Treating cancer and infectious diseases with killed but intact bacteria

Abstract

The present disclosure generally relates to compositions, dosage forms, and methods for preventing and treating cancer and infectious diseases. One example method involves the use of 1 x 10 Escherichia coli cells treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by approximately 70% to 99% compared to untreated wild-type Escherichia coli cells, as measured by a Limulus Amebocyte Lysate (LAL) assay. 7 ~500×10 7 The present invention relates to a method for treating E. coli infection, comprising administering to a patient an effective amount of a composition comprising intact, stabilized, and substantially non-viable E. coli cells, wherein the composition contains 124 to 62,000 endotoxin units (EU) of LPS.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 426,245, filed November 17, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Cancer is the second leading cause of death in the United States. Over the past decade, advances in immunotherapy have transformed cancer treatment, leading to many new therapies. Immune checkpoint blockade with inhibitory antibodies against programmed cell death 1 (PD-1) 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 immunotherapies ultimately progress to disease.

[0003] Tumors evade immune surveillance by suppressing innate and adaptive immune effectors, limiting neoantigen presentation, and impairing the function of infiltrating immune effector cells. Failure of PD-1 / PD-L1 inhibitors may be due to insufficient production of antitumor T cells, clearance of T cells from the tumor, dysfunction of tumor-specific T cells, and / or impaired T cell memory formation.

[0004] Efficient and sustained antitumor immune responses require systemic innate and adaptive immunity. Many of the steps involved in innate and adaptive antitumor immune responses, including immune cell generation, recruitment, migration, activation, and antigen presentation, are carried out outside the tumor environment. These steps are regulated to a significant extent by pattern recognition receptors (PRRs), which recognize a wide variety of endogenous and exogenous danger-, pathogen-, and xenobiotic-associated molecular patterns. Toll-like receptors (TLRs) are the most prominent PRR family and comprise nine functional TLRs in mammals, subsets of which are expressed on nearly 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 (TLRs) can be released by dying normal or malignant human cells, most naturally occurring TLRs are present on bacteria, viruses, and other microorganisms, where they alert the immune system to the presence of pathogens and activate appropriate defensive responses. Activation of TLR signaling leads to the direct activation of immune cell function and indirect activation through the induction of cytokine and chemokine secretion, which act via both autocrine and paracrine mechanisms.

[0006] The role that TLRs play in host-mediated anti-pathogen and anti-tumor immune responses has led to extensive efforts to engineer TLR agonist adjuvants and therapeutics for infectious disease and anti-tumor immunotherapy. A variety of monospecific purified or synthetic TLR agonists have been engineered and tested in preclinical and clinical settings. TLR agonists have been used as adjuvants in prophylactic vaccines. However, despite observed anti-pathogen and anti-tumor activity in therapeutic vaccine settings, these efforts have faced significant challenges. Issues encountered include both lack of efficacy and excessive toxicity, suggesting the need for further refinement of existing TLR agonist approaches to infectious disease and cancer prevention and treatment.

[0007] LPS endotoxin constitutes 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 thought to be a major contributor to both the antitumor activity and intravenous toxicity of Gram-negative bacteria. Summary of the Invention

[0008] This disclosure demonstrates that attenuated, killed, intact, and stabilized bacteria made from non-pathogenic Gram-negative bacterial cells, such as E. coli, exhibit potent effects in inhibiting tumor growth and viral replication and activity. At the same time, these treated bacterial cells are well tolerated in in vivo studies, suggesting that they may have a high therapeutic index suitable for clinical development and use.

[0009] Accordingly, one embodiment of the present disclosure is a method for treating or preventing cancer or an infectious disease in a patient in need thereof, comprising administering to a subject in need thereof 1×10 Escherichia coli cells treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% compared to untreated wild-type Escherichia coli cells, as measured by a Limulus Amebocyte Lysate (LAL) assay. 7 ~500×107 The present invention provides a method for treating a bacterial infection comprising administering to a patient an effective amount of a composition comprising intact, stabilized, and substantially nonviable E. coli cells, 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 a subject in need thereof 1×10 Escherichia coli cells treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% compared to untreated wild-type Escherichia coli cells, as measured by a Limulus Amebocyte Lysate (LAL) assay. 7 ~500×10 7 In one embodiment, a method is provided for administering to a patient an effective amount of a composition comprising intact and substantially non-viable E. coli cells, wherein the composition contains 124 to 62,000 endotoxin units (EU) of LPS.

[0011] In some embodiments, the composition contains 2×10 7 ~200×10 7 In some embodiments, the composition comprises 3 x 10 intact and substantially non-viable E. coli cells. 7 ~100×10 7 In some embodiments, the composition comprises 5 x 10 intact and substantially non-viable E. coli cells. 7 ~50×10 7 In some embodiments, the composition comprises 3 x 10 intact and substantially non-viable E. coli cells. 7 , 7×10 7 , 10×10 7 , 20×10 7 , or 70×10 7 The present invention comprises intact and substantially non-viable E. coli cells.

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

[0013] In some embodiments, the intact and substantially non-viable E. coli cells have been treated to result in about an 85% to 98% reduction in LPS-associated endotoxins, hi some embodiments, the intact and substantially non-viable E. coli cells have been treated to result in about a 90% to 98% reduction in LPS-associated endotoxins.

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

[0015] In some embodiments, the treatment of E. coli cells is with polymyxin, preferably polymyxin B or polymyxin E. In some embodiments, the treatment of E. coli cells is carried out at a temperature of about 2°C to about 10°C, preferably about 4°C. In some embodiments, the treatment of E. coli cells is with polymyxin and glutaraldehyde. In some embodiments, the treatment is with 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 comprises phosphate buffer, Mg 2+ In some embodiments, the composition further comprises 0.3 x 10 9 / mL ~ 5 × 10 91 mL of intact and substantially nonviable 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 monopotassium 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 of 7.0 to 7.7.

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

[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 the group consisting of bladder cancer, gastrointestinal cancer (esophageal, stomach, liver, colon, pancreas), 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 the group consisting of cyclophosphamide, IL-2, a non-steroidal anti-inflammatory drug (NSAID), an anti-PD-1 or anti-PD-L1 antibody, an anti-CTLA-4 antibody, and an anti-CD20 antibody.

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

[0021] Also in one embodiment, a method for providing a therapeutically acceptable composition is provided, comprising administering to at least 1×10 Escherichia coli cells that have been treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% compared to untreated wild-type Escherichia coli cells, as measured by a Limulus Amebocyte Lysate (LAL) assay. 6 to prepare a lyophilized composition; and storing the lyophilized composition (a) at a temperature between 1°C and 10°C for at least two months or (b) at a temperature below -15°C for at least two years, thereby providing a therapeutically acceptable composition suitable for therapeutic use.

[0022] In some embodiments, the solution contains phosphate buffer, Mg 2+ In some embodiments, the solution further comprises 0.3 x 10 9 / mL ~ 5 × 10 9 1 mL of intact and substantially nonviable 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 monopotassium 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 of 7.3 to 7.7.

[0023] Also provided in one embodiment is 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 nonviable E. coli cells that have been treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% compared to untreated wild-type E. coli cells, as measured by a 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 antigen or a bacterial antigen. In some embodiments, the composition comprises 1×10 7 ~500×10 7 The sample contains 124 to 62,000 endotoxin units (EU) of LPS. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows that the manufacturing process of Decoy bacteria stabilizes bacterial cells. [Figure 2] FIG. 1 shows that Decoy bacteria induced human PBMCs to secrete approximately 3,500-fold higher levels of the same cytokines compared to mouse PBMCs, based on a 48-hour in vitro (full) dose-response performed in triplicate. [Figure 3] FIG. 1 shows that Decoy bacteria cooperated with human PBMCs to kill human MDA-MB-231 breast cancer cells in vitro. [Figure 4] FIG. 1 shows that single-agent Decoy inhibited metastasis of orthotopic murine CT26 colon cancer and prolonged mouse survival. [Figure 5]FIG. 1 shows that single-agent Decoy bacteria dose-dependently inhibited the in vivo growth of mouse colon cancer cells without significant toxicity. [Figure 6] FIG. 1 shows that Decoy cooperated with low dose cyclophosphamide (LDC) to suppress the growth of subcutaneous CT26 colon tumors. [Figure 7] This figure shows that Decoy inhibited subcutaneous CT26 colon cancer in combination with low-dose interleukin-2 (IL-2) and / or low-dose indomethacin. Treatment was initiated in 5 mice per group on day 10, when tumors reached 75 mm3. [Figure 8] FIG. 1 shows that Decoy inhibited the growth of subcutaneous CT26 colon cancer cells in mice. [Figure 9] FIG. 1 shows that single-agent Decoy extended survival of mice bearing metastatic murine Pan02 pancreatic cancer. [Figure 10] FIG. 1 shows that Decoy had single-agent activity and cooperated with the oral low-dose NSAID indomethacin in a metastatic murine Pan02 pancreatic cancer model. [Figure 11] FIG. 1 shows that Decoy has single-agent activity and, in combination with an NSAID and anti-PD-1, caused regression of 10 / 12 194 mm subcutaneous H22 hepatocellular carcinoma (HCC) tumors in mice. [Figure 12] FIG. 1 shows that when cured mice were re-implanted with fresh HCC tumor cells, they rejected the tumor (immune memory). [Figure 13] FIG. 1 shows that Decoy, in combination with NSAIDs, caused regression of 4 / 6 of 183 mm 3 subcutaneous H22 hepatocellular carcinoma (HCC) tumors in mice. [Figure 14] FIG. 1 shows that Decoy (once weekly), an NSAID, and anti-PD-1 worked together to induce 100% regression of 183 mm 3 subcutaneous H22 hepatocellular carcinoma (HCC) tumors in mice. [Figure 15]FIG. 1 shows that Decoy, in combination with oral low-dose NSAID indomethacin, caused regression of established subcutaneous H22 hepatocellular carcinoma (HCC) in mice. [Figure 16] This figure shows that Decoy and NSAID, in combination with anti-PD-1, regressed 200 mm3 murine H22 hepatocellular carcinoma with a therapeutic index >33 (the Decoy group also received anti-PD-1 + oral NSAID). [Figure 17] FIG. 1 shows that when cured mice were re-implanted with fresh HCC tumor cells, they rejected the tumor (immune memory). [Figure 18] FIG. 1 shows NanoString gene expression analysis of tumor-derived RNA reveals a treatment-associated increase in HCC tumor inflammation scores (from cold to hot tumors). [Figure 19] FIG. 1 shows that Decoy bacteria, in combination with low-dose cyclophosphamide (LDC), caused 100% regression of established murine A20 non-Hodgkin's lymphoma (NHL) after only two weeks of treatment. [Figure 20] FIG. 1 shows that the cooperative regression of A20 non-Hodgkin's lymphoma (NHL) tumors by Decoy and low-dose cyclophosphamide (LDC) was durable and induced immunological memory. [Figure 21] This figure shows that A20 non-Hodgkin's lymphoma (NHL) tumors that regrew within 1 to 2 weeks after suboptimal Decoy and low-dose cyclophosphamide (LDC) treatment were sensitive to optimal retreatment, and that even very large tumors could regress with Decoy+LDC treatment. [Figure 22] This figure reproducibly shows that Decoy, in combination with low-dose cyclophosphamide (LDC), eradicated 200 mm3 subcutaneous A20 non-Hodgkin's lymphoma (NHL) in mice with immunological memory. [Figure 23]Figure 1 shows that CD4+ and CD8+ T cells (adaptive immunity) and NK cells (innate immunity) are required for high-efficiency eradication of subcutaneous A20 non-Hodgkin's lymphoma (NHL) by Decoy and low-dose cyclophosphamide (LDC), although depletion alone results in transient regression and partial eradication. [Figure 24] FIG. 1 shows that two different strains of Decoy bacteria exhibited similar antitumor activity against murine A20 non-Hodgkin's lymphoma (NHL). [Figure 25] FIG. 1 shows that Decoy and low-dose cyclophosphamide (LDC) synergized with rituximab to induce regression of subcutaneous human Ramos non-Hodgkin's lymphoma (NHL) in SCID mice. [Figure 26] FIG. 1 shows that Decoy and low-dose cyclophosphamide (LDC) can cooperate with rituximab to induce immunological memory via the innate immune system. [Figure 27] This figure shows that expression of exogenous antigens (e.g., HER2, lower panel) significantly enhanced the antitumor effect of single-agent Decoy (upper panel), and after single-agent Decoy treatment, 2 out of 5 mice achieved a complete response (lower panel). [Figure 28] FIG. 1 shows that Decoy suppressed human hepatitis B virus (HBV) replication in a mouse (AAV-HBV) chronic HBV infection model. [Figure 29] FIG. 1 shows that Decoy suppressed human hepatitis B virus (HBV) HBeAg levels in a mouse (AAV-HBV) chronic HBV infection model. [Figure 30] FIG. 1 shows that Decoy suppressed human hepatitis B virus (HBV) HBsAg levels in a mouse (AAV-HBV) chronic HBV infection model. [Figure 31] FIG. 1 shows that Decoy suppressed human hepatitis B virus (HBV) DNA expression in the liver of HBV-infected mice (AAV-HBV model). [Figure 32]FIG. 1 shows that Decoy suppressed human hepatitis B virus (HBV) HBeAg expression in the liver of HBV-infected mice (AAV-HBV model). [Figure 33] FIG. 1 shows that Decoy and ETV suppressed the expression of human hepatitis B virus (HBV) cccDNA-like molecules in the livers of HBV-infected mice (AAV-HBV model). [Figure 34] FIG. 1 shows that entecavir and Decoy, and their combination, suppressed human hepatitis B virus (HBV) replication in a mouse (AAV-HBV) chronic human HBV infection model. [Figure 35] FIG. 1 shows that Decoy suppressed human hepatitis B virus (HBV) HBeAg levels in a mouse chronic HBV infection model. [Figure 36] FIG. 1 shows that Decoy suppressed human hepatitis B virus (HBV) HBsAg levels in a mouse chronic HBV infection model. [Figure 37] FIG. 1 shows that Decoy reduced human HIV virus levels in a (humanized) mouse model of chronic human HIV infection. [Figure 38] Figure 1 shows that Decoy induced transient plasma cytokine, chemokine, and biomarker expression in human subjects. Reference ranges (healthy volunteers, pg / mL or units / mL) are indicated in the figure. Most baseline measurements reflect the lower limit of assay quantitation. [Figure 39] FIG. 1 shows that pharmacokinetic analysis confirmed that systemically administered Decoy was rapidly cleared in human subjects. DETAILED DESCRIPTION OF THE INVENTION

[0026] The following description illustrates example embodiments of the present technology, but it should be understood that the description is not intended to limit the scope of the present disclosure, but is provided solely as an illustration of example embodiments.

[0027] As used herein, the following words, phrases, and symbols are generally intended to have the meanings set forth below, unless the context in which they are used indicates a different meaning.

[0028] Therapeutic Use of Treated Bacteria The presently disclosed experimental examples demonstrate that attenuated, intact, stabilized, and nonviable Gram-negative bacterial cells (e.g., Escherichia coli) treated to significantly reduce LPS-associated endotoxin activity (Decoy bacteria or Decoy) contain agonists of all functional human TLR receptors and receptor heterodimers (TLR2, 2 / 1, 2 / 6, 3, 4, 5, 7, 8, and 9), as well as agonists of NOD-like receptors (NLRs) and stimulator of interferon (IFN) genes (STING) receptors. Surprisingly, Decoy bacteria exhibited reduced in vivo pyrogenicity and acute toxicity, while exhibiting increased ability to induce the secretion of numerous cytokines and chemokines from immune cells compared to untreated parental bacteria. Such engineered bacterial cells are therefore suitable for providing a safe and effective means for stimulating an immune response in a subject and are useful in the treatment of tumors and bacterial, fungal, parasitic or viral infections.

[0029] These engineered bacterial cells, also known as "Decoy bacteria" or simply "Decoy," are 100% killed, stabilized, and intact bacteria derived from nonpathogenic Gram-negative bacterial cells (e.g., Escherichia coli) after attenuation, with approximately 90% reduction in LPS endotoxin activity and pyrogenicity. A comprehensive nonclinical pharmacology program was developed to support Decoy's first-in-human (FIH) studies. Primary pharmacodynamic (PD) studies with Decoy included in vitro evaluation of cytokine and chemokine secretion induction by mouse and human peripheral blood mononuclear cells, as well as in vivo evaluation of IV antitumor activity against established subcutaneous (sc) mouse colon carcinoma, metastatic mouse pancreatic carcinoma, established subcutaneous mouse hepatocellular carcinoma (HCC), and established subcutaneous mouse and human non-Hodgkin's lymphoma (NHL) models. Decoy was also tested against established mouse mammary carcinoma tumors with and without expression of exogenous antigens. Decoy has been tested as a single agent and in combination with certain other therapeutic agents. Significant single-agent antitumor activity was observed in several models, including regression of established mouse breast cancer tumors expressing exogenous antigens. Synergistic effects were observed in combination with low-dose cyclophosphamide (LDC), indomethacin, rituximab, and anti-PD-1 checkpoint therapy, but not with agents such as anti-GITR antibody, INF-γ, phenformin, gemcitabine, or 5-FU. In addition, tumor-regressing treatment was associated with the induction of immunological memory, as demonstrated by the rejection of tumor reimplantation without additional treatment.

[0030] Similarly, in animal models, Decoy, either alone or in combination with other drugs such as entecavir (ETV), has shown potent activity in inhibiting the replication and activity of hepatitis B virus (HBV) and human immunodeficiency virus (HIV).

[0031] The safety profile of Decoy was determined in single-dose, 2-week repeated-dose ranging, and 4-week repeated-dose toxicity studies in New Zealand White (NZW) rabbits after 1-hour IV administration. NZW rabbits are the non-human laboratory animal species considered to be most similar to humans with respect to their sensitivity to the adverse effects of LPS. Studies in mice also provided additional safety information for Decoy.

[0032] The maximum tolerated dose (MTD) of Decoy administered once in rabbits was 1.5 × 10 9 When administered twice weekly for 2 weeks, Decoy was found to kill 6 × 10 killed bacteria (KB) / kg. Four dose levels were tested, and the no-observed-adverse-effect-level (NOAEL) was 6 × 10 7 In the pivotal 4-week repeated-dose toxicity study in rabbits (4 dose levels), the NOAEL for Decoy was 4 x 10 7 Decoy was also 97% less pyrogenic than the parent (untreated) bacteria in rabbits (rectal temperature test) and 3 times less toxic (acute LD ) than the parent (untreated) bacteria. 100 ) was found to be.

