were tolerated. Except for the highest (toxic) dose of the decoy, all groups showed a transient weight loss in the range of 5 - 9% within 1 - 2 days after decoy administration, and less weight loss after repeated dosing, probably due to LPS tolerance. Both regimens of CTX produced significant tumor growth inhibition without any tumor regression. The combination of the decoy with Q4Dx2 CTX produced significant tumor growth inhibition and one complete tumor regression or complete remission until termination on day 91, indicating a potential synergistic effect. Compared with untreated mice, treatment with the decoy as a single agent at all doses ( Figure 8 ), and in combination with the corresponding CTX treatment, effectively inhibited the growth of subcutaneously implanted CT26.WT colorectal cancer tumors (p<0.05). In the Q4D regimen, only the combination treatment of the decoy and CTX was more effective against the growth of CT26.WT colorectal cancer than treatment with CTX or the decoy alone. In vivo activity in a mouse metastatic pancreatic cancer model

[0178] In a primary pharmacodynamic study, the in vivo efficacy of the bait, gemcitabine, low-dose cyclophosphamide (LDC), and bait + LDC was evaluated in female C57BL / 6 mice (7 animals per group) using an intrasplenic Pan02 pancreatic cancer mouse model. On day 0, pancreatic tumor cells were surgically implanted (injected) into the spleens of C57BL / 6 female mice. The mice were administered the bait (5x10 7 and 2x10 8 bacteria / animal, intravenous injection, QDx2 weekly for 3 weeks), gemcitabine (50 mg / kg, intravenous injection, BIWx7), or LDC (20 mg / kg, intravenous injection, QDx4 for 3 weeks). A group of untreated animals was used as a control. Large tumors developed in the spleens, livers, and pancreases of all untreated animals and needed to be terminated around day 30. At a dose of 5x10 7 and 2x10 8 per mouse, the bait significantly increased the median survival time. The maximum transient weight loss (relative to the start of treatment) in the high / low-dose bait groups was 9.8% / 8.2% in the first week of treatment, 2.6% / 2.1% in the second week of treatment, and no weight loss in the third week. The positive control drug gemcitabine significantly improved the median survival rate between the results of the two bait doses. In this model, the combination of the bait and LDC did not result in a higher survival rate than the bait alone ( Figure 9 ).

[0179] In a second experiment, using the same intrasplenic Pan02 pancreatic cancer mouse model, the therapeutic effects of the bait, gemcitabine, and indomethacin, as well as bait + gemcitabine and bait + indomethacin, were evaluated. The mice were administered the bait (2x10 8 bacteria / animal, intravenous injection, QDx2 weekly for 3 weeks), gemcitabine (several different doses and schedules), and low-dose oral indomethacin (10 µg / mL, oral, QD ad libitum for 31 days). A group of animals administered the bait vehicle was used as a control. Monotherapy with the bait and monotherapy with gemcitabine improved survival. Gemcitabine and the bait did not appear to have an additive or synergistic effect. Monotherapy with indomethacin did not significantly improve survival, but in combination with the bait, it appeared to improve survival in a potentially synergistic manner (ILS increased by 38% with the bait alone and extended to 208%). In this combination setting, the maximum transient weight loss in the first week of treatment was 6.4%, 1.5% in the second week of treatment, and no weight loss relative to the start of treatment in all subsequent treatment weeks ( Figure 10 ). In vivo activity of a subcutaneous established mouse hepatocellular carcinoma model

[0180] Using a subcutaneous H22 murine syngeneic hepatocellular carcinoma model, the therapeutic effects of the single bait and its combination with anti-PD-1 or indomethacin and anti-PD-1 were evaluated in female BALB / c mice (6 animals per group). Treatment was initiated when the mean tumor size was 194 mm 3 . Mice were injected intravenously with the single-agent bait at a dose of 2x10 8 bacteria / animal, slowly pushed, QDx2 weekly for 6 weeks. The bait (2x10 8 bacteria / animal, QDx2 weekly and 1x for 6 weeks) was tested in combination with anti-PD-1, and at various dose levels up to 6x10 8 bacteria / animal (once a week for 6 weeks), in combination with indomethacin and anti-PD-1. Indomethacin was administered orally at a dose of 10 µg / mL, ad libitum in drinking water daily for x 6 weeks (used alone and in combination), and anti-PD-1 was injected intraperitoneally at a dose of 10 mg / kg, BIW for 2 weeks (used alone and in combination). A group of untreated animals was used as a control in the study. No bait-related mortality was observed at any dose of the bait or combination. A transient weight loss was observed after each bait treatment. The maximum weekly mean weight loss rates of QDx2 bait during the 6-week treatment period were -6.91%, -5.34%, -5.48%, -3.28%, -0.94%, and 0%, indicating the existence of a recognized tolerance phenomenon of intravenous injection of LPS in mammals (including humans). The combined use of the bait with indomethacin and anti-PD-1 produced a high proportion of durable tumor regressions, but did not result in a significant increase in weight loss or other signs of toxicity. Administering 2x10 8 bacteria / animal for 6 weeks together with indomethacin and anti-PD-1 resulted in a weekly mean group weight loss of -6.37%, -3.81%, -3.86%, -0.58%, -1.49%, and 0%. Administering 6x10 8 bacteria / animal for 6 weeks together with indomethacin and anti-PD1 resulted in a weekly mean group weight loss of -8.30%, -4.99%, -5.35%, -2.94%, -3.70%, and -0.17%.

[0181] Administering the single-agent bait QDx2 weekly for 6 weeks slightly prolonged the lifespan, but it was statistically significant (27.5% ILS). Similar results were observed using the single-agent indomethacin or anti-PD-1 (30% ILS and 37.5% ILS respectively), without any tumor regression. All combined treatments significantly increased the ILS statistically. In addition, the combined use of the bait with anti-PD-1 once a week resulted in 2 / 6 durable complete tumor regressions, and the QDx2 bait twice a week resulted in 1 / 6 durable complete tumor regressions. In those receiving 2x10 8 or 6x108 In each group of the bait, treatment with indomethacin + bait + anti-PD-1 led to tumor regression in 5 / 6. Except for 1 out of 10 regressions, the rest persisted until the end of the experiment on day 143 ( Figure 11 ).

[0182] On day 91, 9 cured animals were subcutaneously re-challenged on the other side of the back at the site of the first tumor attack with fresh tumor cells (without further drug treatment), and an additional 52-day follow-up was performed on the two tumor sites of each animal. Tumors began to grow at all re-challenge sites but then regressed completely spontaneously, showing 100% immune memory. Naïve animals receiving the same tumor cells on the same re-challenge day developed progressively growing tumors that eventually exceeded the humane euthanasia limit of 3000 mm 3 . Figure 12 ).

[0183] In the second study, the therapeutic effects of indomethacin, anti-PD-1, bait + indomethacin, and bait + indomethacin + anti-PD-1 were evaluated in female BALB / c mice (6 animals per group) using a subcutaneous H22 hepatocellular carcinoma murine syngeneic model (Study E0776-U1802). The bait was tested in combination settings of weekly QDx2, weekly 2x, and weekly 1x. In this study, the bait was not tested as a single agent. The bait was tested by slow intravenous injection at 2x10 8 colony-forming units / animal for 6 weeks. Indomethacin was tested in drinking water at a concentration of 10 µg / mL, orally administered daily, ad libitum for 2 or 6 weeks. Anti-PD-1 was tested at a dose of 10 mg / kg, intraperitoneally injected twice a week for 2 weeks. Treatment was initiated when the mean tumor size was 184 mm 3 . A group of untreated animals was used as a control. One death was observed three weeks after administration of bait (weekly QDx2) + indomethacin + anti-PD-1. All other mice tolerated the same combination as well as all other treatments and combinations well. The maximum transient weight loss rate in all groups receiving bait treatment was 7.5% to 10.1%, occurring in the first week of treatment, and the weight loss rate in the triple combination treatment group was not higher compared to the dual combination treatment. In subsequent weeks of treatment, the transient weight loss after bait administration generally decreased, and after the last treatment at week 6, the weight loss in all triple combination treatment groups was less than 1%, indicating tolerance.

[0184] Indomethacin (2 weeks and 6 weeks) and anti-PD-1 produced a statistically significant increase in lifespan (ILS), and there was one regression after two weeks of treatment with indomethacin. Indomethacin (2 weeks) + anti-PD-1 also produced an increase in ILS and 1 regression. Decoy (QDx2 for 6 weeks) + indomethacin (6 weeks) led to an increase in ILS and 4 regressions. Since we have never seen a regression with decoy alone in this model, nor any regression with 6-week indomethacin treatment, the results strongly support synergy. At the end of the experiment on day 91, all regressions were persistent ( Figure 13 ).

[0185] In the same study, decoy administered twice and once weekly in combination with indomethacin produced 1 / 6 and 2 / 6 persistent regressions, respectively. Decoy (QDx2 weekly, twice weekly, and once weekly) in combination with indomethacin (6 weeks) + anti-PD-1 was also tested and produced 4, 5, and 6 regressions or partial regressions, respectively. Once-weekly decoy + anti-PD-1 + 6-week indomethacin obtained the best results, producing 6 complete regressions, 5 of which persisted until termination on day 91 ( Figure 14 ).

