Cancer Combination Therapy

Abstract

The present invention relates to the prevention and / or treatment of cancer in a subject, wherein the subject is administered a Gram-negative bacterium in a first treatment phase and a chemotherapeutic agent in a second treatment phase.

Description

[Technical field]

[0001] The present invention relates to the field of cancer therapy. In particular, the present invention relates to methods of preventing, treating, or inhibiting the development of neoplastic disease in a subject. [Background technology]

[0002] As understanding of the underlying mechanisms involved in cancer formation and progression improves, the field of cancer therapy continues to evolve with new therapies. However, despite the great advances made in the field of cancer, chemotherapy remains the mainstay of cancer treatment.

[0003] Chemotherapy is a term used to encompass drug treatments that use cytotoxic chemicals to kill rapidly growing cells in the body. These drugs are most often used to treat cancer cells, as they have increased growth and proliferation rates compared to most other cells in the body. However, chemotherapy can be associated with serious adverse effects and many other drawbacks, such as insufficient drug concentrations in tumors, the appearance of systemic toxicity, and the induction of drug resistance. Therefore, many methods have been developed that can maintain or improve the effectiveness of chemotherapy while adequately controlling the side effects that occur.

[0004] For example, one such method is the use of chemotherapy drugs in combination with bacteria. It has been shown that such combinations can significantly improve the efficacy of chemotherapy treatment in mouse models (Non-Patent Document 1). However, the bacteria in this study were administered directly to the tumor, i.e., they needed to be in close proximity to the tumor to have an effect. In addition, it was found that to overcome the lethal toxicity of the combination of bacteria and chemotherapy, it was necessary to optimize the administration regimen so that the bacteria were administered 12 days after the chemotherapy treatment. In another method, it has been shown that bacteria, specifically Salmonella typhimurium, can be co-administered with chemotherapy drugs so that a synergistic effect is realized (see Patent Document 1).

[0005] However, despite the shift towards the use of chemotherapy in combination with additional treatments, the currently observed levels of efficacy still leave room for improvement. Thus, there remains a great need in the cancer field for methods to increase the efficacy of chemotherapy. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 106754 [Patent Document 2] International Publication No. 2010 / 079343 [Patent Document 3] International Publication No. 2000 / 68261 [Patent Document 4] International Publication No. 2019 / 110819 [Non-patent literature]

[0007] [Non-Patent Document 1] Jia et al., 2007, int. J. Cancer: 121, 666-674. [Non-Patent Document 2] Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001). [Non-Patent Document 3] Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999). [Non-Patent Document 4] Protein Methods (Bollag et al., John Wiley & Sons 1996). [Non-Patent Document 5] Spiram et al., 2021, ScienceSignaling: 14:705. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides an effective method for treating and / or preventing neoplastic disease, including metastasis, in a subject by administering live attenuated Gram-negative bacteria in combination with chemotherapy. Such methods can improve the efficacy of current chemotherapy regimens and reduce the level of normal tissue damage during / after chemotherapy. [Means for solving the problem]

[0009] In a first aspect of the invention there is a live attenuated gram-negative bacterium for use in the prevention or treatment of a neoplastic disease in a subject undergoing or to undergo chemotherapy with a chemotherapeutic agent, the live attenuated gram-negative bacterium being for administration in a first treatment phase and the chemotherapeutic agent being for administration in a second treatment phase.