[0033] Based on these non-clinical findings, human clinical trials were designed and conducted to develop formulations suitable for clinical use. Example formulations are shown in Table 4. Example doses are shown in Table 5. The starting dose in Part 1 of the clinical trial was 7 x 10 7 KB / patient, which was studied in a 4-week rabbit toxicity study (4 × 10 7This is approximately 1 / 10 of the human equivalent dose (HED) calculated from the no observed adverse effect level (NOAEL) observed twice weekly at 1000 KB / kg. Based on the 4-week Good Laboratory Practice (GLP) study data, and taking into account a 3.1-fold allometric scaling factor (dose reduction) for the HED and a 10-fold dose reduction safety adjustment, the human starting dose is 1.29 × 10 6 KB / kg, or approximately 16 Decoy-associated endotoxin units (EU) / kg, which is 7.74 × 10 7 KB / 60 kg subject, which corresponds to approximately 960 EU / 60 kg subject (instead of the conventional 70 kg, taking into account underweight patients). The starting dose in the study was somewhat low, 7.0 × 10 7 The dose is KB, which corresponds to 868 EU / 60 kg subject or 1.8 ng / kg LPS. Based on published results of systemic clearance of live and killed bacteria in mice, rabbits, and humans, Decoy is expected to be rapidly cleared by the liver and spleen (within minutes to approximately 1 hour), and therefore weight-based dose adjustments are not expected. The starting dose in the study contains less LPS than the maximum dose (4 ng / kg) found to be well tolerated after IV administration of purified LPS to over 1,000 healthy human volunteers. Repeated weekly dosing will be conducted in the later stages of the clinical trial. Barring any safety concerns, subjects will receive continuous weekly administration of Decoy for up to two years.

[0034] As shown in the preliminary results of a Phase I clinical trial (Example 4), 7One-hour intravenous administration of killed Decoy bacteria resulted in disease stabilization in all four cancer patients, including three with tumor progression prior to treatment. Importantly, Decoy bacteria were cleared from the blood within 30–120 minutes after administration ended and caused the transient induction of more than 50 cytokines, chemokines, and biomarkers in the plasma. Many of these are known to be involved in the direct stimulation of innate and / or adaptive immune responses, particularly antitumor responses. The transient induction of cytokines and chemokines is an important and novel feature of the response to Decoy bacteria and contributes to reducing the potential for systemic toxicity known to result from sustained or prolonged systemic exposure to these potent immune-activating molecules.

[0035] Thus, in accordance with one embodiment of the present disclosure, there is provided a method for treating or preventing cancer in a patient in need thereof, the method involving administering to the patient an effective amount of a composition comprising treated bacteria.

[0036] In another embodiment, a method of stimulating an immune response in a subject in need is provided. In another embodiment, a method is provided for preventing or treating an infectious disease in a patient in need thereof. In another embodiment, a method is provided for treating an immunodeficiency in a patient in need thereof. In another embodiment, a method is provided for vaccinating a subject at risk for an infectious disease or cancer.

[0037] In some embodiments, the treated bacterial cells are intact, stabilized, and substantially nonviable 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 nonviable Gram-negative bacterial cells have been treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% compared to untreated wild-type Gram-negative bacteria, as measured by a Limulus Amebocyte Lysate (LAL) assay.

[0038] Candidate bacterial organisms for use in the methods described herein include those that are Gram-negative and have LPS-associated endotoxin activity as wild-type organisms. The term "Gram-negative bacteria" refers to bacteria that do not retain the initial basic dye (e.g., crystal violet) staining that is part of a procedure known as Gram staining. In a typical Gram stain, cells are first fixed to a slide by heating and stained with a basic dye (e.g., crystal violet). This dye is taken up by both Gram-negative and Gram-positive bacteria. The slide is then treated with a mordant (e.g., Gram's iodine), which binds and retains the basic dye (e.g., crystal violet) within the cells. The cells are then washed with acetone or alcohol before being counterstained with a second stain of a different color (e.g., safranin). Gram-positive organisms retain the initial violet stain, while Gram-negative organisms are decolorized by an organic solvent wash, revealing the counterstain. Representative Gram-negative bacteria include, but are not limited to, the genera Escherichia, Shigella, Salmonella, Campylobacter, Neisseria, Haemophilus, Aeromonas, Francisella, Yersinia, Klebsiella, Bordetella, Legionella, Corynebacteria, Citrobacter, Chlamydia, Brucella, Pseudomonas, Helicobacter, and Vibrio.

[0039] Among Gram-negative organisms, there is a large family, the Enterobacteriaceae, which includes many harmless commensals as well as well-known pathogens such as Salmonella, E. coli, Yersinia pestis, Klebsiella, Shigella, Proteus, Enterobacter, Serratia, and Citrobacter. Members of the Enterobacteriaceae family are sometimes called enterobacteria, and some members inhabit the intestines of animals.

[0040] In one embodiment, Escherichia coli (E. coli) is the organism of choice. Particularly contemplated strains include E. coli strain 2617-143-312, (Migula) Castellani and Chalmers (ATCC® 13070™). Additional usable E. coli strains include MG1655 (ATCC® 47076).

[0041] The term "lipopolysaccharide (LPS)" refers to a large molecule composed of covalently bonded lipids and polysaccharides (glycophospholipids). LPS contains 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 comprises the outermost domain of the LPS molecule. The core oligosaccharide is directly attached to lipid A and typically contains sugars such as heptose and 3-deoxy-D-mannooctulosonic acid (KDO, also known as keto-deoxyoctulosonate). Lipid A is a phosphorylated glucosamine disaccharide with multiple fatty acids attached. The fatty acids anchor LPS to the bacterial outer membrane, while the remainder of LPS protrudes from the cell surface.

[0042] Endotoxin activity resides in the lipid A domain of LPS and is also referred to as "LPS-associated endotoxin activity" or "LPS endotoxin activity." Gram-negative bacteria also contain additional TLRs, including agonists for TLR2 / 1, 2 / 6, 2, 3, 5, 7, 8, and 9, as well as other immune stimulatory molecules such as stimulator of interferon genes (STING) and nucleotide-binding oligomerization domain-containing protein (NOD) agonists. Intact bacteria that enter the circulation are rapidly taken up by immune cells in the liver and spleen, leading to direct activation of immune cells / pathways and indirect activation of immune cells / pathways by inducing cytokine and chemokine secretion. Rapid clearance of circulating bacterial cells by immune cells in the liver and spleen helps localize immune activation to key immune organs. Surviving and growing bacteria release or shed large amounts of LPS endotoxin. When live bacteria grow and invade normal tissues and cells, and / or are degraded in the circulation, they can release large amounts of immune activators throughout the body. This can lead to an inappropriate and excessive inflammatory response throughout the body, potentially resulting in fatal shock (known as endotoxic shock or septic shock). Therefore, the invention and use of killed, stabilized bacteria with reduced LPS endotoxin activity can enable short-term or transient immune activation in the liver and spleen after systemic administration, significantly reducing the potential for inappropriate systemic inflammation caused by live bacteria that are capable of invading and growing in normal cells and tissues, growing and degrading in the systemic circulation, and shedding and releasing various immune activators throughout the body. The most potent bacterial-associated immune stimulator, LPS endotoxin, can contribute to both antitumor and antiviral effects and systemic toxicity. LPS-associated endotoxin activity can be measured by methods known to those skilled in the art, such as the Limulus Amebocyte Lysate (LAL) assay. The LAL assay utilizes horseshoe crab blood and is capable of detecting even extremely low concentrations of LPS. The presence of endotoxin activity causes clotting of the Limulus hemolysate via amplification by an enzymatic cascade.Gel-clot, turbidimetric, and colorimetric LAL assays are commercially available.

[0043] Enzyme-linked immunoadsorbent assay (ELISA)-based endotoxin activity assays are also known, such as EndoLISA® from Hyglos, near Munich, Germany. This assay captures LPS using an LPS-specific phage protein bound to a solid phase, and after washing, detects the presence of LPS by adding recombinant Factor C. When activated by LPS, the recombinant Factor C cleaves a fluorescent compound. Factor C is present in LAL, usually as a dimogen, and is the initiator of the coagulation cascade that occurs in the LAL test.

[0044] Pyrogenicity refers to the ability of an agent to induce fever in a subject and can be measured as the increase in rectal temperature in rabbits after intravenous administration of a TLR agonist, microorganism, or derivative thereof.

[0045] Various methods are available for reducing the endotoxin activity and / or pyrogenicity of Gram-negative organisms, including treatment of the organisms with agents that bind to or inhibit the formation of LPS.

[0046] In one embodiment, reduction of endotoxin activity or pyrogenicity is achieved by treating the bacterial organism with an antibiotic that inactivates endotoxins. Suitable antibiotics include polymyxins, including polymyxin B or polymyxin E. One skilled in the art can determine the amount of antibiotic and treatment conditions. In one embodiment, the polymyxin, either polymyxin B or E, may be used at a concentration of about 3 micrograms to 5,000 micrograms per milliliter. In another embodiment, the concentration of the polymyxin may be about 200 micrograms to 5,000 micrograms per milliliter. In one embodiment, the antibiotic is applied to the bacteria for 10 minutes to 4 hours, or about 30 minutes to about 3 hours.

[0047] In one embodiment, the bacteria are grown in the presence of magnesium (Mg) in the form of MgCl. In one embodiment, the bacteria are treated with polymyxin in the presence of MgCl, at a temperature suitable to maintain bacterial integrity. In one embodiment, the MgCl concentration in the growth medium is about 0.5 mM to about 5.0 mM, or about 2 mM, and the MgCl concentration 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 recovery efficiency in a clearly defined pellet after centrifugation at 3,000 x g for 10 minutes, and by electron or light microscopy with Gram staining. In a preferred embodiment, bacterial recovery after treatment and washing is greater than about 80%, and the bacteria appear intact by light or electron microscopy.

[0048] In another embodiment, KDO2-Lipid IV A Reduction of endotoxin activity can be achieved by treating bacterial organisms with antibiotics known to inhibit the biosynthesis of lipid IV. For example, Goldman et al. (J. Bacteriol. 170(5):2185-91, 1988) described antibacterial agents, including Antibacterial Agent III, that specifically inhibit CTP:CMP-3-deoxy-D-manno-octurosonate cytidylyltransferase activity and are useful for inhibiting the incorporation of 3-deoxy-D-manno-octurosonate (KDO) into the LPS of Gram-negative organisms. When LPS synthesis was halted, bacterial growth also ceased. The LPS precursor species Lipid IV A The addition of KDO to ATP is the primary pathway for lipid A-KDO formation in both S. typhimurium and E. coli. In one embodiment, the antibiotic is antimicrobial agent III, and the Gram-negative bacteria are treated with a suitable amount, e.g., 5 micrograms / milliliter to 500 micrograms / milliliter, for a suitable time, e.g., 2 to 8 hours.

[0049] Similarly, α-C-(1,5-anhydro-7-amino-2,7-dideoxy-D-manno-heptopyranosyl)-carboxylate is known to inhibit 3-deoxy-D-manno-octurosonate cytidylyltransferase (CMP-KDO synthetase), a cytoplasmic enzyme that activates 3-deoxy-D-manno-octurosonate (KDO) for incorporation into LPS (Nature. 1987 10-16;329(6135):162-4). Therefore, treating organisms with this compound can also reduce LPS-associated endotoxin activity.

[0050] In another embodiment, reduction of endotoxin activity is achieved by treating the organism with an LPS inhibitor. For example, the bacterial cyclic lipopeptide surfactin has been shown to bind to and inhibit the activity of lipid A (J Antibiot 2006 59(1):35-43).

[0051] In addition to LPS-related endotoxins, various other components of Gram-negative organisms can induce or contribute to fever and septic shock, including outer membrane proteins, fimbriae, pilus, lipopeptides, and lipoproteins (see Jones, M., Int. J. Pharm. Compd., 5(4):259-263, 2001). Pyrogenicity can be measured by rabbit methods known to those skilled in the art, which involve assessment of rectal temperature after intravenous administration of a putative pyrogen.

[0052] Treatment of Gram-negative organisms with a combination of polymyxin B and glutaraldehyde has been found to reduce pyrogenicity by 30-fold as measured in rabbits. In one embodiment, 1,000 micrograms per milliliter (μg / mL) of polymyxin B and 1% glutaraldehyde were used to reduce pyrogenicity by 30-fold as measured in rabbits. Pyrogenicity is reduced by a combination of the reaction of polymyxin B with LPS and the reaction of glutaraldehyde with LPS and other bacterial components. Glutaraldehyde's bifunctional chemical cross-linking activity also contributes to bacterial cell killing and stabilization, fulfilling a triple role in this context: reducing pyrogenicity, killing cells, and stabilizing cells.

[0053] Thus, in one embodiment, a method is provided for reducing endotoxin activity and pyrogenicity of Gram-negative bacterial microorganisms, and killing and stabilizing the Gram-negative bacterial microorganisms by treating the bacteria with a combination of 1,000 μg / mL polymyxin B and 1% glutaraldehyde. In another embodiment, Gram-negative bacteria are treated with a combination of polymyxin B in a dosage range of about 3 μg / mL to about 1,000 μg / mL and glutaraldehyde in a dosage range of about 0.1% to about 1.0%. In a further embodiment, the dosage range of polymyxin B is about 100 μg / mL to about 1,000 μg / mL, and the dosage range of glutaraldehyde is about 0.25% to about 1.0%. Additionally, Gram-negative bacteria may be treated with, for example, a dosage range of polymyxin B of about 1,000 μg / mL to about 3,000 μg / mL and a dosage range of glutaraldehyde of about 0.25% to about 1.0%. In another embodiment, gram-negative bacteria may be treated with, for example, polymyxin B in a dosage range of about 3,000 μg / mL to about 5,000 μg / mL and glutaraldehyde in a dosage range of about 0.25% to about 2.0%.

[0054] In some embodiments, the intact and substantially non-viable Gram-negative bacterial cells have at least about a 70% reduction in LPS-associated endotoxin activity compared to untreated wild-type bacteria (e.g., as measured by an LAL assay). 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 more than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction rate is, but is not limited to, about 70% to about 99.99%, about 80% to about 99.99%, 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%.

[0055] In some embodiments, a particular level of residual active LPS is preferred. For example, in some embodiments, 1×10 8 There is about 1-200 ng of active LPS per 1 x 10 cells. 8 There is approximately 2-200 ng, approximately 5-150 ng, approximately 5-120 ng, approximately 10-120 ng, approximately 20-100 ng, approximately 20-50 ng, and approximately 10-50 ng of active LPS per cell.

[0056] In some embodiments, the intact and substantially non-viable Gram-negative bacterial cells have at least about a 70% reduction in pyrogenicity compared to untreated wild-type bacteria (e.g., as measured in an in vivo rabbit assay). 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 does not exceed about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction rate is, but is not limited to, about 70% to about 99.99%, about 80% to about 99.99%, 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%.

[0057] As noted above, in addition to LPS-related endotoxins, various other components of Gram-negative organisms can induce or contribute to pyrogenicity, such as outer membrane proteins, fimbriae, pilus, lipopeptides, lipoproteins, etc. In some embodiments, intact and substantially non-viable Gram-negative bacterial cells are treated to achieve reduced pyrogenicity by both reducing LPS-related endotoxin activity and reducing non-LPS-related pyrogenicity, such as by inactivating, removing, or blocking outer membrane proteins, fimbriae, pilus, lipopeptides, or lipoproteins. In some embodiments, the reduction in non-LPS-related fever 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 does not exceed 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%.

[0058] Bacteria administered according to the disclosed methods are nonviable or substantially nonviable prior to administration, or are nonviable or substantially nonviable upon administration. "Nonviable" means that the organism is killed by treatment with an exogenous agent and / or that the organism possesses a mutation that prevents it from surviving in a mammalian host. Substantially nonviable bacteria are strains whose viability is reduced by at least 80%, 85%, 90%, 95%, 99% or more.

[0059] Bacteria can be rendered nonviable by treatment with compounds such as polymyxin. Polymyxin binds to LPS, disrupting membrane integrity during bacterial division and reducing viability by increasing the permeability of the cell envelope. If viability is reduced in this way, additional steps are required to prevent cell lysis and keep the cells intact. Another approach involves growing a bacterial strain with a conditional mutation in the LPS biosynthetic pathway, suppressing the mutation during growth, and then shifting to nonpermissive conditions to activate the mutation and inhibit LPS biosynthesis. In either case, the procedure for rendering bacteria nonviable is applied by determining the optimal treatment time or compound dose for each situation, such that bacterial cell integrity is sufficiently maintained while viability is substantially lost. If the non-viability rate is less than 100%, bacteria can be used that have a mutation that prevents further propagation of surviving bacteria in the mammalian host (e.g., a diaminopimelic acid auxotroph as described in Bukhari and Taylor, J. Bacteriol. 105(3):844-854, 1971 and Curtiss et al., Immunol. Invest. 18(1-4):583-596, 1989).

[0060] Dosage and administration schedule For therapeutic and prophylactic uses, appropriate effective amounts (doses) and administration schedules are also determined. In this context, a preferred Gram-negative bacterial species is Escherichia coli. In some embodiments, an effective amount of treated E. coli cells is 1×10 7 ~500×10 7 Intact and substantially non-viable E. coli cells of

[0061] As shown in the accompanying examples, Decoy products are highly effective 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, nonsteroidal anti-inflammatory drugs (NSAIDs, e.g., indomethacin), anti-PD-1 or anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and anti-CD20 antibodies (e.g., rituximab). Thus, an effective dose is 1×10 7treated cells, or alternatively 2 x 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 7×10 7 , 7.74 x 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 x 10 7 The results may be equivalent to (or greater than) the results of the treated cells.

[0062] As shown, Decoy products are sufficiently safe to allow for clinical use at high doses. Therefore, the effective dose is 500 × 10 7 treated cells, or alternatively 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 7×10 7 , 7.74 x 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 , 250×107 , 300×10 7 , or 400 x 10 7 may be equivalent to (or less than) the treated cells.

[0063] In some embodiments, the effective amount is 1×10 7 ~500×10 7 In some embodiments, the effective amount is 1 x 10 treated cells. 7 ~400×10 7 , 1×10 7 ~300×10 7 , 1×10 7 ~200×10 7 , 1×10 7 ~150×10 7 , 1×10 7 ~100×10 7 , 1×10 7 ~70×10 7 , 1×10 7 ~50×10 7 , 1×10 7 ~20×10 7 , or 1 × 10 7 ~10×10 7 In some embodiments, the effective amount is 3 x 10 treated cells. 7 ~400×10 7 , 3×10 7 ~300×10 7 , 3×10 7 ~200×10 7 , 3×10 7 ~150×10 7 , 3×10 7 ~100×10 7 , 3×10 7 ~70×10 7 , 3×10 7 ~50×10 7 , 3×10 7 ~20×10 7 , or 3 × 10 7 ~10×10 7 In some embodiments, the effective amount is 7 x 10 treated cells. 7 ~400×10 7 , 7×10 7~300×10 7 , 7×10 7 ~200×10 7 , 7×10 7 ~150×10 7 , 7×10 7 ~100×10 7 , 7×10 7 ~70×10 7 , 7×10 7 ~50×10 7 , 7×10 7 ~20×10 7 , or 7 × 10 7 ~10×10 7 In some embodiments, the effective amount is 7.74 x 10 treated cells. 7 ~400×10 7 , 7.74 x 10 7 ~300×10 7 , 7.74 x 10 7 ~200×10 7 , 7.74 x 10 7 ~150×10 7 , 7.74 x 10 7 ~100×10 7 , 7.74 x 10 7 ~70×10 7 , 7.74 x 10 7 ~50×10 7 , 7.74 x 10 7 ~20×10 7 , or 7.74 × 10 7 ~10×10 7 These are treated cells.