[0186] In another study using female BALB / c mice (6 animals per group) in a subcutaneous H22 hepatocellular carcinoma mouse model, the therapeutic effects of decoy, low-dose cyclophosphamide (LDC), indomethacin, gemcitabine (GEM), 5-FU, and various combinations were tested. Decoy at 2 x 10 8 bacteria / animal was tested, administered intravenously by slow bolus injection for 4 weeks (monotherapy) or 7 weeks (in combination with indomethacin). Indomethacin at 10 µg / mL was tested, in drinking water, administered ad libitum orally daily for 4 or 7 weeks. A group of animals treated with decoy vehicle was used as a control. Treatment was initiated when the mean tumor size was 123 mm 3 . There was no treatment-related mortality except for GEM, which caused a 20% weight loss when used alone and led to death when combined with decoy. GEM and LDC produced weak but statistically significant antitumor activity as monotherapies. In this study, no antitumor activity of decoy as a monotherapy was found. Combined activity was observed with LDC + indomethacin (no regression) and decoy + indomethacin, the latter leading to 3 / 6 complete regressions (complete remission – CR), which persisted until termination of the study on day 71. There was no transient weight loss after administration of indomethacin. Monotherapy with decoy produced an 8.2% transient weight loss in the first week of treatment, a 5.3% weight loss in the second week of treatment, and no weight loss relative to the start of treatment in weeks 3 and 4 of treatment, indicating tolerance. Combination with indomethacin produced slightly more weight loss in the first 3 weeks of treatment and little weight loss in weeks 4 - 7 of treatment ( Figure 15).

[0187] Since it was found that the decoy + indomethacin synergizes with anti-PD-1 to produce a high percentage of regressions, the therapeutic efficacy of the decoy + indomethacin in combination with anti-PD-1 and the decoy treatment index were evaluated in female BALB / c (6 animals per group) using a subcutaneous H22 murine hepatocellular carcinoma model. Treatment was initiated when the mean tumor size was 205 mm 3 . Mice were administered the decoy (once a week, 3x10 7 , 1x10 8 , 3x10 8 or 1x10 9 bacteria / animal, intravenous injection, slow bolus, for 6 weeks) + indomethacin (10 µg / mL, in drinking water, ad libitum oral administration per day, for 6 weeks) + anti-PD-1 (twice a week, 10 mg / kg, intravenous injection, for 2 weeks). A group of untreated animals was used as a control in the study. The decoy dose response was evaluated weekly with decoy treatment before anti-PD-1 treatment, and the decoy dose response was evaluated weekly with the first week of anti-PD-1 treatment before decoy treatment. In this study, the decoy and anti-PD-1 were also evaluated as single agents against non-established tumors, but not against established tumors. In other studies, these 3 drugs produced minimal or no single-agent activity in this model and no regressions as single agents. Interestingly, when single-agent anti-PD-1 was administered to mice (non-established tumors) one day after tumor inoculation, it ultimately led to tumor regression in 2 / 6 of the cases, highlighting the good activity of anti-PD-1 against non-established tumors, but having no effect on relatively large established tumors (>100 mm 3 ) at the start of treatment.

[0188] Regardless of the treatment sequence of the decoy or anti-PD-1, the triple combination (starting from 205 mm 3 tumors) produced 4 to 6 durable regressions per group of 6 (5-6 / 6 when starting with anti-PD-1, 4-6 / 6 when starting with the decoy). Both regimens produced 6 / 6 regressions at the lowest dose of the decoy (3x10 7 per animal), with no or only slight transient weight loss in the first week (-0.15%, -3.23%), and less or no weight loss during subsequent weeks of treatment. The triple combination with 1x10 8 decoy per animal produced 6 / 6 or 5 / 6 regressions (both regimens), with a maximum transient weight loss of -4.10% or -5.55%, which decreased or was 0% during subsequent weeks of treatment. Compared with 3x10 8The combined use of the baits produced 5 / 6 regression (two regimens), with maximum instantaneous weight loss of -4.40% or -5.35%, which decreased or became 0 during subsequent weeks of treatment. With 1x10 per animal 9 The bait combinations produced 5 / 6 or 4 / 6 regression (two regimens), with maximum instantaneous weight loss of -8.12% or -8.08%, which decreased or became 0 during subsequent weeks of treatment. The continuous weight loss observed after multiple treatments indicated bait tolerance. There were no treatment-related deaths, indicating that the therapeutic index of the bait in the combination treatment group was at least 33-fold. Additionally, no other toxic clinical symptoms were found in this study except for transient weight loss. The results of the regimens in which the mice were first treated with anti-PD-1 are as Figure 16 shown.

[0189] Furthermore, on day 91, 11 mice with tumor regression (1x10 8 and 3x10 8 bait groups) showed 100% immune memory, as re-challenging the mice with the same (fresh, live) tumor cells on the contralateral side (without further treatment) relative to the first tumor attack led to the onset of tumor growth, followed by complete rejection ( Figure 17 ). The initial tumor regression was true tumor eradication, which was demonstrated by following all mice for 91 days after tumor implantation and following the original tumor sites on the re-challenged mice for 143 days.

[0190] The subcutaneous H22 HCC model was used to evaluate the induction of plasma cytokines and chemokines in vivo by oral indomethacin (10 µg / mL, in drinking water, QD, starting from day 0), intravenous injection of the bait (2x10 8 bacteria / animal, once a week, starting from day 1), intraperitoneal injection of anti-PD-1 (10 mg / kg, twice a week, starting from day 0), and various combinations found to induce tumor regression. On day 0, mice with approximately 200 mm 3Female BALB / c mice with H22 tumors were randomly divided into 8 groups, each group containing 3 subgroups. All possible treatment methods or combinations, including no treatment, were implemented. Two subgroups of mice (5 mice per subgroup) in each group were sacrificed at 6 hours and 24 hours respectively after no treatment, single-agent, 2-agent or 3-agent combination treatment. In the case of 2-agent and 3-agent combination treatment, mice were sacrificed at 6 hours and 24 hours after the first treatment of the second or third component of the combination treatment. Plasma was prepared from each mouse and cytokine / chemokine analysis based on 32plex ELISA was performed. The third subgroup of each group (6 mice per group) was treated for one week, the tumor volume was measured at randomization, and once within one week and at the end of one week of treatment. The mice were sacrificed, then the tumors were harvested and RNA was isolated. The expression of 770 genes involved in the immune response pathway in 48 RNA samples was analyzed using NanoString technology.

[0191] Indomethacin induced the expression of only 2 / 32 cytokines / chemokines, and only at 6 hours after the start of treatment. The decoy induced the expression of 9 / 32 cytokines / chemokines, and like indomethacin, at 6 hours, its level was only significant compared to no treatment. Surprisingly, no cytokine / chemokine induction was observed 6 or 24 hours after the start of anti-PD-1 treatment. In the combination setting, under conditions where tumor growth inhibition or the onset of regression was observed, 18 to 28 out of 32 cytokines / chemokines were observed to have a statistically significant induction of cytokine / chemokine expression, including for many cytokines / chemokines at 6 hours and 24 hours. Relative to the day of randomization, the transient weight loss (4 days after randomization) was -1.63% for the non-treatment group, -0.65% for indomethacin, -8.63% for the decoy, and -1.74% for anti-PD-1. Compared to single-agent treatment, the weight loss in any combination treatment group did not increase. After one week of treatment, compared to no treatment, the tumor growth inhibition rate was 17% for indomethacin, 21% for the decoy, 11% for anti-PD-1, 33% for indomethacin + decoy, 36% for indomethacin + anti-PD-1, 26% for decoy + anti-PD-1, and 50% for indomethacin + decoy + anti-PD-1. At termination, only after one week of treatment (day 8), compared to the measurements on day 4, 5 out of 6 tumors in the triple combination group were smaller ( Figure 18 )). Except for transient weight loss, no other toxic clinical symptoms were observed in any mouse in any group. Notably and surprisingly, a significant increase in cytokine and chemokine expression (in plasma) was observed in the combination treatment groups, while there was no increase in weight loss or any other clinical toxic symptoms.

[0192] Among the 32 cytokines / chemokines detected, most (23 / 32) have been shown to stimulate or promote anti-tumor activity in preclinical models and some clinical settings. Many cytokines / chemokines have also been shown to limit or inhibit anti-tumor activity and / or cause toxicity. The outcome of any particular cytokine / chemokine or combination is highly dependent on many variables, including the animal, model, concentration, location, and the timing of cytokine / chemokine expression. Notably and surprisingly, in the H22 HCC anti-tumor efficacy setting, this example observed a high percentage of tumor eradication rates, a treatment index of at least 33-fold, weight loss after several weeks of treatment, and no other signs of toxicity when the bait bacteria administration was combined with anti-PD-1 treatment (plus or minus indomethacin), despite a significantly synergistic induction of cytokine / chemokine expression compared to monotherapy, and no significant increase in the clinical symptoms of toxicity.