[0010] In a second aspect of the invention, there is a method of preventing or treating a neoplastic disease in a subject, the method comprising administering to the subject (i) a live attenuated Gram-negative bacterium in a first treatment phase and (ii) a chemotherapeutic agent in a second treatment phase, the method resulting in increased therapeutic efficacy compared to administration of either the bacterium or the chemotherapeutic agent alone. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a schematic of the mechanism of action of the conditioning effect of Salmonella on chemotherapy. [Figure 2A] Figure 2A shows that orally administered Salmonella induces significant long-term phenotypic changes in systemic myeloid cells. Graph shows median fluorescence intensity of markers CD80, CD86, and PD-L1 on viable CD11chigh, HLA-DR+, CD11b+ / -, PDCA-1- conventional dendritic cells. n=4 or 5 mice / group. [Figure 2B](B) Orally administered Salmonella induces significant long-term phenotypic changes in systemic myeloid cells. Graph shows median fluorescence intensity of markers CD80, CD86, and PD-L1 on viable CD11c- / low, PDCA1+, HLA-DR- / Int, CD11b- plasmacytoid dendritic cells. n=4 or 5 mice / group. [Figure 2C] (C) Orally administered Salmonella induces significant long-term phenotypic changes in systemic bone marrow cells. Graphs show median fluorescence intensity of markers PD-L1, CD80, and HLA-DR on viable CD11c-, CD11b+, Ly6C+, F4 / 80- monocytes. n=4 or 5 mice / group. [Figure 2D] Figure 2D shows that orally administered Salmonella induces significant long-term phenotypic changes in systemic myeloid cells. Graph shows median fluorescence intensity of markers PD-L1, CD80 and HLA-DR on viable CD11c-, CD11b+, Ly6C-, F4 / 80+ macrophages. n=4 or 5 mice / group. [Figure 3A] Figure 3A shows the time course of Salmonella-induced phenotypic changes. A) Experimental diagram detailing the experimental timeline. [Figure 3B] Figure 3B shows the time course of Salmonella-induced phenotypic changes. B) Graph shows median fluorescence intensity of markers CD80 and CD86 as a percentage of the PBS control group mean for viable CD11chigh, HLA-DR+, CD11b+ / -, PDCA-1- conventional dendritic cells (cDCs) and viable CD11c- / low, PDCA1+, HLA-DR- / Int, CD11b- plasmacytoid dendritic cells (pDCs). Shown is the mean of n=4-5 mice / group. [Figure 3C]Figure 3C shows the time course of Salmonella-induced phenotypic changes. C) Graphs show median fluorescence intensity of markers CD80, PD-L1 and HLA-DR as percentage of PBS control mean for viable CD11c-, CD11b+, Ly6C+, F4 / 80- monocytes and CD11c-, CD11b+, Ly6C-, F4 / 80+ macrophages. Shown are the mean of n=4-5 mice / group. [Figure 4A] FIG. 4A shows that oral administration of Salmonella results in an increase in myelopoiesis. A) The graph shows the % of viable lineage negative (CD5-, CD11b-, B220-, GR-1-, Terr-119-, Ly-6B.2-) cells expressing both cKit and Sca-1, termed LKS cells, relative to total bone marrow cells. n=4-5 mice / group. [Figure 4B] Figure 4B shows that oral administration of Salmonella results in increased myelopoiesis. B) Representative flow cytometry plots showing an increase in viable LKS cells in the bone marrow of animals orally treated with Salmonella. [Figure 4C] FIG. 4C shows that oral administration of Salmonella results in increased myelopoiesis. C) % viable LKS cells relative to total bone marrow cells at day 14 after Salmonella treatment was correlated with % splenic monocytes (viable CD11c-, CD11b+, Ly6C+, F4 / 80- cells) at the same time points using Spearman rank correlation. [Diagram 5] Figure 5 shows that orally administered Salmonella induces a systemic dendritic cell hyperresponsive state that lasts for at least 14 days. n=5 mice / group in a single experiment; bars are mean + / - SEM; statistical method shown is the Mann-Whitney test. [Figure 6] FIG. 6 shows in vitro data demonstrating hyperresponsiveness of primary human monocytes to pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), with cells preconditioned with medium alone, Salmonella typhimurium or β-glucan (fungal / bacterial portion). [Figure 7A]Figure 7A shows a schematic demonstration of the timeline of oral systemic administration of Salmonella typhimurium followed by systemic administration of cyclophosphamide in a syngeneic orthotopic 4T1 mouse breast cancer model. [Figure 7B] Figure 7B shows primary tumor volumes (mm3) of 4T1 tumor-bearing mice treated with Salmonella Typhimurium MD58, cyclophosphamide, or both (n=5 mice / group). Female BALB / c mice were pretreated orally with Salmonella MD58 or PBS control. On day 0, mice were inoculated into the mammary fat pad with 