[0064] In some embodiments, the effective amount is 10×10 7 ~400×10 7 , 10×10 7 ~300×10 7 , 10×10 7 ~200×10 7 , 10×10 7 ~150×10 7 , 10×10 7 ~100×10 7 , 10×10 7 ~70×10 7 , 10×10 7~50×10 7 , or 10×10 7 ~20×10 7 In some embodiments, the effective amount is 15×10 7 ~400×10 7 , 15×10 7 ~300×10 7 , 15×10 7 ~200×10 7 , 15×10 7 ~150×10 7 , 15×10 7 ~100×10 7 , 15×10 7 ~70×10 7 , 15×10 7 ~50×10 7 , or 15 × 10 7 ~20×10 7 In some embodiments, the effective amount is 20×10 7 ~400×10 7 , 20×10 7 ~300×10 7 , 20×10 7 ~200×10 7 , 20×10 7 ~150×10 7 , 20×10 7 ~100×10 7 , 20×10 7 ~70×10 7 , or 20 × 10 7 ~50×10 7 These are treated cells.

[0065] In some embodiments, the effective amount is 30×10 7 ~400×10 7 , 30×10 7 ~300×10 7 , 30×10 7 ~200×10 7 , 30×10 7 ~150×10 7 , 30×10 7 ~100×10 7 , 30×10 7 ~70×10 7, or 30 × 10 7 ~50×10 7 In some embodiments, the effective amount is 40×10 7 ~400×10 7 , 40×10 7 ~300×10 7 , 40×10 7 ~200×10 7 , 40×10 7 ~150×10 7 , 40×10 7 ~100×10 7 , 40×10 7 ~70×10 7 , or 40×10 7 ~50×10 7 In some embodiments, the effective amount is 50×10 7 ~400×10 7 , 50×10 7 ~300×10 7 , 50×10 7 ~200×10 7 , 50×10 7 ~150×10 7 , 50×10 7 ~100×10 7 , or 50 × 10 7 ~70×10 7 In some embodiments, the effective amount is 70×10 7 ~400×10 7 , 70×10 7 ~300×10 7 , 70×10 7 ~200×10 7 , 70×10 7 ~150×10 7 , or 70×10 7 ~100×10 7 In some embodiments, the effective amount is 100×10 7 ~400×10 7 , 100×10 7 ~300×10 7 , 100×10 7 ~200×10 7 , or 100 x 107 ~150×10 7 These are treated cells.

[0066] In some embodiments, the effective amount is about 1 (or 0.5-1.5) x 10 7 , 2 (or 1.5 to 2.5) × 10 7 , 3 (or 2 to 4) x 10 7 , 4 (or 3 to 5) x 10 7 , 5 (or 4 to 6) x 10 7 , 7 (or 6 to 8) x 10 7 , 7.74 x 10 7 , 10 (or 8 to 12) x 10 7 , 15 (or 13 to 17) x 10 7 , 20 (or 15-25) x 10 7 , 30 (or 25-35) x 10 7 , 40 (or 30-50) x 10 7 , 50 (or 40-60) x 10 7 , 60 (or 50-70) x 10 7 , 70 (or 60-80) x 10 7 , 80 (or 70-90) x 10 7 , 90 (or 80-100) x 10 7 , 100 (or 80-120) x 10 7 , 150 (or 130-170) x 10 7 , 200 (or 150-250) x 10 7 , 250 (or 200-300) x 10 7 , 300 (or 200-400) x 10 7 , or 400 (or 300-500) x 10 7 These are treated cells.

[0067] In some embodiments, the effective amount is at least 0.02×10 per kilogram (kg) of patient body weight. 7 In some embodiments, the effective amount is at least 0.05 x 10 treated cells per kilogram (kg) of patient body weight. 7 , 0.12×10 7 , 0.13×10 7 , 0.17×107 , 0.33×10 7 , 0.83×10 7 , 1.17×10 7 , 1.67×10 7 , 2.50 x 10 7 , 3.33 × 10 7 , 5.00×10 7 , or 6.67 × 10 7 In some embodiments, the effective amount is at most 0.05 x 10 treated cells per kilogram (kg) of patient body weight. 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 x 10 7 , 3.33 × 10 7 , 5.00×10 7 , 6.67×10 7 , or 8.33 × 10 7 These are treated cells.

[0068] In some embodiments, an effective amount of treated cells ccc comprises a predetermined amount of active LPS, measured in endotoxin units (EU). In some embodiments, an effective amount comprises 124 to 62,000 endotoxin units (EU) of LPS. In some embodiments, an effective amount comprises at least 124, 372, 868, 960, 1240, 2480, 6200, 8680, 12,400, 18,600, 24,800, 37,200, or 49,600 EU of LPS. In some embodiments, an effective amount comprises at most 62,000 endotoxin units (EU) of LPS. In some embodiments, an effective amount comprises at most 372, 868, 960, 1240, 2480, 6200, 8680, 12,400, 18,600, 24,800, 37,200, or 49,600 EU of LPS.

[0069] In some embodiments, an effective amount includes 124-62,000 endotoxin units (EU) of LPS, hi some embodiments, an effective amount includes 372-62,000 EU of LPS, or alternatively 868-62,000 EU, 960-62,000 EU, 1,240-62,000 EU, 2,480-62,000 EU, 6,200-62,000 EU, 8,680-62,000 EU, 12,400-62,000 EU, 18,600-62,000 EU, 24,800-62,000 EU, 37,200-62,000 EU, or 49,600-62,000 EU of LPS. In some embodiments, the effective amount includes 372-49600 EU, 868-49600 EU, 960-49600 EU, 1240-49600 EU, 2480-49600 EU, 6200-49600 EU, 8680-49600 EU, 12400-49600 EU, 18600-49600 EU, 24800-49600 EU, or 37200-49600 EU of LPS. In some embodiments, the effective amount includes 372-37200 EU, 868-37200 EU, 960-37200 EU, 1240-37200 EU, 2480-37200 EU, 6200-37200 EU, 8680-37200 EU, 12400-37200 EU, 18600-37200 EU, or 24800-37200 EU of LPS.

[0070] In some embodiments, an effective amount includes 372-24,800 EU, 868-24,800 EU, 960-24,800 EU, 1,240-24,800 EU, 2,480-24,800 EU, 6,200-24,800 EU, 8,680-24,800 EU, 12,400-24,800 EU, or 18,600-24,800 EU of LPS. In some embodiments, an effective amount includes 372-18,600 EU, 868-18,600 EU, 960-18,600 EU, 1,240-18,600 EU, 2,480-18,600 EU, 6,200-18,600 EU, 8,680-18,600 EU, or 12,400-18,600 EU of LPS. In some embodiments, an effective amount includes 372-12400 EU, 868-12400 EU, 960-12400 EU, 1240-12400 EU, 2480-12400 EU, 6200-12400 EU, or 8680-12400 EU of LPS. In some embodiments, an effective amount includes 372-8680 EU, 868-8680 EU, 960-8680 EU, 1240-8680 EU, 2480-8680 EU, or 6200-8680 EU of LPS. In some embodiments, an effective amount includes 372-6200 EU, 868-6200 EU, 960-6200 EU, 1240-6200 EU, or 2480-6200 EU of LPS. In some embodiments, an effective amount includes 372-6200 EU, 868-2480 EU, 960-2480 EU, or 1240-2480 EU of LPS. In some embodiments, an effective amount includes 372-1240 EU, 868-1240 EU, or 960-1240 EU of LPS. In some embodiments, an effective amount includes 372-960 EU or 868-960 EU of LPS. In some embodiments, an effective amount includes 372-868 EU.

[0071] In some embodiments, an effective amount includes at least 2.07 endotoxin units (EU) of LPS per kilogram (kg) of patient body weight, hi some embodiments, an effective amount includes 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 per kilogram (kg) of patient body weight. In some embodiments, the effective amount includes at most 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 per kilogram (kg) of patient body weight.

[0072] In some embodiments, an effective amount of treated cells comprises a predetermined amount of active LPS, measurable by the amount of active LPS. In some embodiments, an effective amount comprises between 15 ng and 7714 ng of active LPS. In some embodiments, an 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, an effective amount comprises at most 46, 108, 119, 154, 309, 771, 1080, 1543, 2314, 3086, 4629, 6171, or 7714 ng of active LPS.

[0073] The term "active LPS" refers to LPS capable of expressing LPS-associated endotoxin activity in a composition, as measured, for example, by the LAL assay. Based on standard LPS preparations, 5-9 endotoxin units (EU) are considered to correspond to 1 ng of active LPS. The amount of active LPS in a composition can be expressed as the weight of uninhibited LPS capable of expressing the same LPS-associated endotoxin activity as the composition.

[0074] In some embodiments, an effective amount includes between 15 ng and 7714 ng of active LPS, hi some embodiments, an effective amount includes between 46 ng and 7714 ng, 108 ng and 7714 ng, 119 ng and 7714 ng, 154 ng and 7714 ng, 309 ng and 7714 ng, 771 ng and 7714 ng, 1080 ng and 7714 ng, 1543 ng and 7714 ng, 2314 ng and 7714 ng, 3086 ng and 7714 ng, 4629 ng and 7714 ng, or 6171 ng and 7714 ng of active LPS. In some embodiments, an effective amount includes 15 ng to 6171 ng, 46 ng to 6171 ng, 108 ng to 6171 ng, 119 ng to 6171 ng, 154 ng to 6171 ng, 309 ng to 6171 ng, 771 ng to 6171 ng, 1080 ng to 6171 ng, 1543 ng to 6171 ng, 2314 ng to 6171 ng, 3086 ng to 6171 ng, or 4629 ng to 6171 ng of active LPS. In some embodiments, the effective amount includes 15 ng to 4629 ng, 46 ng to 4629 ng, 108 ng to 4629 ng, 119 ng to 4629 ng, 154 ng to 4629 ng, 309 ng to 4629 ng, 771 ng to 4629 ng, 1080 ng to 4629 ng, 1543 ng to 4629 ng, 2314 ng to 4629 ng, or 3086 ng to 4629 ng of active LPS.

[0075] In some embodiments, an effective amount includes 15 ng to 3086 ng, 46 ng to 3086 ng, 108 ng to 3086 ng, 119 ng to 3086 ng, 154 ng to 3086 ng, 309 ng to 3086 ng, 771 ng to 3086 ng, 1080 ng to 3086 ng, 1543 ng to 3086 ng, or 2314 ng to 3086 ng of active LPS. In some embodiments, an effective amount includes 15 ng to 2314 ng, 46 ng to 2314 ng, 108 ng to 2314 ng, 119 ng to 2314 ng, 154 ng to 2314 ng, 309 ng to 2314 ng, 771 ng to 2314 ng, 1080 ng to 2314 ng, or 1543 ng to 2314 ng of active LPS. In some embodiments, the effective amount includes 15 ng to 1543 ng, 46 ng to 1543 ng, 108 ng to 1543 ng, 119 ng to 1543 ng, 154 ng to 1543 ng, 309 ng to 1543 ng, 771 ng to 1543 ng, or 1080 ng to 1543 ng of active LPS.

[0076] In some embodiments, an effective amount includes 15 ng to 1080 ng, 46 ng to 1080 ng, 108 ng to 1080 ng, 119 ng to 1080 ng, 154 ng to 1080 ng, 309 ng to 1080 ng, or 771 ng to 1080 ng of active LPS. In some embodiments, an effective amount includes 15 ng to 771 ng, 46 ng to 771 ng, 108 ng to 771 ng, 119 ng to 771 ng, 154 ng to 771 ng, or 309 ng to 771 ng of active LPS. In some embodiments, an effective amount includes 15 ng to 309 ng, 46 ng to 309 ng, 108 ng to 309 ng, 119 ng to 309 ng, or 154 ng to 309 ng of active LPS. In some embodiments, an effective amount includes 15 ng to 154 ng, 46 ng to 154 ng, 108 ng to 154 ng, or 119 ng to 154 ng of active LPS. In some embodiments, an effective amount includes 15 ng to 119 ng, 46 ng to 119 ng, or 108 ng to 119 ng of active LPS. In some embodiments, an effective amount includes 15 ng to 46 ng of active LPS.

[0077] In some embodiments, an effective amount includes at least 0.26 ng of active LPS per kilogram (kg) of patient body weight. In some embodiments, an effective amount includes 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 per kilogram (kg) of patient body weight. In some embodiments, an effective amount includes at most 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 per kilogram (kg) of patient body weight.

[0078] In some embodiments, the treated cells are administered once, daily, two consecutive days per week, three consecutive days per week, four consecutive days per week, five consecutive days per week, six consecutive days per week, or alternatively daily, every other day, every three days, every five days, weekly, every two weeks, monthly, every two months, every three months, every four months, every six months, or yearly. In preferred embodiments, administration is weekly.

[0079] Diseases and Conditions The intact and substantially non-viable Gram-negative bacterial cells disclosed herein are useful in the treatment or prevention of various cancers, as well as infectious diseases and conditions.

[0080] "Treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibiting a disease or condition (e.g., reducing one or more symptoms resulting from a disease or condition and / or diminishing the extent of the disease or condition); b) slowing or halting the progression of one or more clinical symptoms associated with a disease or condition (e.g., stabilizing the disease or condition, preventing or slowing the worsening or progression of the disease or condition, and / or preventing or slowing the spread (e.g., metastasis) of the disease or condition); and / or c) palliating the disease, i.e., causing regression of clinical symptoms (e.g., improving the disease state, providing partial or complete remission of the disease or condition, enhancing the effect of another drug, slowing disease progression, improving quality of life, and / or prolonging survival).

[0081] "Prevention" or "preventing" refers to any treatment of a disease or condition that keeps the clinical symptoms of the disease or condition from developing. The bacterial cells, in some embodiments, may be administered to subjects (including humans) at risk for or who have a family history of the disease or condition.

[0082] "Subject" refers to an animal, e.g., a mammal (including a human), that has been or will be the object of treatment, observation, or experiment. The methods described herein may be useful 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.

[0083] In some embodiments, the cancer is a solid tumor, including metastatic solid tumors and progressive metastatic solid tumors. In some embodiments, the cancer is leukemia or lymphoma. Non-limiting examples of cancer include bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, hepatocellular carcinoma 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, renal cancer, and small cell lung cancer.

[0084] Additional cancerous diseases or conditions include, but are not limited to, the progression and / or metastasis of malignant tumors and related diseases such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, Heavy Chain's disease, and solid tumors, i.e., sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, , chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal 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, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, germ cell carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0085] In some embodiments, a second agent can be used in combination with the treated cells to treat cancer. The combination can be simultaneous (e.g., at the same time, on the same day, or in the same dosage form) or sequential. Examples of second agents include, but are not limited to, cyclophosphamide, IL-2, nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, anti-PD-1 or anti-PD-L1 antibodies, and anti-CD20 antibodies (e.g., rituximab). Each of these agents has been shown to exhibit synergistic effects in cancer treatment when used in combination with the treated cells.

[0086] In one example, the cancer is colon cancer, and the second agent is cyclophosphamide and / or anti-CTLA-4 antibody. In another example, the cancer is pancreatic cancer, and the second agent is cyclophosphamide, an NSAID (e.g., 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 (e.g., indomethacin), an anti-PD-1 or anti-PD-L1 antibody, or a combination thereof. In another example, the cancer is non-Hodgkin's lymphoma, and the second agent is cyclophosphamide or an anti-CD20 antibody (e.g., rituximab).

[0087] The intact, stabilized, and substantially nonviable Gram-negative bacterial cells disclosed herein are also useful for strengthening a subject's immune system and, therefore, for preventing or treating diseases and conditions through improved immune responses. The intact and substantially nonviable Gram-negative bacterial cells disclosed herein can also be used as vaccines or immune adjuvants for subjects at risk of developing the disease or condition.

[0088] In some embodiments, the disease or condition to be treated is an infectious disease. In some embodiments, the infection is caused by a bacterium, a fungus, a parasite, or a virus. In particular, the bacterial cells disclosed herein are particularly suitable for treating viral infections, optionally in combination with a secondary anti-infective agent.

[0089] In some embodiments, the disease to be treated is HBV infection. In some embodiments, the disease to be treated is HIV infection.

[0090] Administration may begin before actual infection or before infection is diagnosed, as a preventative vaccine or prophylaxis.

[0091] In some embodiments, the one or more additional therapeutic agents may be an inhibitor of the cyclooxygenase (COX) enzyme, such as an NSAID, including 6MNA, aspirin, carprofen, diclofenac, fenoprofen, flufenamate, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, naproxen, niflumic acid, piroxicam, sulindac sulfide, suprofen, and tenida. rap, tolmetin, tomoxiprole, zomepirac, celecoxib, etodolac, meloxicam, nimesulide, diisopropyl fluorophosphate, L745,337, NS398, rofecoxib, SC58125, S-aminosalicylic acid, ampirone, diflunisal, nabumetone, paracetamol, resveratrol, salicin, salicylaldehyde, sodium salicylate, sulfasalazine, sulindac, tamoxifen, ticlopidine, and valeryl salicylate.

[0092] In some embodiments, the one or more additional therapeutic agents may be agonists of stimulatory immune checkpoints, such as CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS, or antagonists of inhibitory immune checkpoints, such as A2AR, B7-H3, B7-H4, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, TIM-3, and VISTA.

[0093] Non-limiting examples of the one or more additional therapeutic agents include abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, baravir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxidine, efavirenz, emtricitabine, enfuvirtide, entecavir, ecoleaver, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon , lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogs, novir, oseltamivir (Tamiflu®), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, raltegravir, ribavirin, rimantadine, ritonavir, pyrimidines, saquinavir, sofosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine. In one embodiment, the additional therapeutic agent is interferon alpha.

[0094] In some embodiments, the second agent is an exogenous antigen. As shown in Example 2, regression of established mouse breast cancer tumors by Decoy was significantly enhanced when the test animals were transfected with an exogenous antigen (HER2). It is thought that Decoy may function as a "super adjuvant" agent that promotes the presentation of antigen molecules. However, tumor cells often acquire the ability to conceal antigens to evade immune responses. When an exogenous antigen is provided, Decoy can promote the presentation of the antigen to the immune system, resulting in a significant immune response.

[0095] Accordingly, another embodiment of the present disclosure provides a method for treating or preventing cancer in a patient in need thereof, comprising administering to the patient (a) an effective amount of a composition comprising intact, stabilized, and substantially non-viable E. coli cells that have been treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% compared to untreated wild-type E. coli cells, as measured by a Limulus amebocyte lysate (LAL) assay; and (b) an exogenous antigen associated with the cancer.