[0193] Mice with 200 mm 3 Subcutaneous HCC tumors (6 mice per group) were untreated or treated as described in the efficacy study and with indomethacin (NSAID), bait, and / or anti-PD-1 treatment for one week as described in the cytokine analysis, then tumors were excised, RNA was isolated, and the expression of 770 immune pathway-related and control genes was analyzed by NanoString gene expression technology. The NanoString analysis included an assessment of the established Tumor Inflammatory Signature (TIS), which provides an indication of the anti-tumor immune environment in the tumor, with lower scores indicating low activation ("cold tumors") and higher scores indicating a higher potential for activation or anti-tumor immune response ("hot tumors"). Figure 18 It was also shown that the treatment increased the TIS signature, which was roughly correlated with the progression from monotherapy to dual combination to triple combination, and this was also associated with the final anti-tumor activity observed in the study with extended treatment for several weeks.

[0194] Additional NanoString gene expression analyses were performed, evaluating a wide variety of anti-tumor related immune system genes, cells, and pathways. The results were validated based on RNA quality and housekeeping gene expression analysis, and the heatmap results represent the Log2-based changes in gene expression relative to the mean of the entire analysis.

[0195] Monotherapy led to a widespread increase in the expression of innate and adaptive immune genes / cells / pathways in 1 or 2 tumors per 6 mice per group, which may be related to some tumor growth inhibition. Dual therapy increased the number of tumors in each group of mice showing widespread innate and adaptive immune gene / cell / pathway activation, which may be related to increased tumor growth inhibition and some tumor regression. The triple-drug combination was associated with widespread innate and adaptive immune gene / cell / pathway activation in essentially all tumors in each mouse, which was highly consistent with the high percentage of regression and tumor eradication observed in this context. Extremely similar results were obtained for general immune gene / cell / pathway activation, cytokine immune gene / pathway activation, chemokine immune gene / pathway activation, innate immune gene / pathway activation, and adaptive immune gene / pathway activation. In vivo activity in a mouse model of non-Hodgkin lymphoma

[0196] Similar to the HCC model, the bait bacteria showed low activity as a single agent against the syngeneic mouse A20 model of NHL. However, the bait bacteria were found to act synergistically with low-dose cyclophosphamide (LDC) to eradicate established tumors in female BALB / c mice. The in vivo therapeutic effects of the bait combined with LDC at different doses and regimens were evaluated in a subcutaneous A20 BALB / c lymphoma syngeneic model. Bait (3x10 7 , 1x10 8 , 3x10 8 , and 1x10 9 bacteria / animal, slow intravenous injection) was administered to BALB / c mice (4 mice per group) twice weekly, QDx2 weekly, QDx3 weekly, or QD4 x 2 weeks, in combination with 20 mg / kg LDC (intraperitoneal administration, QDx4 weekly for two weeks, starting one day before the bait). A group of untreated animals was used as a control. LDC alone was also tested. Treatment started on day 13, when the average tumor size was 158 mm 3 .

[0197] There was one tumor regression with LDC alone, although this was not seen in most studies of LDC alone. The number of partial and complete regressions per group of 4 for each regimen and each bait dose (from low to high) was 2 times weekly (1, 2, 1, 2), QDx2 weekly (2, 1, 4, 4) ( Figure 19), QDx3 per week (4, 3, 3, 3) and QDx4 per week (1, 0, 3, 3), with no deaths (treatment-related or otherwise), although due to excessive transient weight loss (≥20%), for the highest two doses of bait taken QDx4 per week, some of the first-week and all of the second-week administrations were suspended / missed. Due to the observed complete regression in 4 / 4 at the two highest doses, the QDx2 bait per week was considered the optimal regimen. The combination of bait bacteria and LDC produced a higher transient weight loss than the indomethacin / anti-PD-1 combination. During the first week of treatment, the transient weight loss reached 17%, and in the second week, it reached 14% to 17%. The bait + LDC combination generally did not show bait tolerance, which may be because the treatment was only carried out for two weeks. Except for transient weight loss, the only other clinical toxicity symptom was hair ruffling. Surprisingly, the combination consistently did not reduce the maximum tolerated dose of bait bacteria. Mice generally tolerated up to 1x10 9 bacteria of bait combination (QDx2 for two weeks), with no animal loss.

[0198] On day 77, 8 mice with complete tumor regression from the Figure 19 two highest-dose groups were re-challenged on the other side of the back with fresh tumor cells during the first tumor attack (without additional treatment). All new tumor attacks were rejected, and the first attack tumor site remained tumor-free after terminating this part of the experiment on day 123. Tumors grew normally in non-age-matched naive mice that received the same fresh tumor cells on the same day as the re-challenge ( Figure 20 ). Age-matched naive mice were used for most other tumor re-challenge experiments.

[0199] In the same experiment, mice whose tumors initially regressed but began to regrow due to receiving only one week of treatment or a suboptimal dose of bait for two weeks were administered bait at the optimal dose and regimen for two weeks (3x10 8 bacteria / animal, intravenous injection, slow injection, QDx2 x 2 weeks) + LDC, starting at a volume of approximately 100 to 2000 mm 3 . All tumors regressed, and 5 / 8 mice showed durable regression ( Figure 21 ). These results indicate that the sensitivity to the bait technology is not easily lost after initial treatment, and the bait technology can achieve complete regression of very large tumors.

[0200] In another experiment, LDC (as described above, intraperitoneal injection, 20 mg / kg, four times a week for two weeks) was administered one day before, during, and one day after the administration of bait (intravenous injection, slow bolus, QDx2 x 2 weeks). A group of untreated animals was used as a control. When the average tumor size was 212 mm3 Treatment was initiated when. The bait was tested as a single agent and did not produce anti-tumor activity on its own. As in previous experiments, tumor eradication (6 / 6 regression) was induced by bait + LDC, and only two weeks of bait + DLC administration was required ( Figure 22 ). In this study, LDC alone delayed tumor growth and did not produce any regression. Indomethacin was not required to be added to bait + LDC, and 4 / 6 regression was produced. The single-agent bait was administered QDx2 for 2 weeks, and the maximum transient weight loss in the first and second weeks was 9.3% and 3.7% respectively. The combination with LDC produced a 12% transient weight loss in the first week and a 13.6% transient weight loss in the second week. However, surprisingly, despite the increased weight loss, the combination did not consistently reduce the maximum tolerated dose of the bait bacteria. Mice generally tolerated up to 1x10 9 Combination of bait bacteria (QDx2 for two weeks), and there was no animal loss.

[0201] To investigate the anti-tumor mechanism of action of bait treatment, commercially available reagents were used to pre-deplete natural killer (NK) cells, CD4+ T, CD8+ T, or CD4+ and CD8+ T cells in mice. Tumor implantation was performed during the depletion protocol. Then, mice were treated with bait (3x10 8 bacteria / animal, intravenous injection, QDx2 weekly for two weeks) plus LDC (20 mg / kg, intravenous injection, QDx4 weekly for two weeks). At the start of treatment, additional mice were sacrificed in each group to verify immune cell depletion. Figure 23 It was shown that depletion of any immune cell type significantly reduced the anti-tumor activity of the bait regimen. When single immune cell types were depleted, the number of CR / regressions decreased to 1 / 6, and when both CD4+ and CD8+ T cells were depleted, all regressions were eliminated. Positive controls included 2 groups of 6 mice each (one group received indomethacin treatment), with 10 / 12 CR / regressions (6 / 6 regressions without indomethacin and 4 / 6 regressions with indomethacin added). The data indicate that both the innate and adaptive immune systems are involved in the tumor eradication by the bait technology, as expected for products containing multiple TLR agonists and other danger signals of the immune system. For example, TLR4 activated by LPS has been shown to be important (if not essential) for antigen presentation, processing, and dendritic cell activation. Thus, in addition to immune activation associated with innate and adaptive immune cells, LPS provides a key bridging activity between the innate and adaptive immune systems.

[0202] The regression of established A20 mouse syngeneic NHL tumors by bait was repeated using two different bait products representing two different Escherichia coli strains. When the average tumor size was 201 mm 3Treatment was initiated at 8 and 1x10 9 bacteria / animal, intravenous injection) or Batch 2 (3x10 8 and 1x10 9 bacteria / animal, intravenous injection) (QDx2 for 2 weeks) in combination with LDC (20 mg / kg / animal, intraperitoneal injection, QDx4 for 2 weeks), resulting in regression in 4 - 5 / 5 and long - term survival in 3 - 4 / 5 of each group ( Figure 24 ). In this study, the bait as a single agent was not tolerated, with 1x10 9 bacteria administered per animal, QDx2 weekly for two weeks, and all animals died after the third dose. Surprisingly, combination with LDC allowed successful administration of the bait at a dose of 1x10 9 bacteria per animal, QDx2, with the exception of the fourth dose in one animal in each bait group due to weight loss requiring a dosing holiday. Based on the above study, taking the bait twice a week for consecutive days, especially in combination with LDC, resulted in significant transient weight loss. During the two - week treatment period, the maximum transient single - agent weight losses for the 3x10 8 bait doses were 9.8% and 7.0% respectively, and for the single - agent bait at 3x10 8 bacteria per dose were 10.7% and 7.4%. The maximum transient weight losses for the bait in the combination groups were 9.7% and 17.6% respectively, and for the bait were 11% and 16.1%. The maximum transient weight losses for the 1x10 9 bait single - agent dose groups were 12.9% and 15.9% (treatment for one week), and the maximum transient weight losses for the combination groups during two - week treatment were 16.3% to 20.5%.

[0203] If the bait bacteria in combination with LDC can activate or enhance the innate anti - tumor immune response ( Figure 23 ), then it is conceivable that in the context of human tumor xenografts (only innate), the bacteria may act synergistically with therapies mediated by the innate immune system. This was tested in a human Ramos NHL xenograft model in SCID mice lacking B and T cells.