4T1-Luc2-1A4 tumor cells. Primary tumor volumes were measured three times per week for 52 days. Statistical comparisons were MD58+cyclophosphamide vs. PBS+cyclophosphamide using a mixed-effects model and Sidak's multiple comparison test (degrees of significance not shown). [Figure 7C] Figure 7C shows spontaneous lung metastases in 4T1 tumor-bearing mice treated with Salmonella typhimurium MD58, cyclophosphamide, or both (n=10 mice / group). Female BALB / c mice were treated as in Figure 7A and B. Lung tumor burden was measured by bioluminescence imaging (BLI) 31 days after tumor inoculation. Statistical comparisons were individual Mann-Whitney tests for selected comparisons only. [Figure 7D] Figure 7D shows the percent survival of 4T1 tumor-bearing mice treated with Salmonella typhimurium MD58, cyclophosphamide, or both (n=15 mice / group). Female BALB / c mice were treated as in Figures 7A-C. Survival was monitored over a 52-day period. Both cyclophosphamide-treated groups had significantly better survival than the PBS+saline control, but there was no statistical difference between the PBS+cyclophosphamide and MD58+cyclophosphamide groups. The statistical method was the log-rank (Mantel-Cox) test. [Figure 8A] FIG. 8A shows a schematic demonstration of the timeline of oral systemic administration of Salmonella typhimurium followed by intravenous administration of 4T1 mouse mammary carcinoma cells and subsequent systemic cyclophosphamide. [Figure 8B]Figure 8B shows experimental lung metastases in 4T1 tumor-bearing mice treated with Salmonella typhimurium MD58, cyclophosphamide, or both (n=10 mice / group). Female BALB / c mice were treated as in Figure 8A. Lung tumor burden was measured by bioluminescence imaging (BLI) on days 5 and 12 after tumor inoculation. Each bar represents the median value for a group. Statistical comparisons are individual Mann-Whitney tests for selected comparisons only. [Figure 8C] FIG. 8C shows the percentage survival of mice bearing experimental metastases of 4T1 breast cancer cells treated with Salmonella typhimurium MD58, cyclophosphamide, or both (n=15 mice / group). Female BALB / c mice were treated as in FIG. 8A and B. Survival was monitored over a 25-day period. Statistical comparisons shown are the indicated groups vs. PBS+cyclophosphamide by log-rank (Mantel-Cox) test only. [Figure 9A] FIG. 9A shows a schematic demonstration of the timeline of oral systemic administration of Salmonella typhimurium followed by intravenous administration of 4T1 mouse breast cancer cells and subsequent intraperitoneal gemcitabine. [Figure 9B] Figure 9B shows experimental lung metastases in 4T1 tumor-bearing mice treated with Salmonella typhimurium MD58, gemcitabine, or both (n=10 mice / group). Female BALB / c mice were treated as in Figure 9A. Lung tumor burden was measured by bioluminescence imaging (BLI) 6 days after tumor inoculation. Each bar represents the median value for a group. Statistical comparisons are individual Mann-Whitney tests for selected comparisons only. [Figure 10A] FIG. 10A shows a schematic demonstration of the timeline of oral systemic administration of Salmonella typhimurium followed by intravenous administration of LL / 2-Luc-M38 mouse lung cancer cells and subsequent systemic cyclophosphamide. [Figure 10B]Figure 10B shows experimental lung metastases in LL / 2-luc tumor-bearing mice treated with Salmonella typhimurium MD58, cyclophosphamide, or both (n=10 mice / group). Female albino C57BL / 6 mice were treated as in Figure 10A. Lung tumor burden was measured by bioluminescence imaging (BLI) 10 days after tumor inoculation. Each bar represents the median value for the group. Statistical comparisons are individual Mann-Whitney tests for selected comparisons only (ns). [Figure 11A] FIG. 11A shows a schematic demonstration of the timeline of oral systemic administration of Salmonella typhimurium followed by intravenous administration of 4T1 mouse breast cancer cells and subsequent systemic administration of doxorubicin. [Figure 11B] FIG. 11B shows experimental lung metastases in 4T1 tumor-bearing mice treated with Salmonella typhimurium MD58, doxorubicin, or both (n=10 mice / group). Female BALB / c mice were treated as in FIG. 11A. Lung tumor burden was measured by bioluminescence imaging (BLI) 6 days after tumor inoculation. Each bar represents the median value for a group. Statistical comparisons are individual Mann-Whitney tests for selected comparisons only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In order that the present invention may be more readily understood, certain terms are defined first. Additional definitions are set forth throughout the detailed description.