[0096] 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.

[0097] Yet another embodiment of the present disclosure provides a method for treating or preventing an infectious disease in a patient in need thereof, comprising administering to the patient (a) an effective amount of a composition comprising intact, stabilized, and substantially nonviable E. coli cells that have been treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% compared to untreated wild-type E. coli cells, as measured by a Limulus amebocyte lysate (LAL) assay; and (b) an exogenous antigen associated with the infectious disease. In some embodiments, the antigen is a viral or bacterial antigen.

[0098] In some embodiments, the exogenous antigen may be encapsulated within, bound to, or expressed by the E. coli cells. For example, if the antigen is a protein, a vector encoding the antigen can be introduced into the E. coli cells to express and secrete the antigen. In some embodiments, the exogenous antigen is separate from the E. coli cells. In some embodiments, the two may be mixed prior to administration to achieve simultaneous administration. In some embodiments, the two may be administered separately, simultaneously, or sequentially. If administered separately, the administration schedule and frequency can be determined as needed.

[0099] In some embodiments, the second agent is a PBMC or an engineered immune cell. Engineered immune cells are used clinically, for example, immune cells that have been transduced to express a recombinant chimeric antigen receptor (CAR) or T-cell receptor (TCR). In some embodiments, the immune cell is a T cell, macrophage, monocyte, NK cell, or myeloid cell. In some embodiments, the CAR or TCR recognizes a tumor-associated antigen.

[0100] Formulations, dosage forms, and administration modes Formulations and dosage forms of the treated bacterial cells are also provided. In one embodiment, the treated cells are combined with a phosphate buffer, Mg 2+ and trehalose.

[0101] In some embodiments, the phosphate buffer may comprise or be prepared with disodium phosphate dihydrate mixed with monopotassium phosphate, hi some embodiments, magnesium ions may be provided by magnesium chloride hexahydrate.

[0102] 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%, or 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.

[0103] An example formulation contains 0.5 mg / mL to 2 mg / mL disodium phosphate dihydrate, 0.1 mg / mL to 0.4 mg / mL monopotassium phosphate, 3 mg / mL to 12 mg / mL sodium chloride, 0.05 mg / mL to 0.3 mg / mL potassium chloride, 0.15 mg / mL to 0.6 mg / mL magnesium chloride hexahydrate, and 50 mg / mL to 200 mg / mL trehalose dihydrate, and has a pH of 7.3 to 7.7.

[0104] In another embodiment, the formulation comprises 0.8 mg / mL to 1.3 mg / mL disodium phosphate dihydrate, 0.14 mg / mL to 0.25 mg / mL monopotassium phosphate, 4 mg / mL to 8 mg / mL sodium chloride, 0.1 mg / mL to 0.2 mg / mL potassium chloride, 0.2 mg / mL to 0.4 mg / mL magnesium chloride hexahydrate, and 80 mg / mL to 160 mg / mL trehalose dihydrate, and has a pH of 7.3 to 7.7.

[0105] In some embodiments, the formulation comprises 0.1 x 10 9 / mL ~ 20 × 10 9 / mL of treated cells. In some embodiments, the formulation contains 0.2 x 10 9 / mL ~ 10 × 10 9 / mL of treated cells. In some embodiments, the formulation contains 0.5 x 10 9 / mL ~ 5 × 10 9 / mL of treated cells.

[0106] In some embodiments, the intact and substantially non-viable Gram-negative bacterial cells in the dosage form have at least about a 70% reduction in LPS-associated endotoxin activity compared to untreated wild-type bacteria (e.g., as measured by an LAL assay). 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 more than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction rate is, but is not limited to, about 70% to about 99.99%, about 80% to about 99.99%, 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%.

[0107] In some embodiments, the intact and substantially non-viable Gram-negative bacterial cells in the dosage form have at least about a 70% reduction in pyrogenicity compared to untreated wild-type bacteria (e.g., as measured by an in vivo rabbit assay). 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 more than about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.98%, or 99.99%. In some embodiments, the reduction rate is, but is not limited to, about 70% to about 99.99%, about 80% to about 99.99%, 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%.

[0108] In some embodiments, the reduction in non-LPS-related fever 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 does not exceed 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, substantially non-viable bacteria have their viability reduced by at least 80%, 85%, 90%, 95%, 99%, or more.

[0109] As demonstrated in the experimental examples, the produced treated bacterial cells are surprisingly stable. This stability manifests itself as strong resistance to disruption by sonication and retention of integrity during extended storage. Accordingly, one embodiment of the present disclosure provides a method for providing a therapeutically acceptable composition. In some embodiments, the method includes lyophilizing a plurality of treated bacterial cells as disclosed herein to form a lyophilized composition, and storing the lyophilized composition (a) at a temperature between 1°C and 10°C for at least two months, or (b) at or below -15°C for at least two years, thereby providing a therapeutically acceptable composition suitable for therapeutic use.

[0110] In some embodiments, storage is for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 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 at a temperature between 1° C. and 10° C. In some embodiments, storage is for at least 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 3, 4, 5, 6, 7, or 8 years at a temperature of −15° C. or below.

[0111] In some embodiments, the plurality of treated bacterial cells is treated with phosphate buffer, Mg 2+ and trehalose. In one embodiment, the formulated composition comprises 0.3 x 10 9 / mL ~ 5 × 10 9 1 mL of intact and substantially nonviable 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 monopotassium 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 of 7.0 to 7.7.

[0112] The compositions are formulated for pharmaceutical administration to a mammal, preferably a human. The pharmaceutical compositions of the present invention can be administered in a variety of ways, including intravenously, intratumorally, subcutaneously, intradermally, intramuscularly, intravesically, intranasally, or intraperitoneally.

[0113] In one embodiment, the treated cells or compositions (before or after storage as disclosed herein) are administered parenterally. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intravesical, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0114] Sterile injectable compositions may be aqueous or oily suspensions. These suspensions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known to those skilled in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable excipients and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any bland, odorless fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, and natural pharmaceutically acceptable oils, such as olive oil or castor oil, are particularly useful in their polyoxyethylated forms. These oil solutions or suspensions may contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants include Tweens, Spans, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, and can also be used for formulation purposes. The compositions can be formulated for parenteral administration by injection, such as bolus injection or continuous infusion.

[0115] The pharmaceutical composition may be administered in a single dose or multiple doses. The pharmaceutical composition may be administered in various ways, for example, rectally, orally, intranasally, and transdermally. In certain embodiments, the pharmaceutical composition may be administered by intraarterial injection, intravenously, intravesically, intraperitoneally, parenterally, intramuscularly, or subcutaneously.

[0116] An example of an administration form is parenteral administration by injection. Forms into which the pharmaceutical compositions described herein can be incorporated for administration by injection include, for example, aqueous or oily suspensions, or emulsions using sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical excipients.

[0117] Examples of suitable additives include lactose, dextrose, sucrose, sorbitol, mannitol, trehalose, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose.The preparation may additionally contain lubricants such as talc, magnesium stearate, and mineral oil, wetting agents, emulsifying and suspending agents, preservatives such as methyl and propylhydroxybenzoates, sweeteners, and flavorings. [Example]

[0118] 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 are well-functioning techniques in the practice of the present disclosure and are therefore considered to constitute specific modes for the practice of the present disclosure. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made to the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the disclosure.

[0119] Example 1. Characterization of Decoy-treated bacterial products This example summarizes the results of several nonclinical studies on the "Decoy" treated bacteria product. Decoy consists of 100% killed, stabilized, and intact bacteria produced from nonpathogenic E. coli after attenuation, reducing LPS endotoxin activity and pyrogenicity by approximately 90% (also referred to as "treated bacteria," "Decoy bacteria," or simply "Decoy"). Activation or agonist activity of specific human immune receptors (TLR and NOD) was assessed using a panel of human embryonic kidney reporter cell lines (HEK293) transfected with different immune receptors. STING agonist activity was assessed using a human monocyte cell line carrying a luciferase reporter gene that responds to type 1 interferon induction. Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate and in vivo rabbit assays. Bacterial integrity was assessed by electron and light microscopy. Stability was assessed by disruption sensitivity via probe sonication. E. coli was an auxotroph that required diaminopimelic acid (DAP) for growth. Because mammals do not produce DAP, the live strain could not grow in mammals, providing a fail-safe mechanism that increased product safety in addition to 100% kill.

[0120] The viability and endotoxin activity of wild-type E. coli before and after various treatments were assessed using optical density measurements at 600 nm, plating efficiency, and the Endosafe Endochrome K Kinetic LAL assay under various incubation conditions and times. Treatments included polymyxin B, an antibiotic known to lyse Gram-negative bacteria and neutralize endotoxins, and agents that either kill bacteria (phenol) or kill and stabilize bacteria (formaldehyde, glutaraldehyde). Because the antibiotic action of polymyxin B causes cell lysis but is unrelated to the neutralization of endotoxin activity, the goal of exploratory studies was to develop a process with relevant conditions that could significantly reduce endotoxin activity and kill 100% of cells while keeping them intact.

[0121] Incubation of E. coli cells with polymyxin B in the presence of fermentation medium and magnesium chloride at low temperatures was found to achieve the desired reduction in endotoxin activity without cell lysis. Subsequent incubation with glutaraldehyde in phosphate-buffered saline (PBS) achieved 100% cell death. The concentrations and exposure times of polymyxin B and glutaraldehyde were determined using dose-response and time-course experiments. Product integrity was confirmed by light and electron microscopy (after Gram staining). All bacterial cultures used to produce Decoy-treated products were periodically evaluated for DAP dependency and viability before, during, and after the treatment process. Trehalose (final concentration 12%) was added to the product as a cryoprotectant before freezing and storage at -70°C.

[0122] Stabilization of the treated or decoy bacteria compared to the untreated parent bacteria was demonstrated by showing that the untreated parent bacteria were easily disrupted by sonication. In contrast, the treated or decoy bacteria were resistant to disruption by sonication, as shown by light microscopy (following Gram staining) (Figure 1). Sonication was performed using a Fisher Scientific Sonic Dismembrator equipped with a microprobe at setting #3 for 5-10 minutes. Sonication was performed in round-bottom tubes with the probe held just below the surface for efficient sonication of the material.

[0123] The stability of the decoy bacteria was also tested after long-term storage. When frozen and stored at -70°C, the decoy bacteria remained stable for at least two years. Under 4°C storage conditions, the decoy bacteria remained stable for at least six months without detectable degradation.

[0124] The pyrogenicity of untreated bacteria (DB100) and Decoy was evaluated in a dose-ranging study using New Zealand White (NZW) rabbits (4 rabbits / group). 4 bacteria / mL) and Decoy (3 × 10 5 bacteria / mL and 9 x 10 5 The untreated bacteria were administered as a slow intravenous injection (10 mL / dose) via the marginal ear vein of NZW rabbits. 4 A dose of 3 × 10 bacteria / mL increased rectal temperature by 0.7°C, while Decoy increased rectal temperature by 3 × 10 5 A dose of 9 × 10 bacteria / mL increased rectal temperature by 0.1°C. 5 A dose of 100 bacteria / mL increased the temperature by 0.8–1.0°C, representing an approximately 30-fold, or 97%, reduction in fever (Table 1).

[0125] [Table 1]

[0126] Untreated (DB100) E. coli and Decoy were 1 × 10 8 ~3×10 10 Groups of three BALB / c mice were administered a single intravenous dose of 1 × 10 bacteria / mouse and observed for up to 15 days. 10 Administration of DB100 at 100% was fatal (dead or near death), whereas no deaths were observed with the same dose of Decoy. 10 In this and subsequent pharmacological studies, the clinical findings in mice treated with Decoy were mainly transient weight loss and transient ruffled fur. However, when administered in combination, the doses (e.g., 3 × 10) did not cause clinical symptoms. 7 Antitumor activity was observed in decoy bacteria (bacteria / animals). Activation or agonist activity of specific human immune receptors (TLRs and NODs) was assessed using a panel of human embryonic kidney reporter cell lines (HEK293) transfected with different immune receptors. Receptor activation stimulates NF-κB activity, which induces reporter gene expression and produces secreted embryonic alkaline phosphatase (SEAP), which can be quantified. STING agonist activity was assessed using a human monocytic cell line (THP1) carrying a luciferase reporter gene that responds to type 1 interferon induction. Response specificity was determined using a control cell line containing only the reporter gene system without transfected immune receptors. Activity was confirmed when experimental compounds induced reporter gene expression by 2-fold or more. The activity of experimental compounds was also compared to that of a commercially available positive control receptor activator. Treated Decoy bacteria contained all functional TLR receptors and heterodimers, as well as agonists or activators of NOD2 and STING (Table 2).

[0127] The pharmacodynamic effects of decoy on cytokine and chemokine secretion were evaluated using human peripheral blood mononuclear cells (PBMCs). TLR ligands, including Escherichia coli LPS, double-stranded E. coli DNA, E. coli peptidoglycan (PGN), flagellin, R848, polyinosinic-polycytidylic acid (poly I:C), and CpG oligonucleotides (ODN), were also evaluated. Initial experiments were performed for 24, 48, and 72 hours of incubation, with or without anti-CD3 activation. Anti-CD3 activation was not required for the induction of cytokine and chemokine secretion from PBMCs by decoy bacteria. Furthermore, maximum induction of most cytokine and chemokine secretion was observed after 48 hours. Treated Decoy bacteria induced higher levels of most cytokines and chemokines compared with untreated Decoy bacteria at the same dose (Table 2). Essentially all cytokines and chemokines are involved in activating innate and / or adaptive immune (including antitumor and antiviral) pathways, but if expressed in the wrong place, at the wrong time, for too long, or at inappropriately high levels, they can induce significant toxicity.

[0128] [Table 2]

[0129] In addition to endotoxin or LPS, which are outer membrane Toll-like receptor 4 (TLR4) agonists, Gram-negative bacteria such as E. coli contain agonists for various other TLRs, including TLR2, TLR2 / 1, TLR2 / 6, TLR5, and TLR9. Reports have also demonstrated the presence of TLR3, TLR7, and TLR8 agonist RNAs in E. coli. E. coli also contains agonists for the NOD-like receptors (NLRs) NOD1 and NOD2, as well as agonists for the stimulator of interferon (IFN) genes (STING). Because decoy manufacturing modifies the surface but leaves the bacteria intact, it is likely that various 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 individually transfected with different single human or mouse immune receptors or receptor heterodimers, including TLR2, 2 / 1, 2 / 6, 3, 4, 5, 7, 8, 9, NOD1, NOD2, or STING, each coupled with a reporter gene readout. The decoy bacteria demonstrated agonistic activity or activation of all human TLRs, most mouse TLRs, as well as NOD2 and STING (Table 3).

[0130] [Table 3]

[0131] Significant evidence has accumulated that TLR / TLRa signaling can enhance or is required for innate and adaptive immune antitumor responses. Several single, pure, or synthetic TLR agonists have been shown to have antitumor activity in preclinical studies and have been validated in clinical cancer trials. In this example, the induction of cytokine and chemokine secretion from PBMCs by Decoy was compared with that induced by various single TLR agonists. Table 4 shows that Decoy bacteria induced higher levels of cytokines and chemokines than any of four individual TLR agonists, including purified E. coli LPS. These results demonstrate that Decoy bacteria are superior activators of immune cell cytokine and chemokine secretion than many single, pure TLR agonists, supporting the finding that Decoy bacteria contain multiple TLR agonists and other immune stimulators in addition to LPS.

[0132] [Table 4]

[0133] Humans are approximately 500–1,000 times more sensitive than mice to the toxicity of intravenous purified LPS on a ng / kg basis, and this difference in sensitivity has been found to correlate with differences in the ability of LPS to induce cytokine secretion in human and mouse immune cells. Furthermore, this difference in sensitivity has been found to be mediated by factors in the blood or serum. Additional in vitro pharmacological studies have shown that the same number of Decoy bacteria induces 10–3,500 times more cytokine secretion from the same number of human PBMCs (in human serum) compared to mouse PBMCs (in mouse serum) (Figure 2).

[0134] Because cytokines mediate both antitumor activity and toxicity, the finding that human immune cells are more sensitive than mouse immune cells to cytokine induction (including antitumor cytokines) by Decoy bacteria suggests that an acceptable antitumor therapeutic index in mice may also be similar in humans.

[0135] Example 2. Non-clinical trials of Decoy This example summarizes the results of some non-clinical trials of the Decoy product, which consists of treated bacteria.

[0136] In vitro activity in human breast cancer cells The antiproliferative effect and cytotoxicity of Decoy were evaluated in a co-culture model of human PBMCs and the MDA-MB-231 breast tumor cell line. PBMCs (1 × 10 4 cells / well, 1 x 10 5 cells / well, 1 x 10 6 cells / well) alone or with MDA-MB-231 Nuclight Red stable cells (2 × 10 4 ) in a 96-well plate, and decoy (3 × 10 6 The animals were treated with 1 × 10 bacteria for 96 and 168 hours. 5 cells / well and 1 x 10 6 At a concentration of 1 × 10 cells / well, PBMC alone inhibited the proliferation of MDA-MB-231 tumor cells, whereas decoy alone had minimal inhibition of tumor cell proliferation. 4 The combination of 1000 and 1000 cells / well showed a clear synergistic effect with 90% tumor cell growth inhibition and cytotoxicity after 7 days of incubation (Figure 3).

[0137] In vivo activity in a mouse colon cancer model The antitumor activity of Decoy alone was investigated in an orthotopic model of human colon cancer using female BALB / c mice (7 mice / group) bearing green fluorescent protein-labeled CT26 tumor fragments derived from subcutaneous tumors. Five days after cecal implantation (surgery), mice were injected with Decoy vehicle or Decoy (2 × 10 8 The bacteria / animal were administered intravenously twice a week [QD × 2] for 3 weeks. The results are shown in Figure 4. Tumor growth was measured by palpation during in-vivo and tumor weight after necropsy. Metastasis was measured by green fluorescent protein imaging in "laparotomy" animals after necropsy. In this experiment, Decoy alone demonstrated prolonged survival and suppressed metastasis. The maximum transient weight loss during the first week of Decoy treatment was 1.3% compared to the start of treatment, and no weight loss was observed in subsequent weeks of treatment.

[0138] The antitumor efficacy of Decoy was evaluated in a subcutaneous murine syngeneic CT26.WT colon cancer model using female BALB / c mice (8 mice / group). Exploratory studies were also conducted with cyclophosphamide administered orally (po) in drinking water and anti-CTLA-4 antibody alone or in combination with Decoy. Single-agent Decoy was administered at a dose of 5 × 10 per mouse starting 3 days after tumor cell inoculation. 7 or 1×10 9 The 1,000 mm bacterium was administered intravenously twice weekly (Q4D) for 3 weeks. Both doses demonstrated statistically significant tumor growth inhibition compared to vehicle control on days 24 and 28 (low dose) and days 21, 24, and 28 (high dose) by unpaired T-test (Figure 5). At the high dose, 1,000 mm 3 When measured from the time of reaching weight, the rats showed a statistically significant suppression compared to the vehicle control (report). No weight loss was recorded with the low dose of Decoy, and only one transient weight loss (0.56%) was recorded with the high dose of Decoy.