[0204] In an NHL subcutaneous human Ramos mouse xenograft model using CB17 / SCID mice, the anti - tumor activity of the bait (2x10 8 bacteria / animal) as a single agent and in combination with LDC and rituximab was evaluated. Mice were administered the bait (2x10 8Bacteria / animal, intravenous injection, slow injection, QDx2 per week for 3 weeks), indomethacin (14 µg / mL, oral administration as desired, QDx21), LDC (20 mg / kg, QDx4 per week for three weeks) and / or rituximab (100 µg / mouse, intravenous injection, BIW, for three weeks). When the average tumor size was 173 mm 3 Treatment was initiated. Indomethacin at 14 µg / mL in the drinking water did not produce single-agent activity or significantly enhance the anti-tumor activity of any other treatment, and long-term treatment was found to produce some toxicity. All other study compounds, including the bait, produced statistically significant single-agent and combination activity. The maximum transient weight loss in all non-indomethacin groups was generally in the range of 5% to 10%. The growth of established Ramos tumors in this model was inhibited or delayed by the standard-of-care drug rituximab, but there was no regression or cure. The combination of bait and LDC caused regression of established Ramos tumors but did not cure the mice. The combination of bait, LDC, and rituximab produced a relatively durable regression in 5 / 5 mice that lasted until day 85 ( Figure 25 ). Figure 25 Four out of five completely cured tumors in

[0205] grew back after day 85. However, it was possible to cure at least a certain proportion of mice with the triple combination, as repeated administration produced a durable regression in 2 / 4 of the mice. Figure 26 On day 74, five tumor-regressed mice were rechallenged (on the flank opposite the first challenge) with fresh Ramos tumor cells. Only 2 / 5 of the tumors grew out compared to 5 / 5 of naive mice challenged with the same fresh tumor cells ( Effect of single-agent bait on the regression of established murine breast cancer tumors after transfection with exogenous antigen

[0206] Female BALB / c mice bearing approximately 170 mm 3 subcutaneous EMT6 murine breast cancer tumors were injected intravenously with 2 x 10 8 bait bacteria twice a week for 4 weeks. Tumor growth was similar to that of untreated tumors. Treatment of mice bearing approximately 170 mm of tumors expressing the human HER2 receptor3 in mice with EMT6 tumors, resulting in tumor growth inhibition, and complete regression of 2 out of 5 established tumors ( Figure 27 ), indicating that at least in this setting or model, the only aspect of anti-tumor innate / adaptive immunity that the bait fails to induce or provide for some mice is the antigen. Thus, providing antigen can enhance or synergize with bait therapy. In vivo activity of AAV-HBV in a chronic HBV-infected mouse model

[0207] The anti-hepatitis B virus (HBV) efficacy of the bait was evaluated in two studies. A standard preclinical model was used in which the human HBV genome was inserted into adeno-associated virus (AAV). This hybrid construct was able to infect mouse hepatocytes, generating a chronic HBV-like infection with many features of human HBV infection, including high-level replication of HBV in the liver and blood and the production of HBe and HBs antigens (HBeAg and HBsAg). The AAV-HBV virus was administered to mice, and then the blood titers of HBV DNA were monitored. A significant and relatively stable HBV blood level appeared within 28 - 31 days, at which point treatment was initiated. In the first study, many groups received indomethacin and the bait, but not in the second study. It was found that the antiviral activity of the bait did not require the inclusion of indomethacin.

[0208] In the first study, the bait (two different doses) was tested in combination with indomethacin and also in combination with the clinical standard treatment entecavir (ETV). ETV and indomethacin were also tested as single drugs (first study). In the second study, the bait was tested as a single drug to determine whether indomethacin was required for activity. In addition, ETV and mIFNα were tested as single drugs and in combination with the bait (one dose). In the first study, mice received the bait (0.6x10 8 and 2x10 8 bacteria / animal, intravenous injection, QDx2 weekly for 5 weeks), ETV (0.1 mg / kg, oral, QD weekly for 5 weeks), and indomethacin (10 µg / mL, oral ad libitum for 5 weeks). Treatment was initiated 28 days (indomethacin and ETV) or 29 days (bait) after infection. In the study, a bait vector control containing PBS with 2 mM MgCl2 (without Ca 2+ or Mg 2+ ) was used as a control. The higher dose of the bait (as shown below) obtained the best results, although some activity was also observed with the lower dose.

[0209] In the first study, monotherapy with indomethacin did not inhibit HBV replication. Bait bacteria (+ indomethacin) and ETV (± indomethacin) significantly inhibited HBV replication (measured in blood) during and up to 28 weeks after dosing. The combination of bait (+ indomethacin) + ETV also inhibited HBV replication, potentially to a greater extent than bait (+ indomethacin) or ETV (with indomethacin) alone ( Figure 28 ). It is known that ETV does not reduce or inhibit the production of HBeAg or HBsAg, which was confirmed in this study. Bait (+ indomethacin) treatment led to a significant reduction in HBeAg and HBsAg in blood during and up to 28 weeks after dosing ( Figure 29 and Figure 30 ). Bait (+ indomethacin) and bait + ETV (+ indomethacin), but not ETV (± indomethacin), led to a significant reduction in HBV in the liver ( Figure 31 ). Similar results were observed for HBeAg expression in the liver, where inhibition was only observed with bait treatment (+ indomethacin) ( Figure 32 ). Although the exact relationship with HBV covalently closed circular cccDNA in the context of human infection is unclear, bait (+ indomethacin), but not ETV, also reduced the level of cccDNA-like molecules in the AAV-HBV model ( Figure 33 ). Some or slight inhibition of HBcAg by bait (+ indomethacin) was also observed in the liver, but not with ETV. Bait-induced weight loss was mild and transient (6% in the first week of treatment for 12 days) and gradually decreased after the first week of treatment (tolerance phenomenon) ( Figure 28 ). During the period of 28 - 56 days, transiently elevated plasma alanine aminotransferase (ALT) levels occurred 13 times in three mice in the bait (+ indomethacin) group and two mice in the bait + ETV (+ indomethacin) group, and all these levels disappeared after day 56 until the end at day 260. At the end, H&E liver histopathology showed no changes related to bait treatment. In vivo activity in the AAV chronic HBV-infected mouse model

[0210] In the second in vivo secondary pharmacodynamic AAV-HBV study, the anti-HBV efficacy of bait as a single agent (without indomethacin) and in combination with ETV was evaluated in male C57BL / 6 mice (5 animals per group) using the standard AAV-HBV mouse model. Mice were dosed with bait (2x10 8Individual bacteria / animals, intravenous injection, QDx2 per week for 5 weeks), ETV (0.005 mg / kg, oral, QD, 5 weeks) or mIFNα (1000 U / g, subcutaneous injection, TIW x 5 weeks). Combinations of ETV (starting on day 0 or day 7 from 31 days post-infection) + decoy and decoy + mIFNα were also tested. ETV and mIFNα treatment started on day 31 (day 0) post-infection, and decoy treatment started on day 32 (day 1) post-infection. A group of untreated animals was included in the study. AAV HBV-infected mice tolerated all treatments well. Mild, transient weight loss was observed in decoy-treated mice (7% weight loss within 2 days in the first week of treatment, then gradually decreasing with subsequent treatments). One unexplained death occurred in the single-agent decoy group 4 weeks after treatment (after weight loss) and was replaced by a spare mouse (a very rare event).

[0211] The results were similar to the first experiment, indicating that indomethacin is not required for decoy activity in this model. ETV alone, decoy alone, and the combination of ETV and decoy reduced plasma HBV DNA levels from day 31 to day 151 in the absence of indomethacin ( Figure 34 ). Decoy alone and decoy + ETV, but not ETV alone, reduced plasma HBsAg and HBeAg levels ( Figure 35 and Figure 36 ). Decoy alone and the combination of decoy and ETV, but not ETV alone, reduced HBV DNA and HBeAg levels in the livers of mice at termination. The combination of decoy and ETV, but not ETV alone, reduced the HBsAg content in the livers of mice at termination. It was observed that decoy alone could reduce the HBsAg content in the liver, but this finding was not significant. Decoy and ETV, but not ETV alone, significantly reduced the expression of cccDNA-like molecules in the liver. Decoy alone seemed to reduce cccDNA-like molecules in 3 / 5 mice. Decoy induced the long-term production of T cell-mediated anti-HBsAg activity, but not anti-HBsAg B cell activity. During decoy treatment, a transient increase in plasma ALT levels was observed, but it disappeared after treatment was stopped. At termination, H&E liver histopathology showed no changes related to decoy treatment, except for mild perivascular mononuclear cell infiltration in 2 / 10 decoy-treated mice. mIFNα treatment resulted in a transient inhibition of HBV DNA in the plasma, but the effect was lost shortly after treatment was stopped. No inhibitory effect of mIFNα on other infection markers was observed. The combination of mIFNα and decoy did not improve the results obtained with decoy alone. In vivo activity in a chronic human HIV-infected mouse model