[0013] As used herein, the term "attenuated" in the context of the present invention refers to the modification of a microorganism to reduce virulence while maintaining the viability of the microorganism, rendering it harmless to the host. This method is commonly used in the development of vaccines due to its ability to elicit a highly specific immune response while maintaining an acceptable safety profile. The development of such attenuated microorganisms may involve multiple methods, including but not limited to passaging the pathogen under in vitro conditions until toxicity is lost, chemical mutagenesis, and genetic engineering techniques, for example by inactivating or attenuating mutations. The terms "inactivating mutation" and "attenuating mutation" are used interchangeably and refer to the modification of the natural genetic code of a particular gene or the gene promoter associated with that gene, such as by changing the nucleotide code, deleting a section of nucleotides, or adding non-coding or non-natural nucleotides, such that the particular gene is not properly transcribed or translated, or is expressed into an inactive protein, eliminating or reducing to an unmeasurable extent the natural function of the gene. Thus, the mutation of a gene may affect the function of the gene or the tag that the gene encodes. It inactivates the function of proteins.

[0014] By "non-naturally occurring bacterium or bacteria" is meant a bacterial (prokaryotic) cell that has been genetically modified or "engineered" to be altered with respect to a naturally occurring cell. Such genetic modification may be, for example, the incorporation of additional genetic information into the cell, the modification of existing genetic information, or indeed the deletion of existing genetic information. This may be achieved, for example, using transfection of a recombinant plasmid into the cell or direct modifications to the bacterial genome.

[0015] As used herein, the terms "chemotherapy", "chemotherapeutic" and "chemotherapeutic agent" are used interchangeably and refer to anti-cancer drugs used in the prevention and / or treatment of cancer. Specifically, it refers to anti-cancer chemicals that prevent the growth and division of cancer cells. Chemotherapeutic agents include, but are not limited to, alkylating agents, plant alkaloids, antitumor antibiotics, metabolic inhibitors and / or topoisomerase inhibitors, or any combination thereof. Preferably, the chemotherapeutic agent may be selected from the group including cisplatin, gemcitabine, carboplatin, methotrexate, vinblastine, doxorubicin, or any combination thereof.

[0016] The terms "first treatment phase" and "second treatment phase" as used herein refer to a course of treatment in which the first treatment phase and the second treatment phase are separated in time such that there is a gap between the first and second treatment phases during which the patient / subject does not receive the Gram-negative bacteria disclosed herein or the chemotherapy disclosed herein. In other words, the Gram-negative bacteria is given to the subject being treated prior to chemotherapy. In a preferred embodiment, the first treatment phase is initiated at least one week prior to the second treatment phase. The first treatment phase may be initiated at least two weeks, at least three weeks, or at least four weeks prior to the second treatment phase. More preferably, the first treatment phase is initiated at least ten days prior to the second treatment phase. The first treatment phase may be administered to allow sufficient time for a systemic immune response to develop in the subject prior to the second treatment phase.

[0017] The term "heterologous polynucleotide" as used herein refers to a polynucleotide introduced into a Gram-negative bacterium, i.e., the introduction of a polynucleotide not previously present. The polynucleotide may be exogenous to the bacterium, and thus these terms have their usual meaning in the art. In the case of an endogenous polynucleotide, this may include the introduction of additional copies or copies of said endogenous polynucleotide or polynucleotides in a heterologous manner. An endogenous polynucleotide or polynucleotides may include the introduction of a predominant variant of said polynucleotide or polynucleotides into the host bacterium, where "predominant" refers to the ability of the heterologous polynucleotide to functionally outperform the naturally occurring endogenous equivalent. A heterologous polynucleotide in the context of the present invention may code for a target polynucleotide intended for delivery, i.e., transport and secretion in a subject. The resulting polynucleotide is also referred to herein as "cargo" or "cargo molecule". Thus, in addition to the effects disclosed herein, the Gram-negative bacteria may act as a "delivery vehicle" or "carrier" for a selected cargo, if desired. One of skill in the art will readily appreciate that the cargo delivered will depend on a number of factors, including the type and severity of the cancer being treated. Preferably, the heterologous polynucleotide encodes a therapeutic protein, such as a cytokine and / or a chemokine. More preferably, the heterologous polynucleotide encodes IL-15, IL-21, IFNα, CXCL9, CXCL10, IL-18, IL-27, or any combination thereof.

[0018] The terms "tumor," "cancer," and "neoplasm" are used interchangeably and refer to a cell or population of cells whose growth, proliferation, or survival is greater than that of normal counterpart cells, e.g., a cell proliferative or differentiative disorder. Typically, the growth is uncontrolled. The term "malignancy" refers to a tumor that is adjacent to the tumor. The term "metastasis" refers to the spread or propagation of a tumor, cancer or neoplasm to another site, location or region within a subject's body, which site, location or region is distinct from the primary tumor or cancer.