[0139] The antitumor activity of Decoy in combination with low-dose intraperitoneal (ip) cyclophosphamide (LDC) was investigated in a subcutaneous colon cancer model using female BALB / c mice (8 mice / group) bearing established CT26 tumors (identical to CT26.WT). At 11 days post-implantation, the mean tumor size was 202 mm. 3 At 0°C, mice were either untreated or treated with Decoy alone (1.5, 3.0, or 6.0 × 10 8 of bacteria / animal, intravenously, twice a week [QD × 2] for 3 weeks), LDC (20 mg / kg, intraperitoneally, QD × 4 for 3 weeks from the day before Decoy treatment), or 3.0 × 10 8 A combination of Decoy and LDC was administered, and the results are shown in FIG.

[0140] In this test, decoy alone was 1.5 or 3.0 × 10 8 bacteria / mouse did not show statistically significant antitumor effects, whereas 6.0 × 10 8 The 3 × 10 bacteria / mouse group demonstrated a statistically significant 25% increased lifespan (%ILS) tumor growth delay. LDC alone demonstrated a statistically significant 57% ILS. However, the 3 × 10 8 The combination of Decoy and LDC demonstrated a statistically significant ILS of 89%, with one complete and durable tumor regression and long-term survival up to day 81. The combination of 1.5, 3.0, and 6.0 × 10 QD × 2 per week 8 The maximum transient weekly weight loss during Decoy treatment at this dose was 8.9%-11.2% in the first week, 2.2%-5.4% in the second week, and 0%-1.9% in the third week, which is in line with expectations for LPS tolerance, as reported for intravenous LPS administration in mammals, including humans. Decoy+LDC treatment resulted in transient weight losses ranging from 12-16% in each week of the three weeks of treatment, but no animal deaths occurred.

[0141] The antitumor efficacy of Decoy as a single agent or in combination with intraperitoneal IL-2 and oral indomethacin was evaluated in a syngeneic subcutaneous murine CT26.WT colon cancer model using female BALB / c mice (5 mice / group). CT26.WT cells were injected subcutaneously into the right flank of BALB / c mice. At day 10, the average tumor size was 75 mm. 3 Starting at 0°C, groups of mice were either untreated, or received decoy vehicle, decoy (1 × 10 9 Decoy (1,000 U / mouse, intravenously, Q4D × 2, × 2 weeks), IL-2 (2,500 U / animal, intraperitoneally, QD × 28) plus indomethacin (14 μg / mL, orally, ad libitum, QD × 28), or Decoy plus IL-2 plus indomethacin were administered. Single-agent Decoy and IL-2 plus indomethacin both demonstrated statistically significant tumor growth inhibition (median tumor growth delays of 7.0 and 7.1 days, respectively). The combination of Decoy, IL-2, and indomethacin demonstrated a statistically significant median tumor growth delay of 18.9 days and resulted in one tumor regression or complete response with survival to day 60 (Figure 7). The maximum transient weight loss with Decoy alone was 5% during the first week of treatment and 0% during the second week. The maximum transient weight loss with Decoy plus IL-2 plus indomethacin was 7% during the first week of treatment and 3% during the second week.

[0142] The study also tested several additional compounds, including anti-GITR antibodies, INF-γ (which was accidentally administered at 10 times the intended dose, resulting in toxicity), and phenformin, alone, in combination with each other, and / or in combination with Decoy. Some of these groups received these compounds after day 31. No significant additive and / or synergistic antitumor activity was observed with these agents or combinations.

[0143] In an in vivo pharmacodynamic study, the anticancer activity of Decoy (DB104) alone or in combination with cyclophosphamide (CTX) was evaluated in a subcutaneous murine CT26.WT colon cancer model using female BALB / c mice (5 mice / group). Murine CT26.WT colon cancer cells were implanted (injected) into the right flank of BALB / c mice. Animals were randomized, and treatment began at a group mean tumor weight of 75-79 mg (Starting Day = SD). Mice were injected with Decoy (3 × 10 8 of bacteria / animal, intravenous, Q4D × 4 [SD + 1]; Q8D × 4 [SD + 1]), Decoy (1 × 10 8 , 3×10 8 , 1×10 9 , 3×10 9 of bacteria / animal, intravenous, Q4D × 4 [SD + 1]), and CTX (50 mg / kg, intraperitoneal, Q4D × 2 [SD]; Q8D × 2 [SD]). A group of untreated animals served as control. 9 Decoy at the 2000 bacteria / animal dose was not tolerated, resulting in four deaths, whereas all other treatments were well tolerated. Transient weight loss ranging from 5% to 9% was observed in all groups except the highest-dose Decoy group for 1–2 days after Decoy administration. Weight loss decreased after rechallenge, likely due to LPS tolerance. Both CTX schedules demonstrated significant tumor growth inhibition without tumor regression. The combination of Decoy with CTX at Q4D×2 demonstrated significant tumor growth inhibition and one complete tumor regression or complete response, which persisted through day 91, suggesting a potential synergistic effect. Treatment with all doses of Decoy as a single agent (Figure 8) and in combination with the corresponding CTX treatments was effective in inhibiting the growth of subcutaneously implanted CT26.WT colon cancer tumors compared to untreated mice (p<0.05). Only combined treatment with Decoy and CTX on a Q4D schedule was more effective in inhibiting the growth of CT26.WT colon cancer than CTX or Decoy treatment alone.

[0144] In vivo activity in a mouse metastatic pancreatic cancer model In a primary pharmacodynamic study, the in vivo efficacy of Decoy, gemcitabine, low-dose cyclophosphamide (LDC), and Decoy+LDC was evaluated in an intrasplenic Pan02 pancreatic cancer mouse model using female C57BL / 6 mice (7 mice / group). Pancreatic tumor cells were surgically implanted (injected) into the spleen of C57BL / 6 female mice on day 0. Mice were treated with Decoy (5 × 10 7 and 2 × 10 8 The mice were administered 5 × 10 bacteria / animal, intravenously, QD × 2 / week for 3 weeks), gemcitabine (50 mg / kg, intraperitoneally, BIW × 7), or LDC (20 mg / kg, intraperitoneally, QD × 4 × 3 weeks). A group of untreated animals served as a control. All untreated animals developed large tumors in the spleen, liver, and pancreas, leading to death at approximately day 30. Decoy was administered at a dose of 5 × 10 bacteria / animal, intravenously, QD × 2 / week for 3 weeks. 7 and 2 × 10 8 The high-dose and low-dose Decoy groups significantly extended median survival time. The maximum transient weight loss (relative to the start of treatment) in the high-dose and low-dose Decoy groups was 9.8% / 8.2% in the first week of treatment, 2.6% / 2.1% in the second week of treatment, and there was no weight loss in the third week of treatment. The positive control drug gemcitabine significantly extended median survival time between the results obtained at the two Decoy doses. The combination of Decoy and LDC did not extend survival beyond Decoy alone in this model (Figure 9).

[0145] In a second experiment using the same intrasplenic Pan02 pancreatic cancer mouse model, the therapeutic effects of decoy, gemcitabine, and indomethacin, as well as decoy plus gemcitabine and decoy plus indomethacin, were evaluated. Mice were treated with decoy (2 × 10 8Mice were administered 1000 bacteria / animal, intravenously, QD x 2 / week for 3 weeks, gemcitabine (at several different doses and schedules), and low-dose oral indomethacin (10 μg / mL, orally, ad libitum for 31 days). A group of animals administered Decoy vehicle served as a control. Single-agent Decoy and single-agent gemcitabine extended survival. Gemcitabine did not appear to exhibit additive or synergistic activity in combination with Decoy. Single-agent indomethacin did not significantly extend survival, but its combination with Decoy enhanced survival, suggesting a potential synergistic effect (a 38% increase in ILS with Decoy alone expanded to 208% with the combination). In this combination setting, the maximum transient weight loss during the first week of treatment was 6.4%, followed by 1.5% during the second week of treatment, with no weight loss relative to the start of treatment in subsequent weeks of treatment (Figure 10).

[0146] In vivo activity in a subcutaneously established mouse hepatocellular carcinoma model The therapeutic efficacy of Decoy alone and in combination with anti-PD-1 or indomethacin plus anti-PD-1 was evaluated in a subcutaneous H22 mouse syngeneic hepatocellular carcinoma model using female BALB / c mice (6 mice / group). The mean tumor size was 194 mm. 3 Treatment was initiated with 2 × 10 8 Decoy was administered as a single agent intravenously, slow-push, QD x 2 / week for 6 weeks. 8 of bacteria / animal, QD x 2 and 1 x / week for 6 weeks) in combination with anti-PD-1 and up to 6 x 10 8Various dose levels of bacteria / animal were tested in combination with indomethacin and anti-PD-1, once per week for 6 weeks. Indomethacin was administered orally at 10 μg / mL in drinking water ad libitum daily for 6 weeks (alone and in combination), and anti-PD-1 was administered intraperitoneally at 10 mg / kg BIW for 2 weeks (alone and in combination). A group of untreated animals served as the study control. No decoy-related mortality was observed with any of the decoy or combination treatments. Transient weight loss was observed after each decoy treatment. The maximum weekly mean group weight losses during 6 weeks of decoy treatment QD x 2 were -6.91%, -5.34%, -5.48%, -3.28%, -0.94%, and 0%, demonstrating the established resistance phenomenon seen with intravenous LPS administration in mammals, including humans. Combining Decoy with indomethacin and anti-PD-1 resulted in a high rate of durable tumor regression without significant increases in weight loss or other signs of toxicity. 8 In a 6-week treatment with 6 × 10 bacteria / animal indomethacin and anti-PD-1, the mean weekly group weight loss was −6.37%, −3.81%, −3.86%, −0.58%, −1.49%, and 0%. 8 In the 6-week treatment of bacteria / animal with indomethacin and anti-PD-1, the mean weekly group weight loss was −8.30%, −4.99%, −5.35%, −2.94%, −3.70%, and −0.17%.

[0147] Single-agent Decoy administered QD x 2 per week for 6 weeks resulted in a small but statistically significant increase in lifespan (27.5% ILS). Single-agent indomethacin or anti-PD-1 produced similar results (30% ILS and 37.5% ILS, respectively) and no tumor regression. All combination treatments showed statistically significant increases in ILS. In addition, the combination of Decoy once per week with anti-PD-1 resulted in 2 / 6 durable complete tumor regressions, and Decoy QD x 2 per week resulted in 1 / 6 durable complete tumor regression. Treatment with indomethacin, Decoy, and anti-PD-1 resulted in a 2x10 8 or 6×10 8Tumor regression was achieved in 5 / 6 cases in each of the Decoy groups. The regression in all but one of the 10 cases continued until the end of the experiment on day 143 (FIG. 11).

[0148] Nine cured animals were reimplanted subcutaneously on the dorsum contralateral to the primary tumor implantation (without further drug treatment) with fresh tumor cells on day 91, and both tumor sites on each animal were followed for an additional 52 days. Tumor growth initiated at all reimplantation sites but subsequently regressed spontaneously and completely, demonstrating 100% immunological memory. Naive animals implanted with the same tumor cells on the same implantation day experienced gradual tumor growth, eventually reaching 3,000 mm 3 The human sacrifice criteria were exceeded (Figure 12).

[0149] The therapeutic effects of indomethacin, anti-PD-1, Decoy + indomethacin, and Decoy + indomethacin + anti-PD-1 were evaluated in a second study in a subcutaneous H22 hepatocellular carcinoma mouse syngeneic model (Study No. E0776-U1802) using female BALB / c mice (6 mice / group). Decoy was tested in combination settings of QD × 2 / week, 2 times / week, and 1 time / week; Decoy was not tested as a single agent in this study. Decoy was administered at 2 × 10 8 1000 bacteria / animal, intravenous, slow push, 6 weeks. Indomethacin was tested at 10 μg / mL in drinking water, orally, ad libitum daily, for 2 or 6 weeks. Anti-PD-1 was tested at 10 mg / kg, intraperitoneally, twice weekly, for 2 weeks. Mean tumor size was 184 mm. 3 Treatment began at 1 week. A group of untreated animals served as a control. One death was observed after 3 weeks of treatment with Decoy (QD × 2 / week) + indomethacin + anti-PD-1. This combination, as well as all other treatments and combinations, was well tolerated in all other mice. Maximum transient weight loss in all Decoy-treated groups ranged from 7.5% to 10.1% and occurred during the first week of treatment. The triple-drug combination was never higher than the dual-drug combination. Transient weight loss after Decoy treatment generally decreased as the treatment weeks progressed, remaining less than 1% after the final treatment in all triple-drug combination groups at week 6, indicating tolerance.

[0150] Indomethacin (2 and 6 weeks) and anti-PD-1 resulted in a statistically significant increase in lifespan (ILS), with one regression observed after 2 weeks of indomethacin treatment. Indomethacin (2 weeks) plus anti-PD-1 also resulted in an increase in ILS and one regression. Decoy (2 x QD for 6 weeks) plus indomethacin (6 weeks) resulted in an increase in ILS and four regressions. These results support a strong synergistic interaction, as no regressions have ever been observed with decoy alone in this model, and no regressions were observed after 6 weeks of indomethacin treatment. All regressions persisted until the end of the experiment on day 91 (Figure 13).

[0151] In the same study, Decoy was tested in combination with indomethacin at doses of 2x / week and 1x / week, resulting in 1 / 6 and 2 / 6 sustained regressions, respectively. Decoy was also tested in combination with indomethacin (6 weeks) plus anti-PD-1 at doses of 2x / week, 2x / week, and 1x / week, resulting in 4, 5, and 6 regressions or partial regressions, respectively. The best results were achieved with Decoy at doses of 1x / week plus anti-PD-1 plus indomethacin for 6 weeks, resulting in complete regressions in all 6 cases, 5 of which persisted through the 91-day follow-up (Figure 14).

[0152] The therapeutic effects of decoy, low-dose cyclophosphamide (LDC), indomethacin, gemcitabine (GEM), 5-FU, and various combinations were examined in an additional study using a subcutaneous H22 hepatocellular carcinoma mouse model in female BALB / c mice (6 mice / group). 8 The study included 1000 bacteria / animal administered intravenously by slow push for 4 weeks (single agent) or 7 weeks (combined with indomethacin). Indomethacin was administered orally in drinking water at 10 μg / mL ad libitum daily for 4 or 7 weeks. A group of animals administered the decoy vehicle served as a control. The mean tumor size was 123 mm. 3Treatment was initiated with 100 mg / kg / day. No treatment-related deaths were observed except for GEM. GEM alone resulted in a 20% weight loss, and the combination with Decoy resulted in death. GEM and LDC demonstrated weak but statistically significant single-agent antitumor activity. No single-agent antitumor activity was observed with Decoy in this study. Combination activity was observed with LDC plus indomethacin (no regression) and Decoy plus indomethacin. Decoy plus indomethacin resulted in complete regression (complete response [CR]) in 3 / 6 patients, and complete regressions persisted until the end of the study on day 71. No transient weight loss was observed with indomethacin administration. Single-agent Decoy resulted in transient weight loss of 8.2% in the first week of treatment and 5.3% in the second week of treatment. There was no weight loss compared to the start of treatment in weeks 3 and 4 of treatment, indicating tolerance. The indomethacin combination resulted in slightly greater weight loss during the first 3 weeks of treatment, but little or no weight loss occurred between weeks 4 and 7 of treatment (Figure 15).

[0153] Since Decoy plus indomethacin was found to synergize with anti-PD-1 and produce a high rate of regression, the therapeutic efficacy of the combination of Decoy plus indomethacin and anti-PD-1 and the therapeutic index of Decoy were evaluated in a subcutaneous H22 mouse hepatocellular carcinoma model using female BALB / c mice (6 mice / group). The average tumor size was 205 mm. 3 Treatment was initiated with 3 × 10 7 , 1×10 8 , 3×10 8 , or 1 × 10 9Decoy was administered intravenously, slow-push, to animals for 6 weeks, plus indomethacin (10 μg / mL in drinking water, orally, ad libitum, daily for 6 weeks) plus anti-PD-1 (10 mg / kg, twice weekly, intraperitoneally) for 2 weeks. A group of untreated animals served as study controls. Decoy dose-response was assessed by treatment with Decoy prior to each week of anti-PD-1 treatment and by treatment with anti-PD-1 for the first week prior to each week of Decoy treatment. Decoy and anti-PD-1 have been evaluated as single agents against non-established tumors, but not against established tumors in this study. In other studies, all three agents showed minimal or no single-agent activity in this model, and no regressions were observed as single agents. Interestingly, single-agent anti-PD-1 ultimately led to tumor regression in 2 / 6 cases when administered to mice the day after tumor inoculation (non-established tumors), highlighting the established activity of anti-PD-1 against non-established tumors, but not in established tumors (>100 mm) that were relatively large at the start of treatment. 3 ) showed no activity against

[0154] Triple drug combination (205mm 3 The first-dose regimen (starting with the tumor) resulted in sustained regression in 4-6 of 6 mice / group, regardless of the order of treatment with decoy or anti-PD-1 (5-6 / 6 with anti-PD-1 first, 4-6 / 6 with decoy first). 7 / animal) resulted in 6 / 6 regressions with no or minimal transient weight loss (-0.15%, -3.23%) in the first week and reduced or no weight loss in subsequent weeks of treatment. 8 Triple therapy with Decoy per animal resulted in 6 / 6 or 5 / 6 regressions (two regimens), with a maximum transient weight loss of -4.10% or -5.55%, with a decrease or 0% in subsequent weeks of treatment. 8 The combination of Decoy / animals resulted in regression in 5 / 6 cases (both regimens), with a maximum transient weight loss of -4.40% or -5.35%, with a decrease or 0 in subsequent weeks of treatment. 9The Decoy / animal combination resulted in regressions in 5 / 6 or 4 / 6 cases (both regimens), with a maximum transient weight loss of -8.12% or -8.08%, with a decrease or no weight loss in subsequent weeks of treatment. The consistent decrease in weight loss across multiple treatments indicates the phenomenon of resistance with Decoy. No treatment-related deaths were observed, demonstrating a therapeutic index of at least 33-fold for Decoy in the combination setting. In addition, no clinical signs of toxicity of any kind were observed in this study other than transient weight loss. The results of the regimen in which mice were given anti-PD-1 first are shown in Figure 16.

[0155] Furthermore, tumor regression was observed in 11 mice (1 × 10 8 and 3 × 10 8 The mice (Decoy group) were re-implanted with the same (fresh, viable) tumor cells on the contralateral side of the primary tumor implant (without further treatment), and tumor growth was initiated, followed by complete rejection, demonstrating 100% immunological memory (Figure 17). The initial tumor regression represented true tumor eradication, as evidenced by a follow-up of all mice for 91 days after tumor implantation, and a follow-up of the re-implanted mice, including the original tumor site, for a total of 143 days.