[0212] In this additional pharmacodynamic study, the effects of bait alone and in combination with indomethacin or the human standard of care “highly active antiretroviral therapy” (HAART) (a mixture of raltegravir, tenofovir disoproxil fumarate, and lamivudine) on HIV plasma viremia and immune cell population counts were evaluated in female NOD / Shi-scid / IL-2Rγ null mice (4–6 animals per group) using a chronic human immunodeficiency virus (HIV) humanized mouse model. Immunodeficient mice reconstituted with the human immune system and infected with HIV-1 were dosed with bait (6x10 7 bacteria / animal, intravenous injection, BIW x 5 weeks), indomethacin (10 µg / mL in drinking water, 5 weeks), and / or HAART (oral, ad libitum x 5 weeks). A bait vector control was used in the study. HAART treatment significantly reduced the HIV viral load in plasma within 2 weeks of starting treatment and continued for 3 weeks after treatment was stopped. Bait did not significantly reduce the viral load during treatment, but a significant reduction was observed starting at approximately 2–3 weeks after treatment was stopped and continued for approximately 10 weeks (not significant at all time points) ( Figure 37 ). Indomethacin suppressed the HIV viral load, but only after 1 week and 2 weeks of treatment during the 18-week treatment period. Combination therapy did not appear to significantly improve monotherapy. By week 24, the levels of human CD4+ T cells had significantly decreased in all mice. None of the drugs restored the CD4+ T cell levels to normal, although an increase in CD4+ T cells relative to untreated mice was observed in mice treated with HAART alone or in combination with bait, indomethacin, and HAART. Summary

[0213] A comprehensive nonclinical pharmacology program has been developed to support the first-in-human (FIH) oncology study of bait. The primary pharmacodynamic (PD) studies with bait included in vitro evaluations of cytokine and chemokine secretion in murine and human peripheral blood mononuclear cells, and in vivo evaluations of the anti-tumor activity of intravenous injection against established subcutaneous (s.c.) murine colorectal cancer, orthotopic murine colorectal cancer, metastatic murine pancreatic cancer, established subcutaneous murine hepatocellular carcinoma (HCC), established subcutaneous murine non-Hodgkin lymphoma (NHL), and established subcutaneous human non-Hodgkin lymphoma models, all in mice. Bait was tested as a single agent and in combination with low-dose cyclophosphamide (LDC), oral low-dose non-steroidal anti-inflammatory drug (NSAID / indomethacin), murine anti-PD-1 checkpoint therapy, and / or rituximab. Bait was also tested against established murine breast cancer tumors with and without exogenous antigen expression.

[0214] Although the LPS endotoxin activity was reduced by approximately 90%, the induction of cytokine and chemokine secretion in bait-mediated mouse and human peripheral blood mononuclear cells was largely unaffected. The induction of multiple cytokines and chemokines is responsible for activating innate and adaptive immune cells and pathways, including those required for known anti-tumor and anti-viral responses. This very surprising observation may be related to changes in the mechanism and / or time course by which immune cells process glutaraldehyde-chemically stabilized bacteria.

[0215] In a mouse subcutaneous colorectal cancer model and a mouse metastatic pancreatic cancer model, statistically significant anti-tumor activity was observed using bait as a single agent, and in a mouse subcutaneous HCC model and a mouse subcutaneous NHL model, synergistic tumor eradication was observed in combination with LDC, indomethacin, and / or anti-PD-1 therapy. None of the drugs tested produced consistent regression or eradication as a single agent. Up to 100% of the animals in each group observed combination-based tumor eradication (no tumors for at least 3 - 5 months after tumor implantation), which was associated with the induction of 100% immune memory, as demonstrated by the rejection of 100% tumor rechallenge without additional treatment. Bait produced anti-tumor activity as a single agent, and after only 2 to 6 weeks of weekly intravenous injection once or twice (depending on the model), the combination-based tumor eradication rate was 80 - 100%, including in one combination therapy model (HCC) producing a ≥33-fold therapeutic index at well-tolerated doses without clinical toxicity symptoms. Bait also produced single-dose regression in a mouse model of breast cancer transfected with foreign antigen.

[0216] The anti-tumor efficacy study of bait was extended to a human NHL tumor xenograft model in severe combined immunodeficient mice lacking an adaptive immune system. The bait + LDC combination produced a high percentage of regression in established tumors, which was not persistent. Adding the standard-of-care targeted antibody rituximab produced a persistent, complete regression, which is difficult to achieve in a purely innate environment. Mice with regressed tumors were rechallenged with fresh tumor cells without further treatment. A subset of the new tumors was rejected, showing partial innate immune memory, which has been reported but is also considered difficult to achieve. Combining the high percentage of immune memory observed in a syngeneic environment, these results further support the dual innate + adaptive mechanism of action of the killed bait bacteria in a combination environment.

[0217] In a mouse NHL model, depletion of NK or CD4+ T cells or CD8+ T cells before starting bait + LDC combination therapy led to almost complete loss of anti-tumor activity of tumor eradication, further demonstrating the role of activation of innate and adaptive immune pathways. Bait + LDC was also able to eradicate very large tumors, up to 2000 mm 3Established subcutaneous tumors. In another HCC tumor eradication model based on a triple combination (decoy + indomethacin + anti-PD-1), 770-gene NanoString gene expression analysis was performed on subcutaneous tumors isolated from mice one week after single-agent, dual, or triple combination treatment, including a single intravenous injection of the decoy dose. The progression from single-agent (no tumor regression or eradication) to dual-agent (1 - 2 out of 6 eradications) to triple-agent (5 - 6 out of 6 eradications) treatment was associated with a progressive increase in tumor expression of cytokines, chemokines, innate and adaptive immune pathway genes, including an increase in the tumor inflammation signature score (from cold to hot tumors). The tumor eradication combination treatment was also associated with a synergistic induction of plasma cytokine and chemokine expression compared to single-agent decoy treatment, without any increase in toxic clinical symptoms.

[0218] Preliminary pharmacological studies of the decoy have shown significant single-agent and combination-based anti-tumor activity against multiple tumor types and support a mechanism of action based on priming or activating innate and adaptive immune cells and pathways. The high native level of LPS endotoxin activity was significantly reduced (by approximately 90%), which may improve the safety of intravenous injection, while the remaining activity may be sufficient to promote the innate and adaptive immune-stimulatory properties of LPS and other TLR agonists found in Gram-negative bacteria, as well as complementary or synergistic effects with other immune-activating molecules in bacteria, such as NOD and STING agonists. Secondary pharmacological studies were also conducted with the decoy, demonstrating significant single-agent antiviral activity in non-clinical models of chronic hepatitis B virus (HBV) infection and chronic human immunodeficiency virus (HIV) infection.

[0219] The safety of the decoy was determined after a 1-hour intravenous injection in single-dose, two-week repeated-dose range finding, and four-week repeated-dose toxicology studies in New Zealand white rabbits (NZW), a non-human laboratory species considered to be most similar to humans in terms of sensitivity to adverse reactions to LPS. Additional safety information was obtained using the decoy in studies conducted in mice. In the proposed Phase 1 clinical study, safety data from a key two-week, twice-weekly repeated-dose toxicology study were used to support the starting dose of the decoy, including once-weekly dosing.

[0220] Due to one death at a dose level of 1.5x10 9 The maximum tolerated dose (MTD) of the single-dose decoy was determined to be 1.5x10 9 killed bacteria [KB] / kg in rabbits at 4 dose levels tested. Four dose levels were tested for twice-weekly decoy administration for two weeks, resulting in 6x10 7No Observed Adverse Effect Level (NOAEL) of 7 KB / kg / dose. The study also found that the pyrogenicity (rectal temperature test) of the bait in rabbits was 97% lower than that of the parental (untreated) bacteria, and the toxicity (acute LD100) was also 3 times lower than that of the parental (untreated) bacteria.

[0221] Intravenous infusion of the bait induced minor clinical signs and symptoms, most of which were transient and reversible. These included increases in body temperature and spleen weight, changes in hematological parameters (such as decreases in platelets, red blood cells, hematocrit, and hemoglobin) and increases in white blood cells (mainly neutrophils). Clinical chemistry parameters also changed (for example, increases in alanine aminotransferase, aspartate aminotransferase, triglycerides, and cholesterol, and decreases in albumin and albumin / globulin ratio), as well as increases in C-reactive protein and fibrinogen. A dose-dependent increase in plasma cytokine levels of interleukin- (IL-) 6 was observed shortly after dosing (1.5 hours), and it returned to baseline levels within 24 hours after the first dose and within 6 hours after the last dose. Among the 13 tested, IL-6 was the only cytokine or chemokine that increased in plasma. For various molecules associated with cytokine release syndrome, only transient induction or no induction was observed, and these molecules represent the severe toxicities associated with many immunotherapies. Stabilizing the bacteria to prevent breakdown in circulation, combined with the rapid clearance of bacteria by immune cells in the liver and spleen, can reduce the risk of cytokine release syndrome associated with immunotherapies that rely on continuous exposure doses.

[0222] Compared with the same time points after the first dose of the bait, at different time points after subsequent doses, the changes in bait-mediated platelets, white blood cells (WBC), neutrophils, albumin, cholesterol, triglycerides, body temperature, and IL-6 either did not occur (platelets, WBC, neutrophils, and albumin) or decreased (cholesterol, triglycerides, body temperature, and IL6). These results are consistent with the phenomenon of LPS tolerance, which has been well documented in mice, rabbits, and humans. This phenomenon should also improve the safety of the product.