[0019] The term "effective amount" or "pharmaceutical effective amount" refers to a sufficient amount of an agent to provide a desired biological or therapeutic result. The result may be the reduction, amelioration, remission, reduction, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In the context of cancer, an effective amount may include an amount sufficient to shrink a tumor and / or reduce the rate of growth of a tumor (such as to inhibit tumor growth), or prevent or delay other inappropriate cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay the progression or prolong survival, or induce stabilization of a cancer or tumor.

[0020] In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay recurrence. A therapeutically effective amount can be administered in one or several doses. A therapeutically effective amount of a drug or combination can result in one or more of the following: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) inhibiting, delaying, slowing, or preferably stopping, to some extent, cancer cell invasion into surrounding organs; (iv) inhibiting (i.e., slowing, to some extent, or preferably stopping) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the appearance and / or recurrence of tumors; and / or (vii) relieving, to some extent, one or more symptoms associated with cancer.

[0021] For example, in treating tumors, a "therapeutically effective dose" may induce tumor shrinkage of at least about 5% compared to baseline measurements, such as at least about 10%, or about 20%, or about 60% or more. Baseline measurements may be obtained from untreated subjects.

[0022] A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such an amount based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0023] The term "immune response" in the context of the present invention refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (such as antibodies, cytokines, and complement) produced by the above cells or the liver, which results in the selective damage, destruction, or elimination of cancerous cells from the human body. Preferably, the immune response generated by the Gram-negative bacteria disclosed herein is systemic. As used herein, the terms "systemic" and "activating the whole body" are used interchangeably and, in the context of the present invention, refer to a broad immune response that extends throughout the subject's body, as opposed to a local, spatially restricted response. Preferably, the systemic immune response includes activation and / or maturation of myeloid cells, such as dendritic cells, monocytes, and / or macrophages, and, in the context of the present invention, is believed to assist in the "conditioning" of the subject's immune system so that the subject becomes more responsive to cancer treatment, such as chemotherapy. Thus, the Gram-negative bacteria may act to "prime," "boost," "amplify," "augment," "improve," "promote," "preactivate," or "promote" the immune response of a subject prior to the administration of chemotherapy. The foregoing terms are used interchangeably with the term "conditioned."

[0024] The terms "treatment" or "therapy" refer to the administration of an active agent for the purpose of curing, healing, alleviating, mitigating, altering, relieving, ameliorating, or affecting a condition (e.g., a disease), the symptoms of a condition, or preventing or delaying the onset of symptoms, complications, or biochemical indicators of a disease, or otherwise halting or inhibiting further development of a disease, condition, or disorder in a statistically significant manner.

[0025] The term "subject" as used herein includes humans and non-human animals.Preferred subjects include human patients who need to increase the effectiveness of any given chemotherapy.The method is particularly suitable for treating human patients with disorders that can be treated by enhancing immune response.In a particular embodiment, the method is particularly suitable for treating cancer in vivo.

[0026] It should be understood that the use of the alternative (e.g., "or") means either one, both, or any combination thereof of the alternatives. The indefinite article "a" or "an" as used herein should be understood to refer to "one or more" of any listed or sequenced components.

[0027] As used herein, "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how it is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice of the art. Alternatively, "about" can mean within a range of 20% or less.

[0028] Where a specific value is provided in this application and claims, unless otherwise indicated, the meaning of "about" should be presumed to be within an acceptable error range for the specific value.

[0029] As used herein, the terms "recombinant" and "recombinant strain(s)" are used interchangeably and refer, in the context of the present invention, to a Gram-negative bacterial strain that has been genetically engineered such that the bacterial DNA is modified by the introduction of new DNA. Recombinant DNA methods typically involve the introduction of new DNA via a vector, e.g., a plasmid. Such methods are well known to those skilled in the art. The use of recombinant strains of bacteria may confer advantageous properties to the strain, such as prolonged activity, eliciting a stronger immune response in the subject, or the introduction of a desired molecule.

[0030] As used herein, the terms "non-recombinant" and "non-recombinant strain(s)" are used interchangeably and refer to the fact that, in the context of the present invention, these strains do not contain eukaryotic genes or gene fragments. Thus, the non-recombinant strains disclosed herein do not act as "carrier strains" intended to deliver therapeutic molecules to a subject / patient. Thus, the non-recombinant strains disclosed herein do not encode eukaryotic heterologous DNA, or eukaryotic heterologous DNA encoding a therapeutic molecule, or eukaryotic DNA encoding a protein or fragment thereof destined to be an antigen.