[0156] A subcutaneous H22HCC model was used, and oral indomethacin (10 μg / mL in drinking water, QD, starting on day 0), intravenous decoy (2 × 10 8 In vivo plasma cytokine and chemokine induction was assessed with intravenous administration of intravenous anti-PD-1 (10 mg / kg, 2 times / week, starting on day 0), intraperitoneal anti-PD-1 (10 mg / kg, 2 times / week, starting on day 1), and various combinations that were found to induce tumor regression. 3Female BALB / c mice bearing H22 tumors were randomized on day 0 into eight groups with three subgroups per group. All potential treatments or combinations, including no treatment, were administered. Mice from two subgroups (five mice per subgroup) within each group were sacrificed 6 and 24 hours after no treatment, single-agent, dual-agent, or triple-agent combination treatment. For dual-agent and triple-agent combination treatments, mice were sacrificed 6 and 24 hours after the first dose of the second or third agent in the combination. Plasma was prepared from each mouse and subjected to cytokine / chemokine analysis by 32-plex ELISA. A third subgroup (six mice per group) within each group was treated for one week, and tumor volumes were measured at randomization, once a week, and at the end of the first week of treatment. After mice were sacrificed, tumors were excised and RNA was isolated. Forty-eight RNA samples were analyzed for the expression of 770 genes related to immune response pathways using NanoString technology.

[0157] Indomethacin induced expression of only 2 / 32 cytokines / chemokines, but only 6 hours after the start of treatment. Decoy induced expression of 9 / 32 cytokines / chemokines, and like indomethacin, significant levels compared with no treatment were only observed 6 hours after the start of 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 initiation of regression was observed, statistically significant induction of cytokine / chemokine expression was observed for 18–28 of the 32 cytokines / chemokines, with many cytokines / chemokines observed at both 6 and 24 hours. Transient weight loss (4 days after randomization) relative to the day of randomization was -1.63% in the no-treatment group, -0.65% in indomethacin, -8.63% in decoy, and -1.74% in anti-PD-1. None of the combination groups demonstrated increased weight loss compared with monotherapy. After one week of treatment, tumor growth inhibition rates compared to no treatment were 17% for indomethacin, 21% for 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 the end of the one-week treatment period (day 8), five of six mice in the triple combination group had reduced tumor size compared to measurements taken on day 4 (Figure 18). Other than transient weight loss, no other clinical signs of toxicity were observed in any of the mice in either group. It is noteworthy and surprising that despite the significant increase in cytokine and chemokine expression (in plasma) in the combination group, there was no increase in weight loss or other clinical signs of toxicity.

[0158] Of the 32 cytokines / chemokines measured, the majority (23 / 32) have been shown to stimulate or contribute to antitumor activity in preclinical models and in some clinical settings. Many cytokines / chemokines have also been shown to limit or inhibit antitumor activity and / or contribute to toxicity. The outcome of a particular cytokine / chemokine or combination thereof is highly dependent on many variables, including the animal, model, concentration, site, and timing of cytokine / chemokine expression. Remarkable and surprising findings regarding the antitumor efficacy of H22HCC include a high rate of tumor eradication with a therapeutic index of at least 33-fold in this example, reduced weight loss without an increase in other signs of toxicity after multiple weeks of treatment, and no increase in clinical signs of toxicity despite the clear synergistic induction of cytokine / chemokine expression compared to monotherapy.

[0159] 200mm 3 Mice (6 / group) bearing subcutaneous HCC tumors were either untreated or treated with indomethacin (NSAID), decoy, and / or anti-PD-1 for one week as described above for efficacy testing and cytokine analysis. Subsequently, tumors were excised, RNA was isolated, and the expression of 770 immune pathway-related and regulatory genes was analyzed using NanoString gene expression technology. NanoString analysis also assessed the known tumor inflammation signature (TIS), which is indicative of the intratumor anti-tumor immune environment; lower scores indicate less activation ("cold tumors"), whereas higher scores indicate a more likely activation or anti-tumor immune response ("hot tumors"). Figure 18 also shows that treatment increases the TIS signature, which roughly correlates with progression from single-agent to double- and triple-drug combinations and with the ultimate anti-tumor activity observed in studies in which treatment was extended for multiple weeks.

[0160] Additionally, NanoString gene expression analysis was performed to evaluate a variety of anti-tumor-related immune system genes, cells, and pathways. Results were validated by RNA quality and housekeeping gene expression analysis, and heatmap results represent the Log2-based change in gene expression relative to the mean value across the entire analysis.

[0161] With single-agent treatment, widespread increases in innate and adaptive immune gene / cell / pathway expression were observed in one or two tumors / mouse / group of six, which may be associated with some tumor growth inhibition. With double-agent treatment, an increased number of tumors / mouse / group showed widespread innate and adaptive immune gene / cell / pathway activation, which may be associated with increased tumor growth inhibition and some tumor regression. With triple-agent combination treatment, widespread innate and adaptive immune gene / cell / pathway activation was observed in nearly all tumors / mouse, which is highly consistent with the high rates of regression and tumor eradication observed under these conditions. Very similar results were also observed 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.

[0162] In vivo activity in a murine non-Hodgkin's lymphoma model Similar to the HCC model, Decoy bacteria showed poor activity as a single agent in the syngeneic murine A20 NHL model. However, Decoy bacteria were found to synergize with low-dose cyclophosphamide (LDC) to eradicate established tumors in female BALB / c mice. The in vivo therapeutic efficacy of various doses and regimens of Decoy in combination with LDC was evaluated in a subcutaneous A20 BALB / c lymphoma syngeneic model. Decoy (3 × 10 7 , 1×10 8 , 3×10 8 , and 1 × 10 9BALB / c mice (4 animals / group) were administered 2 doses of bacteria / animal (intravenous slow injection) twice weekly, QD x 2, QD x 3, or QD x 4 / week for 2 weeks, and 20 mg / kg LDC was administered intraperitoneally QD x 4 / week for 2 weeks, starting 1 day before decoy. Untreated animals served as controls. LDC alone was also tested. Treatment resulted in a mean tumor size of 158 mm. 3 It started on the 13th day.

[0163] Although one tumor regression was observed with LDC alone, most studies of LDC alone did not result in tumor regression. The number of partial and complete regressions per group of four mice for each regimen and each Decoy dose (from low to high) was 2x / week (1, 2, 1, 2), QD x 2 / week (2, 1, 4, 4) (Figure 19), QD x 3 (4, 3, 3, 3), and QD x 4 / week (1, 0, 3, 3), with no deaths (treatment-related or unrelated). However, at the two highest doses of QD x 4 / week Decoy, part of week 1 and all of week 2 were discontinued / missed due to excessive transient weight loss (≥20%). QD x 2 / week Decoy was considered the optimal regimen because 4 / 4 complete regressions were observed at the two highest doses. The combination of Decoy bacteria and LDC resulted in greater transient weight loss than the indomethacin / anti-PD-1 combination. Transient weight loss reached 17% in the first week of treatment and 14-17% in the second week. Decoy resistance was generally not observed with the Decoy+LDC combination, likely because treatment was only for 2 weeks. The only clinical sign of toxicity other than transient weight loss was ruffled fur. Surprisingly, this combination did not consistently reduce the maximum tolerated dose of Decoy bacteria. Mice received a maximum of 1 × 10 9 The combination of decoy bacteria (QD × 2, 2 weeks) was always tolerated, and no animals died.

[0164] The eight mice that experienced complete tumor regression in the two highest-dose groups in Figure 19 were reimplanted with fresh tumor cells on the dorsal side opposite the original tumor implantation on day 77 (without further treatment). All new tumor implants were rejected, and the original tumor site remained tumor-free by the end of this portion of the experiment on day 123. In age-unmatched naive mice that received the same fresh tumor cells on the same day as the reimplantation, tumors grew normally (Figure 20). In most other tumor reimplantation experiments, age-matched naive mice were used.

[0165] In the same experiment, mice bearing tumors that initially regressed but began to regrow after only 1 week of treatment or after 2 weeks of suboptimal doses of Decoy were treated with the optimal dose and regimen of Decoy (3 × 10 8 of bacteria / animal, intravenous slow injection, QD x 2 x 2 weeks) + LDC was administered to tumors with a volume of approximately 100-2,000 mm 3 The treatment was continued for two weeks starting from the time of initial treatment. All tumors regressed, and sustained regression was observed in 5 out of 8 mice (Figure 21). These results demonstrate that sensitivity to the Decoy technology is not easily lost after the initial treatment and that the Decoy technology can completely regress even very large tumors.

[0166] In another experiment, LDC was administered intraperitoneally at 20 mg / kg, 4 times per week for 2 weeks, 1 day before, during, and 1 day after intravenous slow-push administration of Decoy (QD x 2 / week for 2 weeks). Untreated animals served as controls. Treatment resulted in a mean tumor size of 212 mm. 3Decoy was also tested as a single agent, but showed no antitumor activity on its own. Similar to the previous experiment, Decoy+LDC induced tumor eradication (6 / 6 regressions), and 2 weeks of Decoy+LDC administration was sufficient (Figure 22). LDC alone only delayed tumor growth in this study, not causing regressions. The addition of indomethacin to Decoy+LDC was unnecessary and resulted in 4 / 6 regressions. Two weeks of administration of single-agent Decoy QD × 2 resulted in a maximum transient weight loss of 9.3% at week 1 and 3.7% at week 2. The LDC combination resulted in a transient weight loss of 12% at week 1 and 13.6% at week 2. However, surprisingly, despite the increased weight loss, this combination did not consistently reduce the maximum tolerated dose of Decoy bacteria. Mice were administered 1 × 10 9 The combination of up to 1000 mg of decoy bacteria (QD × 2, 2 weeks) was always tolerated, and no animals died.

[0167] To investigate the mechanism of antitumor activity of Decoy treatment, mice were pre-depleted of natural killer (NK) cells, CD4+ T cells, CD8+ T cells, or both CD4+ and CD8+ T cells using commercially available reagents. Tumors were implanted during the depletion regimen. Mice were treated with Decoy (3 × 10 8The mice were administered a combination of 1000 bacteria / animal, intravenously, QD × 2 / week for 2 weeks, and LDC (20 mg / kg, intraperitoneally, QD × 4 / week for 2 weeks). Surplus mice from each group were sacrificed at the start of treatment for immune cell depletion verification. Figure 23 shows that depletion of any immune cell type significantly reduced the antitumor activity of the Decoy regimen, with a 1 / 6 reduction in CR / regression rate after single immune cell depletion and abolishing all regressions when both CD4+ and CD8+ T cells were depleted. Two positive control groups of six mice each (one group administered indomethacin) achieved 10 / 12 CR / regressions (6 / 6 regressions without indomethacin, 4 / 6 regressions with indomethacin). These data demonstrate that tumor eradication by Decoy technology involves both the innate and adaptive immune systems, as expected for a product containing multiple TLR agonists and other immune system danger signals. For example, TLR4, activated by LPS, has been shown to be important, if not essential, for antigen presentation, therapy, and dendritic cell activation. Thus, in addition to immune activation associated with innate and adaptive immune cells, LPS provides an important bridging function between the innate and adaptive immune systems.

[0168] Decoy-induced regression of established A20 mouse syngeneic NHL tumors was replicated with two Decoy preparations derived from two different E. coli strains. Treatment resulted in a mean tumor size of 201 mm. 3 Decoy batch #1 (3 x 10 8 and 1 × 10 9 of bacteria / animal, intravenous) or batch #2 (3 × 10 8 and 1 × 10 9 In this study, Decoy was administered as a single agent at a dose of 1×10 bacteria / animal, intravenously (QD×2 for 2 weeks) in combination with LDC (20 mg / kg / animal, intraperitoneally, QD×4 for 2 weeks), resulting in 4-5 / 5 regressions and 3-4 / 5 long-term survival in each group (Figure 24). 9 The administration of 1 × 10 bacteria / animal, QD × 2 / week for 2 weeks was not tolerated, and all animals died after the third administration. Surprisingly, with the combination of LDC, the number of bacteria / animal was 1 × 10 except for one animal in each decoy group, which had to be discontinued after the fourth administration due to weight loss.9 Decoy was successfully administered twice daily in 3 × 10 bacteria / animals. Throughout the above studies, significant transient weight loss was observed when Decoy was administered twice weekly, especially when combined with LDC. 8 The maximum transient weight loss with Decoy alone was 9.8% and 7.0%, respectively, at 3 × 10 per dose. 8 The maximum transient weight loss in the Decoy-combined group was 9.7% and 17.6%, while that in the Decoy-combined group was 11% and 16.1%. 9 The maximum transient weight loss in the Decoy monotherapy group was 12.9% and 15.9% (after one week of treatment), while in the combination therapy group it was 16.3%-20.5% after two weeks of treatment.

[0169] If the combination of Decoy bacteria and LDC can activate or enhance the innate immune system's antitumor response (Figure 23), this bacterium may be synergistic with innate immune system-mediated therapy in the setting of human tumor xenografts (innate immunity only). This was verified in the human Ramos NHL xenograft model in SCID mice deficient in B and T cells.

[0170] Decoy (2×10 8 The antitumor activity of Decoy (2 × 10 bacteria / animal) as a single agent and in combination with LDC and rituximab was evaluated in a subcutaneous human Ramos mouse xenograft NHL model using CB17 / SCID mice. 8 The patients received 100 μg / mouse of bacteria / animal, intravenous slow infusion, QD × 2 / week, for 3 weeks), indomethacin (14 μg / mL, oral, ad libitum, QD × 2), LDC (20 mg / kg, intraperitoneal, QD × 4 / week, for 3 weeks), and / or rituximab (100 μg / mouse, intraperitoneal, twice weekly (BIW), for 3 weeks). Treatment resulted in a mean tumor size of 173 mm. 3Treatment was initiated at 8 days. Indomethacin at 14 μg / mL in drinking water showed no single-agent activity, did not significantly enhance the antitumor activity of other treatments, and exhibited some toxicity with long-term treatment. All other test compounds, including Decoy, demonstrated statistically significant single-agent and combination activity. Maximum transient weight loss in all non-indomethacin groups generally ranged from 5% to 10%. Growth of established Ramos tumors in this model was inhibited or delayed by the standard treatment rituximab, but regression or cure was not observed. The combination of Decoy and LDC regressed established Ramos tumors but did not cure the mice. The combination of Decoy, LDC, and rituximab resulted in relatively durable regression in 5 / 5 mice, which persisted until day 85 (Figure 25). Four of the five tumors that completely regressed in Figure 25 regrew after day 85. However, retesting of the triple-drug combination resulted in durable regression in 2 / 4 mice, and cure was possible in at least some mice.

[0171] Five tumor-regressed mice were reimplanted with fresh Ramos tumor cells on day 74 (on the opposite side from the first implantation). Tumors grew in only 2 / 5 mice, compared with 5 / 5 naive mice implanted with the same fresh tumor cells (Figure 26). Therefore, this example demonstrates immunological memory under conditions of innate immunity alone. While this has been previously reported with a few other drugs, it is considered extremely difficult to achieve. Tumor regression accompanied by immunological memory via the innate immune system alone is extremely rare in preclinical models, but is consistent with multiple TLR agonist mechanisms. These results suggest that Decoy technology may exhibit synergistic effects with other commercially available targeted antibody therapeutics based on antibody-dependent cellular cytotoxicity (ADCC or ADCP) mechanisms, which act to a significant extent through activation of the innate immune system.

[0172] Regression of established mouse mammary carcinoma tumors by single-agent Decoy after transfection with exogenous antigens Approximately 170mm 3 Female BALB / c mice bearing subcutaneous EMT6 mouse mammary carcinoma tumors were treated with 2 × 10 8Decoy bacteria were administered intravenously twice a week for 4 weeks. Tumor growth was similar to that of untreated tumors. 3 Treatment of mice bearing EMT6 tumors resulted in tumor growth inhibition and complete regression of established tumors in two out of five cases (Figure 27). This indicates that, at least in this condition or model, the only component of anti-tumor innate / adaptive immunity that Decoy cannot induce or provide in some mice is the antigen. Therefore, providing antigen may enhance or have a synergistic effect with Decoy therapy.

[0173] In vivo activity of AAV-HBV in a mouse model of chronic HBV infection The anti-hepatitis B virus (HBV) efficacy of Decoy was evaluated in two studies. Using a standard preclinical model, the human HBV genome was inserted into an adeno-associated virus (AAV). This hybrid construct was able to infect mouse hepatocytes and produce a chronic HBV-like infection with many characteristics of human HBV infection. This includes high levels of HBV replication in the liver and blood, and production of HBe and HBs antigens (HBeAg and HBsAg). Mice were administered the AAV-HBV virus, and blood HBV DNA titers were subsequently monitored. Significant and relatively stable blood HBV levels emerged within 28–31 days, at which point treatment was initiated. In the first study, many groups received Decoy in combination with indomethacin, whereas in the second study, indomethacin was not used. The inclusion of indomethacin was found not to be essential for Decoy's antiviral activity.

[0174] In the first study, Decoy (at two different doses) was tested in combination with indomethacin and in combination with the clinical standard of care, entecavir (ETV). ETV and indomethacin were also tested as single agents (Study 1). In Study 2, Decoy was tested as a single agent to determine whether indomethacin was required for activity. In addition, ETV and mIFN-α were also tested as single agents and in combination with Decoy (at one dose). In Study 1, mice were administered Decoy (0.6 × 10 8 and 2 × 10 8 The bacteria / animal were administered intravenously (QD x 2 / week x 5 weeks), ETV (0.1 mg / kg, orally, QD x 5 weeks), and indomethacin (10 μg / mL, orally, ad libitum x 5 weeks). Treatment began on day 28 (indomethacin and ETV) or day 29 (decoy) post-infection. The control group in this study was (Ca 2+ and Mg 2+ A Decoy vehicle control of 2 mM MgCl2 in PBS (without MgCl2) was used. Best results were obtained with high doses of Decoy (shown in the figures below), although some activity was observed at lower doses.

[0175] In the first study, single-agent indomethacin did not suppress HBV replication. Decoy bacteria (+ indomethacin) and ETV (± indomethacin) significantly suppressed HBV replication (measured in the blood) during treatment and for up to 28 weeks after treatment ended. The combination of Decoy (with indomethacin) and ETV also suppressed HBV replication, potentially providing a stronger inhibitory effect than Decoy (with indomethacin) or ETV (with indomethacin) alone (Figure 28). ETV is known not to reduce or suppress the production of HBeAg or HBsAg, and this was confirmed in this study. Decoy (+ indomethacin) treatment resulted in a significant reduction in both blood HBeAg and HBsAg during treatment and for up to 28 weeks after treatment ended (Figures 29 and 30). Decoy (+ indomethacin) and Decoy + ETV (+ indomethacin) significantly reduced intrahepatic HBV levels, but ETV (± indomethacin) did not (Figure 31). Similar results were obtained for intrahepatic HBeAg expression; suppression was observed only with Decoy treatment (+ indomethacin) (Figure 32). Although the precise relationship to HBV covalently linked circular cccDNA in human infection is unclear, Decoy (+ indomethacin) also reduced the levels of ccc-DNA-like molecules in the AAV-HBV model, but ETV did not (Figure 33). Decoy (+ indomethacin) also partially or slightly suppressed intrahepatic HBcAg levels, but ETV did not. Decoy-induced weight loss was mild and transient (6% over the first 12 days of treatment) and gradually decreased after the first week of treatment (tolerance phenomenon) (Figure 28). Three mice in the Decoy (+ indomethacin) group and two mice in the Decoy + ETV (+ indomethacin) group showed transient elevations in plasma alanine aminotransferase (ALT) levels 13 times between days 28 and 56, but all of these events resolved after day 56 and by the end of treatment on day 260. H&E liver histopathology at the end of treatment revealed no changes related to Decoy treatment.