[0223] The incidence of macroscopic (such as increased spleen weight and splenomegaly [2 / 40 animals participated in the key study]) and microscopic (such as mild lymphoid hyperplasia in the spleen and mild mononuclear cell infiltration in the liver) was very low. The macroscopic and microscopic findings observed in the non-clinical rabbit model were considered to be without adverse reactions and were related to the proposed mechanism of action of the product.

[0224] Non-clinical studies have shown that the safety of the bait is acceptable both in terms of the therapeutic index in murine pharmacological studies and in rabbit toxicology studies. Similar to the well-documented results of the tolerance (decreased toxicity) of repeated intravenous injection of LPS in mice, rabbits, and humans, the bait containing LPS exhibited the same phenomenon in mice and rabbits. Example 3. Clinical Trials of the Bait

[0225] This example describes the proposed clinical trials of the bait.

[0226] The bait drug product consists of an attenuated, stable, 100% killed suspension of non-pathogenic Gram-negative bacterial cells formulated with trehalose for cryoprotection. After dilution of the cell suspension with trehalose, the final formulated drug product also contains 75% phosphate-buffered saline (pH 7.5), 1.5 mM MgCl2, and 12% trehalose. The bait formulated drug product is filled into 2 mL (2R) vials to a volume of approximately 1.0x10 9 killed bacteria (KB) per mL to a volume of 0.7 mL, resulting in an extractable volume of 0.5 mL, and stored as a frozen liquid at ≤ -60 °C. The target total cell count is 0.3 – 3.0 x 10 9 cells per mL. The composition of the bait drug product is shown in Table 5. Table 5. Composition of the Bait Drug Product Ph.Eur. = European Pharmacopoeia; NF = National Formulary; USP = United States Pharmacopeia.

[0227] The bait will be diluted with sterile 0.9% saline (saline) for injection and administered as a 250 mL intravenous infusion over approximately 1 hour. This study in the first phase will consist of three parts. In Part 1, a single escalating dose will be given, and in Part 2 (Parts 2a and 2b), weekly consecutive doses will be given as described below. Part 1. Single Escalating Dose

[0228] Part 1 will be a single escalating dose test. Subjects will receive a single dose of the bait at the designated dose level on Week 1 (W) and Day 1 (D). The administered doses for each single-dose cohort are described in Table 6 below. Table 6. Dose Levels * In the case where no dose-limiting toxicity (DLT) occurs in cohort 4.

[0229] The starting dose for Part 1 of this study (cohort 1 = 7 x 107 (KB) will be approximately 1 / 10 of the human equivalent dose (HED), which is determined based on the no-observed-adverse-effect level (NOAEL) observed in a 4-week rabbit toxicology study with twice-weekly dosing (4 x 10 7 KB / kg dose). Rabbits are the closest substitute to humans in terms of immune and toxicological responses to intravenously administered purified LPS. Based on the data from a 4-week Good Laboratory Practice (GLP) study, including an allometric scaling factor (dose reduction) of 3.1 times the HED plus a 10-fold dose reduction safety adjustment, the starting human dose is 1.29 x 10 6 KB / kg or approximately 16 endotoxin units (EU) / kg related to the bait, i.e., 7.74 x 10 7 KB per 60 kg subject; equivalent to approximately 960 EU per 60 kg subject (not the conventional 70 kg to account for lower patient weights). The starting dose in the study will be slightly lower, at 7.0 x 10 7 KB: equivalent to 868 EU or 1.8 ng / kg LPS per 60 kg subject. Based on the results of systemic clearance tests of live and inactivated bacteria in mice, rabbits, and humans, rapid clearance of the bait by the liver and spleen is expected (within ~15 - 30 minutes), so dose adjustment based on body weight is not considered necessary. The starting dose of LPS in the study is lower than the well-tolerated maximum dose (4 ng / kg) determined after intravenous injection of purified LPS in over 1000 healthy human volunteers. Part 2: Weekly consecutive dosing

[0230] When the single-dose recommended Phase 2 dose (RP2D) in Part 1 is determined, Part 2a will begin. The first 3 subjects participating in Part 2a will receive 4 doses of the bait at the RP2D on W1D1, W2D1, W3D1, and W4D1. The safety data of these 3 subjects will be reviewed 4 weeks (W8D1) after the administration of the 4th dose of the bait. If there are no safety issues, the subjects will start taking the bait continuously weekly starting from W9D1. Once the 3 subjects complete this dosing schedule, with acceptable toxicity and approval from the Safety Review Committee (SRC), 3 more subjects will be recruited to receive the bait with weekly consecutive dosing. When the 6th subject completes at least 4 weeks of dosing, the SRC will review the cumulative safety data of all 6 subjects in order to include subjects in Part 2b at the same or lower dose.

[0231] In the dose expansion study in Part 2b, the subjects will receive the bait continuously weekly according to the dose and schedule determined by the SRC based on the data obtained from Part 1 and Part 2a.

[0232] This study will recruit subjects histologically diagnosed with incurable advanced metastatic solid tumors who have exhausted all available treatment options and demonstrated clinical benefit from their malignancy.

[0233] In Part 1, the expected treatment duration for each subject is 1 day (single intravenous injection of approximately 1 hour), plus a 28-day dose-limiting toxicity (DLT) observation period. After confirmation of the continuous weekly RP2D and dosing regimen in Part 2a, subjects who participated in Part 1 may receive the bait as continuous weekly dosing at the RP2D for up to 2 years, at the discretion of the investigator and medical monitor, until disease progression, intolerable toxicity, or subject withdrawal, provided they completed the Part 1 DLT observation period without DLT, meet all eligibility criteria at re-enrollment, and the investigator determines that continued dosing with the bait is in the best interest of the subject. Additionally, the protocol allows for dose adjustment to a lower level or skipping of doses as needed.

[0234] The first 3 subjects in Part 2a (safety run-in) will receive the bait for 4 weeks, followed by a 4-week safety observation period without dosing. Based on acceptable safety, these subjects will continue to receive the bait continuously weekly for up to 2 years. Another 3 subjects will receive the bait continuously weekly for up to 2 years.

[0235] Subjects in Part 2 (Parts 2a and 2b) will receive the bait continuously weekly for 2 years until disease progression, intolerable toxicity, or subject withdrawal, whichever occurs first.

[0236] Subjects who have not had documented disease progression, unacceptable toxicity, or withdrawal of consent and who have benefited from the bait after 2 years may continue to receive the bait, subject to the agreement of the investigator and medical monitor. Subjects continuing treatment may be required to re-sign the informed consent form (ICF) in a potential new treatment extension protocol. Inclusion and Exclusion Criteria

[0237] To be considered eligible to participate in this study, subjects in Part 1 (subjects in the single ascending dose part, or subjects selected in Part 1 who are eligible to rejoin the study after establishment of the RP2D), Part 2a, and Part 2b will follow the following criteria. Inclusion Criteria: 1. Must provide a written informed consent form signed by the subject and approved by the institutional review board. 2. Male or female, 18 years of age or older. 3. Histologically diagnosed with advanced metastatic solid tumors. 4. Subjects received at least 1 and up to 3 lines of prior treatment in a metastatic setting, then progressed (recurred, relapsed or refractory) or were intolerant. Prior treatment in the metastatic setting included chemotherapy, targeted (known molecular alteration) therapy, immunotherapy and antibody therapy. Subjects must have exhausted all available treatment methods and demonstrated clinical benefit for their malignancies. Subjects who have received >3 lines of prior treatment may be eligible after discussion with and approval by the sponsor. 5. Measurable disease (at least 1 measurable lesion) as defined by tumor type and according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. 6. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. 7. Life expectancy of at least 3 months. 8. Female subjects must be non-fertile (surgically sterile or postmenopausal for at least 2 years), or agree to use highly effective contraception during receipt of the investigational product and for 30 days after the last dose of the investigational product. a. Fertile females must have a negative serum pregnancy test at screening and a negative serum or urine test at W1D1 prior to receipt of the investigational product. 9. Male subjects must use reliable contraception during receipt of the investigational product and for 30 days after the last dose of the investigational product. 10. Normal organ function confirmed by the following laboratory values at screening: a. Neutrophils ≥1200 / μL b. Platelets ≥100,000 / μL (transfusions and / or growth factor support permitted) c. Hemoglobin ≥8.0 g / dL (transfusions and / or growth factor support permitted) d. Estimated creatinine clearance ≥50 mL / min calculated using the Cockcroft-Gault formula, not dependent on dialysis. e. Aspartate aminotransferase (AST) ≤2.5 x upper limit of normal (ULN) (if liver metastasis or liver involvement - AST ≤5 x ULN) f. Alanine aminotransferase (ALT) ≤2.5 x ULN (if liver metastasis or liver involvement - ALT ≤5 x ULN) g. Bilirubin ≤1.5 x ULN (except in patients with Gilbert's syndrome) h. Ferritin ≤4 x ULN, C-reactive protein (CRP) ≤6 x ULN. 11. Left ventricular ejection fraction (LVEF) ≥45% shown by echocardiogram (ECHO) or multiple gated acquisition scan (MUGA) at screening. 12. Recovered to ≤ Grade 1 from the toxicities of previous treatment, except for peripheral neuropathy, according to the NCI CTCAE v5.0 criteria or the subject's previous baseline. 13. Willing and able to comply with all scheduled visits, laboratory tests, and other study procedures. Exclusion criteria: 1. Females who are pregnant or lactating. 2. Have an active systemic (viral, bacterial, or fungal) infection or require treatment. The infection should be treated, and the subject should recover before enrollment in the study. 3. Received radiotherapy within 28 days after the first dose of the bait. The subject must have recovered from all radiation-related toxicities, not require corticosteroids, and have no radiation pneumonitis. 4. Received chemotherapy, immunotherapy, or major immunomodulatory therapy within 28 days or 5 half-lives after W1D1. If treated with a programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitor (such as nivolumab, pembrolizumab, atezolizumab, and / or durvalumab, etc.) within 2 months before the investigator confirmed eligibility. 5. Received systemic corticosteroid treatment > 5 mg / day of prednisone or an equivalent dose of another corticosteroid within 1 week or 5 half-lives (whichever is shorter) starting from W1D1, or are expected to require (local and inhaled corticosteroids are allowed) or obtain approval from the medical supervisor during the study. Systemic corticosteroids are prohibited after receiving the bait treatment to manage adverse events outside of specific study needs. 6. Radiological examination reveals primary central nervous system (CNS) or CNS metastases or symptomatic CNS involvement (including leptomeningeal carcinomatosis, cranial neuropathy, or mass lesions causing spinal cord compression). 7. Clinical evidence of severe coagulation disorders (such as deep vein thrombosis or pulmonary embolism) during screening or a history of severe uncontrolled coagulation disorders. Subjects with superficial vein thrombosis and visceral / visceral vein thrombosis mainly related to underlying diseases, or with controllable coagulation conditions are eligible. 8. Have an active secondary malignancy in addition to the primary malignancy, excluding low-risk tumors determined by the investigator (such as non-metastatic basal cell carcinoma or squamous cell skin cancer). 9. A history of human immunodeficiency virus (HIV) type 1 or 2 or a history of active infection, positive detection of hepatitis B virus (HBV) antibody or surface antigen (indicating acute HBV or chronic HBV), or positive for hepatitis C (hepatitis C virus [HCV] ribonucleic acid [RNA] detected by qualitative analysis). Subjects with negative hepatitis C antibody testing do not require hepatitis C RNA testing. Subjects with negative HBV surface antigen do not require HBV antibody testing. 10. Known intolerance to non-steroidal anti-inflammatory drugs (NSAIDs). 11. A history of known genetic susceptibility to HLH / MAS. 12. Having undergone splenectomy, having active chronic liver disease, alcoholic liver disease, Wilson's disease, hemochromatosis, primary biliary cirrhosis, primary sclerosing cholangitis, hereditary hemochromatosis, a history of liver transplantation for end-stage liver disease of any etiology or planned liver transplantation, a history of advanced liver fibrosis or cirrhosis and / or a history of liver decompensation, including ascites, hepatic encephalopathy or variceal bleeding. 13. Having received a live vaccine within 28 days after W1D1. 14. Having active autoimmune diseases (including but not limited to psoriasis, multiple sclerosis, lupus and rheumatoid arthritis). 15. A history of severe CNS diseases, such as stroke (a history of transient ischemic attack more than 6 months ago, which can be controlled) or uncontrolled and unstable epilepsy. 16. Having severe interstitial lung disease and / or indoor air oxygen saturation < 92%. 17. Baseline Q-T correlation (QTc) interval calculated using the Fridericia formula > 470 milliseconds in females and > 450 milliseconds in males. 18. New York Heart Association class III or IV heart disease, or myocardial ischemia or infarction within 180 days after screening, severe unstable angina, coronary / peripheral artery bypass grafting, worsening / decompensated heart failure within the past 6 months, or any other clinically significant cardiac abnormality that the investigator deems to pose a health risk to the subject. 19. Having had major surgery within 4 weeks before the first dose of the bait during the study period or anticipating the need for major surgery. (Note: Placement of a central venous access catheter [such as a port or the like] is not considered major surgery). 20. Any other acute or chronic medical or mental illness that may increase the risk associated with participating in the study or taking the bait, or that, in the judgment of the investigator or sponsor, makes the subject ineligible for the study, including pre-existing diseases that may increase the susceptibility to expected toxicity or cytokine-induced inflammation, including abnormal blood chemistry. 21. Received investigational treatment within 28 days or 5 half-lives of W1D1, whichever is shorter. 22. Unwilling or unable to comply with the procedures specified in this protocol. 23. Known allergy or hypersensitivity to the bait or one of the bait components.

[0238] All subjects in Parts 1 and 2 will have a 1-year long-term survival follow-up after the last dose of bait. These assessments can be done by phone or at the clinic every 3 months after the last dose of bait. Information on the start of other anticancer treatments (including start date, treatment type / name, and treatment response) can be collected. Example 4. Preliminary Results of a Phase 1 Study of the Bait

[0239] This example describes the preliminary results of an ongoing first-in-human Phase 1 study of the bait, which is an intravenous, killed multi-immune receptor agonist bacterial product for patients with advanced solid tumors.

[0240] This is a first-in-human, open-label, single-dose escalation and multi-dose expansion multicenter Phase 1 trial of the bait in patients with advanced / metastatic solid tumors, with an initial dose-limiting toxicity (DLT) period of 28 days.

[0241] Primary objective: Safety / tolerance. Secondary objectives: Anti-drug immunogenicity, pharmacokinetics (PK), and preliminary anti-tumor activity. Exploratory objective: Activation of systemic immunity through immune biomarkers. Eligible patients must have measurable tumor recurrence or be refractory to standard therapies. The single-dose escalation (SAD) cohort assessment preceded the multi-dose (MD) cohort and used the standard statistical 3+3 design. The starting dose was 7 x 10 7 killed bait bacteria intravenously for 1 hour, based on the no-observed-adverse-effect level in rabbits, which is the relevant non-clinical toxicology species for LPS.

[0242] Plasma biomarkers were measured by Meso Scale Discovery electrochemiluminescence ( Figure 38 ), or the Luminex platform ( Figure 39 ). Bait pharmacokinetics (PK) were determined by digital droplet (dd) PCR method.

[0243] Ethical approval: This study has been approved by the following institutional ethics committees: WIRB / Copernicus, covering Atlantic and Komanos; and USC.

[0244] The trial enrolled 4 patients, whose characteristics are shown in Table 7. Table 7. Patient and Disease Characteristics Results

[0245] Treatment-related adverse events are shown in Table 8. After bolus injection of saline, paracetamol, pethidine, and oxygen, a dose-limiting toxicity of grade (G) 3 bradycardia occurred and resolved within < 30 minutes; G3 discomfort in the same patient disappeared within 2 days. Two patients had G3 elevation of AST, which improved to G1 within 1 - 2 days. Overall, G1 chills, fatigue, fever, G2 vomiting, hypotension, and G1 - 2 elevation of ALT resolved within 1 - 2 days, and G4 lymphopenia resolved within 2 - 3 days, all of which may have occurred after exposure to LPS (a TLR4 agonist), the active ingredient of the decoy. Table 8. Treatment-related adverse events * SAE due to hospitalization required Note: Data are from an open database and may change

[0246] It was observed that treatment with the decoy induced the expression of transient plasma cytokines, chemokines, and biomarkers ( Figure 38 ). Analyses were performed before dosing, and at 4, 24, 48, 72 hours, 3 weeks (504 hours), and 4 weeks (672 hours) after the end of infusion. The peak induction of cytokines and chemokines occurred within ~4 to 24 hours and returned to baseline levels within 24 - 48 hours. Soluble IL-2 receptor (sIL-2r), a marker of T cell activation, peaked at approximately 24 hours and remained elevated for at least 72 hours.

[0247] Table 9 presents the results of an extended single-time point plasma cytokine, chemokine, and biomarker analysis. Analyses were performed before dosing, and at 0.5, 1, 2, 4, 6, 24, 48, 72 hours, and 4 weeks after the end of infusion. Most induced analytes peaked within 2 - 4 hours and resolved within 24 - 48 hours. The data in Table 9 represent the maximum fold induction or reduction. Table 9. Extended single-time point plasma cytokine, chemokine, and biomarker analysis No data entry indicates no induction ≥4-fold N / A - Not yet analyzed Values in parentheses for the Mesoscale Discovery electrochemiluminescence panel

[0248] Table 10 summarizes cytokines and chemokines (not exhaustive) associated with innate and adaptive anti-tumor immune responses. A single intravenous dose of the decoy induces cytokines and chemokines transiently ≥4-fold, as underlined / bolded in Table 10. Table 10. Cytokines and Chemokines Associated with Innate and Adaptive Anti-Tumor Immune Responses

[0249] Pharmacokinetic analysis confirmed that the systemically administered decoy was rapidly cleared. A ddPCR method with a lower limit of detection / quantitation of 10 / 89 decoy bacteria per milliliter of blood was developed to determine decoy levels in the blood of subjects before dosing and at 5, 10, 30, 120, 240 minutes, 24 hours, and 4 weeks after the end of the infusion. The decoy was cleared from the blood within 30 to 120 minutes after the end of the infusion ( Figure 39 ). Due to the peak concentration within 5 minutes after the end of the infusion and the steep elimination slope, differences in peak height among subjects may reflect minor differences in clearance times during the infusion.