[0176] In vivo activity of AAV in a mouse model of chronic HBV infection In a second in vivo secondary pharmacodynamic AAV-HBV study, the anti-HBV efficacy of Decoy alone (without indomethacin) and in combination with ETV was evaluated in male C57BL / 6 mice (5 mice / group) using a standard AAV-HBV mouse model. Mice were administered Decoy (2 × 10 8 Bacteria / animal were administered intravenously (QD x 2 / week x 5 weeks), ETV (0.005 mg / kg, orally, QD x 5 weeks), or mIFN-α (1000 U / g, subcutaneously, TIW x 5 weeks). Combinations of ETV (started on day 0 or 7, 31 days after infection) plus Decoy and Decoy plus mIFN-α were also tested. ETV and mIFN-α treatment was initiated on day 0, 31 days after infection, and Decoy treatment was initiated on day 1, 32 days after infection. Untreated groups of animals were also included. All treatments were well tolerated in AAV-HBV-infected mice. Decoy-treated mice experienced mild, transient weight loss (7% over 2 days during the first week of treatment, followed by a gradual decrease during subsequent treatments). One mouse in the single-drug Decoy group died of unknown causes after 4 weeks of treatment (following weight loss), and backup mice were replenished (an extremely rare event).

[0177] The results were similar to those of the first study, demonstrating that indomethacin was 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 days 31 to 151 without indomethacin (Figure 34). Decoy alone and Decoy plus ETV reduced plasma HBsAg and HBeAg levels, but ETV alone did not (Figures 35 and 36). Decoy alone and the combination of Decoy and ETV reduced HBV DNA and HBeAg levels in mouse livers at the end of treatment, but ETV alone did not. The combination of Decoy and ETV reduced HBsAg content in mouse livers at the end of treatment, but ETV alone did not. Decoy alone also reduced HBsAg content in the liver, but this finding was not significant. Decoy and ETV significantly reduced cccDNA-like molecule expression in the liver, but ETV alone did not. Decoy alone appeared to reduce cccDNA-like molecules in 3 of 5 mice. Decoy induced long-lasting T cell-mediated anti-HBsAg activity but not anti-HBsAg B cell activity. A transient elevation of plasma ALT levels was observed during Decoy treatment but resolved after treatment was discontinued. H&E liver histopathology at the end of treatment revealed no changes related to Decoy treatment, except for minimal perivascular mononuclear cell infiltration in 2 of 10 Decoy-treated mice. Treatment with mIFN-α resulted in transient suppression of plasma HBV DNA, but the effect disappeared soon after treatment was discontinued. mIFN-α did not suppress other indicators of infection. Combination of mIFN-α and Decoy did not provide superior results compared with Decoy alone.

[0178] In vivo activity in a mouse model of chronic human HIV infection In this additional pharmacodynamic study, the effects of Decoy alone and in combination with indomethacin or the human standard-of-care "highly active retroviral therapy (HAART)" cocktail consisting of raltegravir, tenofovir, disproxil, and lamivudine on HIV plasma viremia and immune cell populations were evaluated in female NOD / Shi-scid / IL-2Rγnull mice (4-6 mice / group) using a chronic human immunodeficiency virus (HIV) humanized hu-mouse model. Immunodeficient mice reconstituted with a human immune system and infected with HIV-1 were administered Decoy (6 × 10 7 Mice were administered 100 μg / animal of bacteria (intravenously, BIW × 5 weeks), indomethacin (10 μg / mL in drinking water, 5 weeks), and / or HAART (oral, ad libitum × 5 weeks). A Decoy vehicle control was used. HAART treatment significantly reduced plasma HIV viral load within 2 weeks of treatment initiation and persisted for 3 weeks after treatment discontinuation. Decoy did not significantly reduce viral load during treatment, but a significant reduction was observed approximately 2–3 weeks after treatment discontinuation and persisted for approximately 10 weeks (not significant at all time points) (Figure 37). Indomethacin suppressed HIV viral load, but only for 1 week during treatment and 2 weeks after treatment discontinuation, out of a total of 18 weeks. The combination did not appear to significantly improve monotherapy. Human CD4+ T cell levels were significantly reduced in all mice by week 24. Although neither drug normalized CD4+ T-cell levels, mice treated with HAART alone or a combination of Decoy, indomethacin, and HAART showed some increases in CD4+ T-cells compared with untreated mice.

[0179] summary A comprehensive nonclinical pharmacology program is being developed to support Decoy's first-in-human (FIH) oncology trials. Primary pharmacodynamic (PD) studies with Decoy included in vitro evaluation of cytokine and chemokine secretion induction by mouse and human peripheral blood mononuclear cells, as well as in vivo evaluation of IV antitumor activity against established subcutaneous (sc) mouse colon carcinoma, orthotopic mouse colon carcinoma, metastatic mouse pancreatic carcinoma, established subcutaneous mouse hepatocellular carcinoma (HCC), established subcutaneous mouse non-Hodgkin's lymphoma (NHL), and established subcutaneous human NHL models, all of which were conducted in mice. Decoy was tested as a single agent and in combination with low-dose cyclophosphamide (LDC), oral low-dose nonsteroidal anti-inflammatory drugs (NSAIDs / indomethacin), mouse anti-PD-1 checkpoint therapy, and / or rituximab. Decoy was also tested against established mouse mammary carcinoma tumors with and without expression of exogenous antigens.

[0180] Despite a 90% reduction in LPS endotoxin activity, Decoy-mediated induction of cytokine and chemokine secretion by mouse and human peripheral blood mononuclear cells was largely intact, including the induction of multiple cytokines and chemokines involved in the activation of innate and adaptive immune cells and pathways, including those known to be required for antitumor and antiviral responses. This striking observation may be related to alterations in the mechanism and / or time course by which immune cells process bacteria chemically stabilized with glutaraldehyde.

[0181] Decoy alone demonstrated statistically significant antitumor activity in mouse subcutaneous colon cancer and metastatic pancreatic cancer models, and its combination with LDC, indomethacin, and / or anti-PD-1 therapy resulted in synergistic tumor eradication in mouse subcutaneous HCC and NHL models. None of the tested agents consistently resulted in tumor regression or eradication as single agents. Tumor eradication (tumor-free for at least 3-5 months after tumor implantation) with the combination was observed in up to 100% of animals per group and was associated with the induction of 100% immunological memory, as evidenced by 100% rejection of tumor reimplantation without additional treatment. Decoy achieved single-agent antitumor activity and 80-100% tumor eradication in combination with once-weekly or twice-weekly IV administration (depending on the model) for 2-6 weeks, including at well-tolerated doses without clinical signs of toxicity, with a therapeutic index of over 33-fold in one combination model (HCC). Decoy also induced regression as a single agent in a mouse breast cancer model transfected with an exogenous antigen.

[0182] The antitumor efficacy of Decoy was also extended to a human NHL tumor xenograft model in severe combined immunodeficient mice lacking the adaptive immune system. The combination of Decoy and LDC resulted in a high rate of regression of established tumors, but the regression was not durable. The addition of rituximab, a standard-of-care targeted antibody, resulted in durable and complete tumor regression, which is difficult to achieve in the setting of innate immunity alone. Mice with tumor regression were reimplanted with fresh tumor cells without additional treatment. Some of the new tumors were rejected, demonstrating partial innate immune memory, which has been reported but is considered difficult to achieve. Together with the high rate of immune memory observed in a syngeneic setting, these results further support a dual mechanism of innate and adaptive immunity when combined with killed Decoy bacteria.

[0183] In a murine NHL model, depletion of NK cells, CD4+ T cells, or CD8+ T cells before initiating Decoy+LDC combination therapy almost completely abolished the antitumor activity of tumor eradication, further demonstrating a role for activation of both innate and adaptive immune pathways. 3 We were able to eradicate even very large, established subcutaneous tumors. In another triple-drug HCC tumor eradication model (Decoy + indomethacin + anti-PD-1), 770-gene Nano-String gene expression analysis was performed on subcutaneous tumors isolated from mice 1 week after single-, double-, or triple-drug combination treatment, including a single IV dose of Decoy. Treatment progression from single-agent (no tumor regression or eradication) to double-agent (eradication in 1–2 of 6 patients) to triple-agent (eradication in 5–6 of 6 patients) was associated with a progression from low to high levels of tumor expression of cytokine, chemokine, innate immune, and adaptive immune pathway genes, as well as an increase in tumor inflammatory signature score (from cool to hot tumors). Compared with single-agent Decoy treatment, tumor-eradicating combination treatment was also associated with a synergistic induction of plasma cytokine and chemokine expression without increased clinical signs of toxicity.

[0184] Primary pharmacology studies with Decoy demonstrated significant single-agent and combination antitumor activity against multiple tumor types, supporting a mechanism of action based on priming or activation of innate and adaptive immune cells and pathways. The significant (approximately 90%) reduction in naturally occurring LPS endotoxin activity may enhance safety during IV administration, while residual activity is sufficient to promote the well-known 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 within bacteria, such as NOD and STING agonists. Secondary pharmacology studies with Decoy also demonstrated significant single-agent antiviral activity in preclinical models of chronic hepatitis B virus (HBV) infection and chronic human immunodeficiency virus (HIV) infection.

[0185] The safety profile of Decoy was determined in single-dose, 2-week repeat-dose ranging, and 4-week repeat-dose toxicity studies in New Zealand White (NZW) rabbits after 1-hour IV administration. NZW rabbits are the nonhuman laboratory animal species considered most similar to humans in terms of sensitivity to the adverse effects of LPS. Studies in mice also provided additional safety information for Decoy. Safety data from the twice-weekly, 4-week pivotal repeat-dose toxicity study were used to support the starting dose of Decoy in the proposed Phase 1 clinical trial, which involves once-weekly dosing.

[0186] The single-dose maximum tolerated dose (MTD) of Decoy in rabbits was tested at four dose levels, with a maximum of 5 × 10 9 One death was observed at the KB / kg dose level, so the 1.5 x 10 9 When Decoy was administered twice weekly for two weeks, four dose levels were tested and the no observed adverse effect level (NOAEL) was 6 x 10 7 In the pivotal 4-week repeated-dose toxicity study in rabbits (4 dose levels), the NOAEL for Decoy was 4 x 10 7 Decoy was also 97% less pyrogenic than the parent (untreated) bacteria in rabbits (rectal temperature test) and 3 times less toxic (acute LD ) than the parent (untreated) bacteria. 100 ) was found to be.

[0187] IV administration of Decoy induced mild, primarily transient, and reversible clinical signs and symptoms. These included increases in body temperature and spleen weight, changes in hematological parameters (e.g., decreases in platelets, red blood cells, hematocrit, and hemoglobin), and increases in white blood cells (primarily neutrophils). Changes in clinical chemistry parameters (e.g., 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, were also observed. A dose-dependent increase in interleukin (IL)-6 plasma cytokine levels was observed shortly after administration (1.5 hours), returning to baseline within 24 hours after the first dose and within 6 hours after the final dose. Of 13 cytokines or chemokines tested, only IL-6 was elevated in plasma. Various molecules associated with cytokine release syndrome were either transiently induced or not induced at all. Cytokine release syndrome represents a serious toxicity associated with many immunotherapies. Stabilization of bacteria to prevent degradation in the circulation, combined with rapid clearance of bacteria by immune cells in the liver and spleen, may reduce the risk of cytokine release syndrome associated with immunotherapies that rely on sustained exposure.

[0188] Decoy-mediated changes in platelets, white blood cells (WBCs), neutrophils, albumin, cholesterol, triglycerides, body temperature, and IL-6 were either absent (platelets, WBCs, neutrophils, and albumin) or decreased (cholesterol, triglycerides, body temperature, and IL-6) at each time point after subsequent administrations compared to the same time point after the first administration of Decoy. These results are consistent with the well-documented phenomenon of LPS tolerance in mice, rabbits, and humans, which should also improve the safety of this product.

[0189] Gross findings (e.g., increased spleen weight and splenomegaly [2 of 40 cases in the pivotal study]) and microscopic findings (e.g., minimal to mild lymphoid hyperplasia in the spleen and minimal to mild mononuclear cell infiltration in the liver) occurred infrequently. The gross and microscopic findings observed in the non-clinical rabbit model are considered non-adverse and related to the proposed mechanism of action of this product.

[0190] Non-clinical studies have shown that Decoy has an acceptable safety profile, both in terms of its therapeutic index in pharmacological studies conducted in mice and in toxicity studies conducted in rabbits. Similar to the well-documented findings of tolerance (reduced toxicity) following repeated IV LPS administration in mice, rabbits, and humans, LPS-containing Decoy also demonstrated a similar phenomenon in mice and rabbits.

[0191] Example 3. Clinical trial of Decoy This example describes a proposed clinical trial of Decoy.

[0192] The decoy preparation consists of a 100% dead, attenuated, and stabilized suspension of non-pathogenic Gram-negative bacterial cells, formulated with trehalose as a cryoprotectant. After diluting the cell suspension with trehalose, the final preparation also contains 75% phosphate-buffered saline (pH 7.5), 1.5 mM MgCl2, and 12% trehalose. The decoy preparation is dispensed in 0.7 mL aliquots into 2 mL (2R) vials, with a concentration of approximately 1.0 x 10 per mL. 9 It contains killed bacteria (KB), has an extractable volume of 0.5 mL, and is stored as a frozen liquid at -60°C or below. The target total cell count is 0.3–3.0 × 10 per mL. 9 The composition of the decoy preparation is shown in Table 5.

[0193] [Table 5]

[0194] Decoy will be diluted in sterile 0.9% saline for injection (normal saline) and administered as a 250mL IV infusion over approximately one hour. This Phase 1 study consists of three parts: Part 1 will involve a single ascending dose, and Part 2 (Part 2a and Part 2b) will involve continuous weekly dosing, as follows:

[0195] Part 1. Single Ascending Dose Part 1 is a single ascending dose study. Subjects will receive a single dose of Decoy at their assigned dose level on Day (D) 1 of Week (W). The doses administered in each single dose cohort are listed in Table 6 below.

[0196] [Table 6]

[0197] The starting dose in Part 1 of this study (Cohort 1 = 7 × 10 7 KB) was a rabbit 4-week toxicity test (4 × 10 7 This is approximately one-tenth of the human equivalent dose (HED) calculated from the no observed adverse effect level (NOAEL) observed twice weekly at 1000 KB / kg. Rabbits are the closest surrogate for humans with respect to immunological and toxicological responses to IV administered purified LPS. Based on 4-week Good Laboratory Practice (GLP) study data including a 3.1-fold allometric scaling factor (dose reduction) for the HED and a 10-fold dose reduction for safety adjustment, the human starting dose was 1.29 x 10 6 KB / kg, or approximately 16 Decoy-associated endotoxin units (EU) / kg, which is 7.74 × 10 7 KB / 60 kg subject, which corresponds to approximately 960 EU / 60 kg subject (instead of the conventional 70 kg, taking into account underweight patients). The starting dose in the study was somewhat low, 7.0 × 10 7KB, which corresponds to 868 EU / 60 kg subject or 1.8 ng / kg LPS. Based on the results of studies on the systemic clearance of live and killed bacteria in mice, rabbits, and humans, Decoy is expected to be rapidly cleared by the liver and spleen (within approximately 15-30 minutes), and therefore dose adjustment based on weight is not expected to be necessary. The starting dose in this study contains a lower dose of LPS than the maximum dose (4 ng / kg) determined to be well tolerated after IV administration of purified LPS to over 1,000 healthy human volunteers.

[0198] Part 2: Continuous weekly dosing Part 2a will begin once the single-dose recommended Phase 2 dose (RP2D) is identified in Part 1. The first three subjects enrolled in Part 2a will receive four doses of Decoy at RP2D on W1D1, W2D1, W3D1, and W4D1. Safety data for each of these three subjects will be reviewed four weeks after the fourth Decoy dose (W8D1). If no safety issues are identified, subjects will receive continuous weekly Decoy doses from W9D1 onward. If three subjects complete this dosing schedule, toxicity is acceptable, and Safety Review Committee (SRC) approval is obtained, three additional subjects will be enrolled and receive continuous weekly Decoy doses. Once the sixth subject has completed at least four weekly doses, the accumulated safety data for all six subjects will be reviewed by the SRC, and enrollment of subjects in Part 2b will be permitted at the same or lower dose.

[0199] Part 2b is a dose-expansion study in which subjects will continue to receive weekly Decoy at a dose and schedule determined by the SRC based on data from Parts 1 and 2a.

[0200] The study will enroll subjects with a histologically confirmed diagnosis of incurable, advanced metastatic solid tumors who have exhausted all available treatment options with proven clinical benefit for their malignancy.

[0201] The expected treatment duration for each subject in Part 1 is 1 day (a single IV infusion over approximately 1 hour) and a 28-day dose-limiting toxicity (DLT) observation period. After the continuous weekly RP2D and dosing regimen are established in Part 2a, if a subject enrolled in Part 1 completes the DLT observation period without a DLT, meets all eligibility criteria at the time of re-enrollment, and the investigator determines that continued Decoy administration is in the subject's best interest, they may continue to receive weekly Decoy at the RP2D for up to 2 years until disease progression, unacceptable toxicity, or subject withdrawal, at the discretion of the investigator and medical monitor. Additionally, the protocol allows for adjustments regarding dose reductions or dose skips, if necessary.

[0202] The first three subjects in Part 2a (safety run-in) will receive four weekly doses of Decoy, followed by a four-week treatment-free safety observation period. If the safety profile is acceptable, these subjects will then continue receiving continuous weekly Decoy for up to two years. An additional three subjects will receive continuous weekly Decoy for up to two years.

[0203] Subjects in Part 2 (Part 2a and Part 2b) will receive continuous weekly Decoy administration for up to 2 years until disease progression, unacceptable toxicity, or subject withdrawal, whichever occurs first.

[0204] Subjects who have benefited from Decoy without disease progression, unacceptable toxicity, or consent withdrawal may continue to receive Decoy after 2 years, with the consent of the investigator and medical monitor. Subjects who continue treatment may be required to re-sign the Informed Consent Form (ICF) in a new extension protocol.

[0205] Inclusion and Exclusion Criteria To be eligible to participate in the study, subjects in Part 1 (subjects in the single ascending-dose portion or Part 1-selected subjects who are eligible to re-enroll in the study after the RP2D is established), Part 2a, and Part 2b must adhere to the criteria outlined below. Selection criteria: 1. Must provide written informed consent by signing an Institutional Review Board-approved informed consent document. 2. Be male or female and be 18 years of age or older. 3.Have a histologically confirmed diagnosis of advanced metastatic solid tumor. 4. Subjects must have progressed (relapsed, relapsed, or refractory) or been intolerant to at least one and up to three prior lines of therapy for metastatic disease. Prior therapy for metastatic disease includes chemotherapy, targeted therapy (with known molecular alterations), immunotherapy, and antibody therapy. Subjects must have exhausted all available therapies with proven clinical benefit for their malignancy. Subjects who have received more than three lines of prior therapy may be eligible upon consultation with and approval by the sponsor. 5. Have measurable disease (at least one measurable lesion) according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, as defined by tumor type. 6.Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. 7. Prognosis is expected to last at least 3 months. 8. Female subjects must be non-childbearing potential (surgically sterile or at least 2 years postmenopausal) or agree to use highly effective contraception during treatment with Decoy and for 30 days after the final dose of Decoy. a. Females of childbearing potential must have a negative serum pregnancy test at screening and a negative serum or urine test on W1D1 prior to administration of Decoy. 9. Male subjects must use reliable contraception during Decoy treatment and for 30 days after the final dose of Decoy. 10. Adequate organ function as determined by the following laboratory values ​​at screening: a.Neutrophil count is 1200 / μL or more b. Platelet count ≥ 100,000 / μL (transfusions and / or growth factor supportive care are acceptable) c. Hemoglobin ≥ 8.0 g / dL (transfusions and / or growth factor supportive care are acceptable) d. The estimated creatinine clearance calculated using the Cockcroft-Gault formula is 50 mL / min or more, and the patient is dialysis-independent. e. Aspartate aminotransferase (AST) is 2.5 times or less than the upper limit of normal (ULN) (AST is 5 times or less than the ULN if there is liver metastasis or liver damage) f. Alanine aminotransferase (ALT) is 2.5 times or less than the ULN (ALT is 5 times or less than the ULN if there is liver metastasis or liver damage) g. Bilirubin ≤1.5x ULN (except for subjects with Gilbert syndrome) h. Ferritin <4x ULN and C-reactive protein (CRP) <6x ULN 11. Left ventricular ejection fraction (LVEF) of 45% or more by echocardiogram (ECHO) or multi-gated acquisition scan (MUGA) at screening. 12.Recovery from prior treatment toxicities, with the exception of peripheral neuropathy, is ≤ Grade 1 per NCI CTCAE v5.0 criteria or has resolved to the subject's prior treatment baseline. 13. Willing and able to comply with all scheduled clinic visits, laboratory tests, and other study procedures. Exclusion criteria: 1. Pregnant or breastfeeding women. 2. Has or requires treatment for an active systemic infection (viral, bacterial, or fungal). The infection must be treated and the subject must recover before enrolling in the study. 3. If the subject has received radiation therapy within 28 days of the first dose of Decoy, the subject must have recovered from all radiation-related toxicities, not require corticosteroids, and not have radiation pneumonitis. 4. Received prior chemotherapy, immunotherapy, or major immunomodulatory therapy within 28 days or 5 half-lives of W1D1. Prior treatment with a programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitor (e.g., nivolumab, pembrolizumab, atezolizumab, and / or durvalumab) was administered within 2 months prior to investigator confirmation of eligibility. 5. Received systemic corticosteroid therapy with >5 mg / day prednisone or equivalent dose of another corticosteroid within 1 week or 5 half-lives (whichever is shorter) of W1D1, or if anticipated to be required during the study (topical and inhaled corticosteroids are permitted), or with approval of the medical monitor. Systemic corticosteroids are contraindicated after Decoy administration, except for study-specific adverse event management. 6. Radiographic evidence of primary central nervous system (CNS) disease or CNS metastases or symptomatic CNS disorders (including meningeal carcinomatosis, cranial neuropathy, or mass lesions causing spinal cord compression). 7. Clinical evidence of significant coagulopathy (e.g., deep vein thrombosis or pulmonary embolism) at screening or a history of significant uncontrolled coagulopathy. Subjects with superficial venous thrombosis and visceral / splanchnic venous thrombosis primarily related to underlying disease, or subjects with a controlled coagulation profile, are eligible. 8. Active secondary malignancies in addition to the primary tumor, except for tumors deemed low risk by the investigator (e.g., non-metastatic basal cell carcinoma or squamous cell skin cancer). 9. History or active human immunodeficiency virus (HIV) type 1 or 2 infection, positive hepatitis B virus (HBV) antibody or surface antigen (suggesting acute or chronic HBV), or positive hepatitis C virus (ribonucleic acid (RNA) detected by [HCV] qualitative test) at screening. Hepatitis C RNA testing is not required for subjects with a negative hepatitis C antibody test. HBV antibody is not required for subjects with a negative HBV surface antigen test. 10. If you have a known intolerance to nonsteroidal anti-inflammatory drugs (NSAIDs). 11. If you have a known genetic predisposition to HLH / MAS. 12. History of splenectomy, active chronic liver disease, alcoholic liver disease, Wilson's disease, hemochromatosis, primary biliary cirrhosis, primary sclerosing cholangitis, hereditary hemochromatosis, history or planned liver transplant for end-stage liver disease from any cause, documented history of progressive liver fibrosis, or history of cirrhosis and / or liver failure including ascites, hepatic encephalopathy, or variceal bleeding. 13. If a live vaccine was administered within 28 days of W1D1. 14. Have an active autoimmune disease (including but not limited to psoriasis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis). 15. History of significant CNS disease such as stroke (transient ischemic attack >6 months prior and controlled is acceptable) or uncontrolled, unstable epilepsy. 16. Severe interstitial pulmonary disease and / or oxygen saturation <92% on room air. 17. Baseline QT correlated (QTc) interval calculated using the Fridericia formula is greater than 470 ms for women and greater than 450 ms for men. 18. New York Heart Association class III or IV cardiac disease, or myocardial ischemia or myocardial infarction within 180 days of screening, severe unstable angina, coronary / peripheral artery bypass graft, worsening heart failure / decompensated heart failure within the past 6 months, or other clinically significant cardiac abnormality that, in the investigator's judgment, poses a health risk to the subject. 19. Have undergone major surgery within 4 weeks prior to the first administration of Decoy, or are expected to require major surgery during the study period (Note: placement of a central venous access catheter [e.g., port, etc.] is not considered major surgery). 20. Has any other acute or chronic medical or psychiatric illness that, in the opinion of the investigator or sponsor, may increase the risks associated with study participation or Decoy administration or that makes the subject unsuitable for study participation, including pre-existing conditions that may increase vulnerability to expected toxicity or cytokine-induced inflammation, including abnormal blood chemistry values. 21. If you received experimental treatment within 28 days or 5 half-lives (whichever is shorter) of W1D1. 22. If you do not agree or are unable to comply with the procedures required by this Protocol. 23. If you have a known allergy or hypersensitivity to Decoy or any of its ingredients.

[0206] All subjects in Part 1 and Part 2 will undergo long-term survival follow-up for one year after their last dose of Decoy. These assessments may be conducted by phone or in clinic every three months after their last dose of Decoy. Information regarding initiation of other anti-cancer treatments may be collected, including start date, type / name of treatment, and response to treatment.

[0207] Example 4. Preliminary Results of a Phase 1 Study of Decoy This example describes preliminary results from an ongoing first-in-human Phase 1 trial of Decoy, an intravenously administered killed multi-immunoreceptor agonist bacterial product, in patients with advanced solid tumors.

[0208] This is a first-in-human, open-label, single-dose escalation and multiple-dose expansion, multicenter, Phase 1 study of Decoy in patients with advanced / metastatic solid tumors, with an initial dose-limiting toxicity (DLT) period of 28 days.

[0209] Primary objective: safety / tolerability. Secondary objectives: anti-drug immunogenicity, pharmacokinetics (PK), and preliminary anti-tumor activity. Exploratory objective: systemic immune activation by immune biomarkers. Eligible patients must have measurable tumors that are relapsed or refractory to standard therapy. Evaluation of single-ascending dose (SAD) cohorts precedes multiple-dose (MD) cohorts, using a standard statistical 3+3 design. The starting dose is 7 x 10 LPS administered by 1-hour intravenous infusion, based on the no-observed-adverse-effect level (NOAEL) in rabbits, a species of relevant nonclinical toxicity for LPS. 7 The dead decoy bacteria.

[0210] Plasma biomarkers were measured by Mesoscale Discovery electroluminescence (Figure 38) or Luminex platform (Figure 39). Pharmacokinetics (PK) of Decoy was measured by digital drop (dd) PCR.

[0211] Ethics approval: The study was approved by the ethics committees of the following institutions: WIRB / Copernicus, which covers Atlantic and Karmanos, and USC.

[0212] Four patients were enrolled in this study, and their characteristics are shown in Table 7.

[0213] [Table 7]

[0214] result Treatment-related adverse events are listed in Table 8. One patient experienced dose-limiting toxicity due to grade (G) 3 bradycardia, which resolved within 30 minutes after administration of a saline bolus, acetaminophen, meperidine, and oxygen. The same patient also experienced grade (G) 3 fatigue, which resolved within 2 days. Two patients experienced elevated G3 AST levels, which improved to grade (G) 1 within 1–2 days. Overall, chills, fatigue, and fever (G) 1, vomiting and hypotension (G) 2, and elevated ALT (G) 1–2 resolved within 1–2 days, and lymphopenia (G) 4 resolved within 2–3 days. All of these could be expected after exposure to LPS (a TLR4 agonist), the active ingredient in Decoy.

[0215] [Table 8]

[0216] We observed that treatment with Decoy induced transient plasma cytokine, chemokine, and biomarker expression (Figure 38). Analyses were performed pre-administration, 4, 24, 48, and 72 hours, and 3 weeks (504 hours), and 4 weeks (672 hours) after the end of the infusion. Peak induction of cytokines and chemokines occurred within approximately 4 to 24 hours and returned to baseline within 24 to 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.

[0217] Table 9 shows the results of the expanded single-timepoint plasma cytokine, chemokine, and biomarker analyses. Analyses were performed pre-dose and 0.5, 1, 2, 4, 6, 24, 48, and 72 hours and 4 weeks after the end of the infusion. Most inducers peaked within 2-4 hours and resolved within 24-48 hours. The data in Table 9 represent the maximum fold induction or reduction.

[0218] [Table 9-1] [Table 9-2] [Table 9-3]

[0219] Table 10 summarizes (not exhaustive) cytokines and chemokines associated with innate and adaptive immune anti-tumor responses. A single intravenous administration of Decoy resulted in a transient induction of 4-fold or greater of the cytokines and chemokines underlined / bold in Table 10.

[0220] [Table 10]

[0221] Pharmacokinetic analysis confirmed the rapid clearance of systemically administered Decoy. A ddPCR assay with a lower limit of detection / quantitation of 10 / 89 Decoy bacteria / mL blood was developed and used to measure Decoy levels in the subjects' blood before administration and at 5, 10, 30, 120, and 240 minutes, 24 hours, and 4 weeks after the end of the infusion. Decoy disappeared from the blood within 30 to 120 minutes after the end of the infusion (Figure 39). Peak concentrations were reached within 5 minutes after the end of the infusion and showed a steep elimination slope; therefore, differences in peak heights between subjects may reflect slight differences in clearance times during the infusion.

[0222] Tumor restaging 4 weeks after treatment showed stable disease in all four subjects, three of whom had progressive disease prior to treatment with Decoy.

[0223] A single intravenous dose of Decoy cleared from the blood within 30–120 minutes and caused the transient induction of more than 50 biomarkers in the plasma, many of which are associated with the stimulation of innate and / or adaptive immune responses. While most cytokines and chemokines have been shown to play a positive role in immune responses, their prolonged presence at abnormally high levels can also result in toxicity. The transient induction of cytokines and chemokines is an important and novel feature of the response to Decoy bacteria and helps reduce the potential for systemic toxicity, which is known to result from sustained or prolonged systemic exposure to these potent immune-activating molecules.

[0224] Additionally, blood immune cell profiling showed a rapid increase in neutrophils and a rapid decrease in nearly all other leukocytes, with all cell types recovering within approximately 72 hours, indicating that Decoy induced a transient but significant leukocyte trafficking or redistribution event.

[0225] In summary, adverse events were generally tolerable and resolved within 30 minutes to 3 days, regardless of treatment.In terms of efficacy, a single dose of Decoy resulted in initial stable disease in all four subjects, including three who had progressed prior to Decoy administration.

[0226] 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.

[0227] The invention illustratively described herein may suitably be practiced in the absence of any element or limitation not specifically disclosed herein. Thus, for example, terms such as "comprise," "include," and "contain" should be interpreted broadly and without limitation. Additionally, the terms and expressions used herein are for purposes of description rather than limitation, and the use of these terms and expressions is not intended to exclude any equivalents of the illustrated or described features or portions thereof, but recognizes that various modifications are possible within the scope of the claimed invention.

[0228] Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, modifications, improvements, and variations of the invention embodied and disclosed herein may be practiced by those skilled in the art, and such modifications, improvements, and variations are considered to be within the scope of the present invention. The materials, methods, and examples provided herein are representative of preferred embodiments, but are illustrative and not intended to limit the scope of the invention.

[0229] The invention has been described broadly and generically. Each of the narrower species and subgeneric groupings falling within this generic disclosure also constitutes part of the invention. This includes any generic description of the invention with a proviso or negative limitation excluding any subject matter from the genus, whether or not the excluded matter is specifically set forth herein.

[0230] Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of members of the Markush group.

[0231] All publications, patent applications, patents, and other documents mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0232] While the present disclosure has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate, but not limit, the scope of the 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. 1. A method for treating or preventing cancer or an infectious disease in a patient in need thereof, comprising: 1 x 10 cells treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by approximately 70% to 99% compared to untreated wild-type E. coli cells, as measured by the Limulus Amebocyte Lysate (LAL) assay. ７ ~500 x 10 ７ administering to the patient an effective amount of a composition comprising intact and substantially non-viable E. coli cells, wherein the composition contains 124 to 62,000 endotoxin units (EU) of LPS.

2. The composition is 2×10 ７ ~200 x 10 ７ 2. The method of claim 1, comprising the intact and substantially non-viable E. coli cells.

3. The composition is 3×10 ７ ~100 x 10 ７ 2. The method of claim 1, comprising the intact and substantially non-viable E. coli cells.

4. The composition is 5×10 ７ ~50 x 10 ７ 2. The method of claim 1, comprising the intact and substantially non-viable E. coli cells.

5. The composition is 3×10 ７ , 7 x 10 ７ , 10 x 10 ７ , 20 x 10 ７ , or 70 x 10 ７ 2. The method of claim 1, comprising the intact and substantially non-viable E. coli cells.

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

7. The method of claim 6, wherein the composition contains 372 EU to 8680 EU of LPS.

8. The method of claim 6, wherein the composition contains 868 EU to 2480 EU of LPS.

9. 9. The method of any one of claims 1 to 8, wherein the intact and substantially non-viable E. coli cells have been treated to result in about 80% to 98% reduction in LPS-associated endotoxins.

10. 10. The method of claim 9, wherein the intact and substantially non-viable E. coli cells have been treated to result in about a 90% to 98% reduction in LPS-associated endotoxins.

11. 11. The method of any one of claims 1 to 10, wherein the administration is performed daily, every other day, every 3 days, every 5 days, every 6 days, weekly, twice a week, three times a week, four times a week, five times a week, six times a week, every two weeks, every three weeks, monthly, every two months, every three months, every four months, every six months, every nine months, or yearly.

12. The method according to any one of claims 1 to 11, wherein said treatment of E. coli cells is with a polymyxin, preferably polymyxin B or polymyxin E.

13. 13. The method of claim 12, wherein said treating of E. coli cells is carried out at a temperature of about 2°C to about 10°C, preferably at about 4°C.

14. The method of any one of claims 1 to 13, wherein the treatment of E. coli cells is with polymyxin and glutaraldehyde.

15. 15. The method of claim 14, wherein the treatment is with 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 composition contains phosphate buffer, Mg ２＋ and trehalose.

17. The composition is 0.3×10 ９ / mL to 5 x 10 ９ 17. The method of claim 16, wherein the solution comprises 0.5 mg / mL to 2 mg / mL of the intact and substantially nonviable 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 monopotassium 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, and has a pH of 7.0 to 7.

7.

18. 18. The method of any one of claims 1 to 17, wherein the administration is intravenous, intratumoral, subcutaneous, intramuscular, intrahepatic, intravesical, intranasal, or intraperitoneal administration.

19. The method of any one of claims 1 to 18, wherein the patient has a solid tumor.

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

21. The method of any one of claims 1 to 20, wherein the cancer is selected from the group consisting of 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. 22. The method of any one of claims 1 to 21, further comprising administering to the patient a second agent selected from the group consisting of cyclophosphamide, IL-2, a non-steroidal anti-inflammatory drug (NSAID), an anti-PD-1 or anti-PD-L1 antibody, an anti-CTLA-4 antibody, and an anti-CD20 antibody.

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

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

25. 1. A method for providing a therapeutically acceptable composition, comprising: At least 1 x 10 cells treated to reduce lipopolysaccharide (LPS)-associated endotoxin activity by about 70% to 99% compared to untreated wild-type E. coli cells, as measured by the Limulus Amebocyte Lysate (LAL) assay. ６ freeze-drying a solution containing intact and substantially non-viable E. coli cells to prepare a freeze-dried composition; storing the freeze-dried composition (a) at a temperature between 1°C and 10°C for at least two months or (b) at a temperature below -15°C for at least two years to provide a therapeutically acceptable composition suitable for therapeutic use. A method comprising:

26. The solution contains phosphate buffer, Mg ２＋ 26. The method of claim 25, further comprising:

27. The solution contains 0.3 x 10 ９ / mL to 5 x 10 ９ / mL of the intact and substantially non-viable E. coli cells, 0.5 mg / mL to 2 mg / mL disodium phosphate dihydrate, 0.1 mg / mL to 0.4 mg / mL monopotassium phosphate, 3 mg / mL to 12 mg / mL sodium chloride, 0.05 mg / mL to 0.3 mg / mL potassium chloride, 0.15 mg / mL to 0.6 mg / mL magnesium chloride hexahydrate, and 50 mg / mL to 200 mg / mL trehalose dihydrate, and having a pH of 7.0 to 7.

7.

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

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

30. 29. The method of claim 28, wherein the antigen is a viral antigen or a bacterial antigen.

31. The composition is 1×10 ７ ~500 x 10 ７ 31. The method of any one of claims 28 to 30, comprising the intact and substantially non-viable E. coli cells and containing 124 to 62,000 endotoxin units (EU) of LPS.

32. The method of 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 within the E. coli cell or on the surface of the E. coli cell.

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