[0250] Tumor restaging 4 weeks after dosing showed stable disease in all 4 subjects. Three of these subjects had progressive disease before taking the decoy.

[0251] A single intravenous dose of the decoy was cleared from the blood within 30 - 120 minutes and produced a transient induction of over 50 biomarkers in plasma, many of which are related to stimulating innate and / or adaptive immune responses. Most cytokines and chemokines have been shown to play a positive role in immune responses, but can also be toxic if present at abnormally high levels for extended periods. The transient induction of cytokines and chemokines is an important and novel feature of the response to decoy bacteria, helping to reduce the likelihood of systemic toxicity known to result from continuous or long-term systemic exposure to these potent immune activating molecules.

[0252] In addition, blood immune cell analysis showed a rapid increase in neutrophils and a rapid decrease in essentially all other white blood cells, with all cell types recovering within approximately 72 hours, indicating that the decoy induced a transient but significant event of white blood cell trafficking or redistribution.

[0253] In summary, adverse reactions were generally tolerable and resolved within 30 minutes to 3 days regardless of treatment. In terms of efficacy, a single dose of the decoy produced initial stable disease in all 4 subjects, 3 of whom were progressing before taking the decoy. ***

[0254] 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 belongs.

[0255] The inventions illustratively described herein can be practiced appropriately without any one or more elements, limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. should be read broadly and without limitation. Further, the terms and expressions used herein are for descriptive purposes only and not limiting, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications are possible within the scope of the invention as claimed.

[0256] Accordingly, it should be understood that although the invention has been specifically disclosed by way of preferred embodiments and optional features, those skilled in the art can make modifications, improvements, and variations to the invention disclosed herein, and such modifications, improvements, or variations are considered to be within the scope of the invention. The materials, methods, and examples provided herein represent preferred embodiments and are exemplary and are not intended to limit the scope of the invention.

[0257] The invention has been described herein in a broad and general manner. Each narrower species and subgeneric grouping falling within the genus disclosed also forms part of the invention. This includes the general description of the invention, with the proviso or negative limitation that any subject matter is excised from the genus, whether or not the excised material is specifically recited herein.

[0258] In addition, where the invention is described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any single member or subgroup of members of the Markush group.

[0259] All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification (including definitions) will control.

[0260] It should be understood that while the present disclosure has been described in conjunction with the above embodiments, the above description and examples are intended to illustrate and not limit the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

Claims

1. A method for treating or preventing cancer or an infectious disease in a patient in need thereof, comprising administering to the patient an effective amount of a composition comprising 1 × 10 7 to 500 × 10 7 intact and substantially non-viable Escherichia coli cells, which Escherichia coli cells have been treated to result in a reduction of about 70% to 99% in lipopolysaccharide (LPS)-associated endotoxin activity as measured by the Limulus amebocyte lysate (LAL) assay compared to untreated wild-type Escherichia coli cells, and wherein the composition contains 124 to 62,000 endotoxin units (EU) of LPS.

2. The method according to claim 1, wherein the composition comprises 2 × 10 7 to 200 × 10 7 intact and substantially non-viable Escherichia coli cells.

3. The method according to claim 1, wherein the composition comprises 3 × 10 7 to 100 × 10 7 intact and substantially non-viable Escherichia coli cells.

4. The method according to claim 1, wherein the composition comprises 5 × 10 7 to 50 × 10 7 intact and substantially non-viable Escherichia coli cells.

5. The method according to claim 1, wherein the composition comprises 3 × 10 7 , 7 × 10 7 , 10 × 10 7 , 20× 10 7 or 70 × 10 7 intact and substantially non-viable Escherichia coli cells.

6. The method according to any one of claims 1 to 5, wherein the composition contains from 372 EU to 24,800 EU of LPS.

7. The method according to claim 6, wherein the composition contains from 372 EU to 8,680 EU of LPS.

8. The method according to claim 6, wherein the composition contains from 868 EU to 2,480 EU of LPS.

9. The method according to any one of the preceding claims, wherein the intact and substantially non-viable Escherichia coli cells have been treated to result in a reduction of LPS-related endotoxin by about 80% to 98%.

10. The method according to claim 9, wherein the intact and substantially non-viable Escherichia coli cells have been treated to result in a reduction of LPS-related endotoxin by about 90% to 98%.

11. The method according to any one of the preceding claims, wherein the administration is once a day, once every other day, once every 3 days, once every 5 days, once every 6 days, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, once every 9 months, or once a year.

12. The method according to any one of the preceding claims, wherein the treatment of the Escherichia coli cells is carried out using polymyxin, preferably polymyxin B or polymyxin E.

13. The method according to claim 12, wherein the treatment of the Escherichia coli cells is carried out at a temperature of about 2°C to about 10°C, preferably at about 4°C.

14. The method according to any one of the preceding claims, wherein the treatment of the Escherichia coli cells is carried out using polymyxin and glutaraldehyde.

15. The method according to claim 14, wherein the treatment is carried out using polymyxin B in a dose range of about 3 mg / mL to about 1,000 mg / mL and glutaraldehyde in a dose range of about 0.1% to about 1.0%.

16. The method according to any one of the preceding claims, wherein the composition further comprises a phosphate buffer, Mg 2+ and trehalose.

17. The method according to claim 16, wherein the composition comprises 0.3 × 10 9 / mL to 5 × 10 9 / mL of intact and substantially non-viable Escherichia coli cells, 0.5 mg / mL to 2 mg / mL of disodium phosphate dihydrate, 0.1 mg / mL to 0.4 mg / mL of potassium dihydrogen phosphate, 3 mg / mL to 12 mg / mL of sodium chloride, 0.05 mg / mL to 0.3 mg / mL of potassium chloride, 0.15 mg / mL to 0.6 mg / mL of magnesium chloride hexahydrate, and 50 mg / mL to 200 mg / mL of trehalose dihydrate, with a pH value of 7.0 to 7.

7.

18. The method according to any one of the preceding claims, wherein the administration is intravenous, intratumoral, subcutaneous, intramuscular, intrahepatic, intravesical, intranasal, or intraperitoneal.

19. The method according to any one of the preceding claims, wherein the patient has a solid tumor.

20. The method according to claim 19, wherein the solid tumor is a metastatic solid tumor.

21. The method according to any one of the preceding claims, wherein the cancer is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

22. The method according to any one of the preceding claims, the method further comprising administering to the patient a second agent selected from cyclophosphamide, IL-2, non-steroidal anti-inflammatory drugs (NSAIDs), anti-PD-1 or anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and anti-CD20 antibodies.

23. The method according to any one of claims 1 to 18, wherein the patient has an infection.

24. The method according to claim 23, wherein the infection is caused by hepatitis B virus (HBV) or human immunodeficiency virus (HIV).

25. A method for providing a therapeutically acceptable composition, comprising: A lyophilization solution for preparing a lyophilized composition, the solution comprising at least 1 × 10 6 intact and substantially non-viable Escherichia coli cells, the Escherichia coli cells having been treated to result in a reduction of about 70% to 99% in lipopolysaccharide (LPS)-associated endotoxin activity as measured by the Limulus amebocyte lysate (LAL) assay compared to untreated wild-type Escherichia coli cells; and storing the lyophilized composition (a) at a temperature of 1 °C to 10 °C for at least 2 months or (b) at a temperature of -15 °C or below for at least 2 years, thereby providing a therapeutically acceptable composition suitable for therapeutic use.

26. The method according to claim 25, wherein the solution further comprises a phosphate buffer, Mg 2+ and trehalose.

27. The method according to claim 25 or 26, wherein the solution comprises 0.3 × 10 9 / mL to 5 × 10 9 / mL of intact and substantially non-viable Escherichia coli cells, 0.5 mg / mL to 2 mg / mL of disodium phosphate dihydrate, 0.1 mg / mL to 0.4 mg / mL of potassium dihydrogen phosphate, 3 mg / mL to 12 mg / mL of sodium chloride, 0.05 mg / mL to 0.3 mg / mL of potassium chloride, 0.15 mg / mL to 0.6 mg / mL of magnesium chloride hexahydrate, and 50 mg / mL to 200 mg / mL of trehalose dihydrate, with a pH value of 7.0 to 7.

7.

28. A method for treating or preventing cancer or an infectious disease in a patient in need thereof, comprising administering to the patient (a) an effective amount of a composition comprising intact and substantially non-viable Escherichia coli cells that have been treated to result in a reduction in lipopolysaccharide (LPS)-associated endotoxin activity of about 70% to 99% as measured by the Limulus amebocyte lysate (LAL) assay compared to untreated wild-type Escherichia coli cells, and (b) an exogenous antigen associated with the cancer or infectious disease.

29. The method according to claim 28, wherein the antigen is a tumor-associated antigen.

30. The method according to claim 28, wherein the antigen is a viral or bacterial antigen.

31. The method according to any one of claims 28 - 30, wherein the composition comprises 1 × 10 7 to 500 × 10 7 intact and substantially non-viable Escherichia coli cells and contains 124 to 62,000 endotoxin units (EU) of LPS.

32. The method according to any one of claims 28 to 31, wherein the exogenous antigen is administered simultaneously or sequentially with the composition.

33. The method according to any one of claims 28 to 31, wherein the exogenous antigen is expressed in or on the Escherichia coli cells.

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