[0031] Therefore, in any embodiment of the invention, the Gram-negative bacteria may be non-recombinant and do not act as a "carrier" strain for the purposes of delivery of a therapeutic molecule or delivery of DNA encoding a therapeutic molecule.

[0032] The present invention provides an effective and safe method that can increase the efficacy of chemotherapy drugs, thus improving cancer outcomes in subjects with various doses of chemotherapy.Therefore, the methods disclosed herein may also provide a method that minimizes the side effects of chemotherapy due to improved efficacy, thus reducing the length of time that a subject may need to undergo cancer treatment.

[0033] Thus in a first aspect there is a live attenuated gram-negative bacterium for use in the prevention or treatment of a neoplastic disease in a subject undergoing or to undergo chemotherapy with a chemotherapeutic agent, the live attenuated gram-negative bacterium being for administration in a first treatment phase and the chemotherapeutic agent being for administration in a second treatment phase.

[0034] Chemotherapeutic drugs are intentionally cytotoxic to allow the destruction of cancerous cells. During the process of destruction of cancerous cells, tissue damage-associated compounds (DAMPs) are released in a process called immunogenic cell death. Examples of DAMPs include heat shock proteins and HMGB1. Immunogenic cell death involves changes in the composition of the cell surface and the release of soluble mediators, which occur in a defined time sequence. Such signals are known to aid in the activation of the immune system against cancer.

[0035] The inventors of the present invention have surprisingly found that administration of Gram-negative bacteria prior to chemotherapy administration results in significant changes in bone marrow cells of the immune system of a subject, thus inducing high levels of bone marrow cell responsiveness to tissue damage-related compounds released after chemotherapy. As a result, enhanced antitumor effects are generated. Thus, live attenuated Gram-negative bacteria are considered as a "conditioning" agent for the immune system of a subject, which results in a systemic immune response, and subsequently results in the ability of the immune system of a subject to mount a more effective innate immune response and an effective adaptive immune response against neoplastic disease after chemotherapy treatment.

[0036] The live attenuated gram negative bacteria will be administered to elicit a systemic immune response, therefore the preferred routes of administration are oral, intravenous, subcutaneous, intradermal and intramuscular.

[0037] The live attenuated gram-negative bacteria may be preferably formulated for oral delivery. Formulations suitable for these delivery routes will be apparent to those skilled in the art. Preferably, the live attenuated gram-negative bacteria are liquid frozen formulations or lyophilized by a process such as freeze-drying and stored accordingly. Alternatively, the live attenuated gram-negative bacteria may be distributed in enteric coated capsules. If an encapsulated formulation is used, the lyophilized bacteria may be mixed with a bile-adsorbing resin such as cholestyramine to increase survival rate when released from the capsule into the small intestine (see US Pat. No. 5,399,323 for further details). The specific formulation of the bacteria may vary depending on various factors, such as the target patient population, i.e., young children, adolescents, or adults. The live attenuated gram-negative bacteria may be formulated in a composition with any other suitable adjuvant, diluent, or excipient. Suitable adjuvants, diluents or excipients include, but are not limited to, disodium hydrogen phosphate, soybean peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, calcium chloride, sucrose, sterile saline, and sterile water.

[0038] Oral administration of live attenuated gram-negative bacteria has numerous advantages over other routes of administration. First, oral administration is a non-invasive method of administration, which is an important factor in improving and maintaining patient compliance. This is especially true in cancer subjects who regularly undergo procedures that are most likely invasive and uncomfortable. Second, oral administration of the live attenuated gram-negative bacteria disclosed herein provides the beneficial effect of improved efficacy of chemotherapy without the need for intratumoral delivery of the live attenuated gram-negative bacteria. Thus, the utility of the present invention is not limited by the location of the tumor to be treated, for example, when the tumor is in a location that is difficult to reach.

[0039] It is anticipated that any live attenuated gram-negative bacteria capable of generating the required immune response in a subject may be used in the present invention. Examples of gram-negative bacteria for use in the present invention include, but are not limited to, Escherichia coli, Salmonella, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia, and Yersinia, or any combination thereof. Gram-negative bacteria can be readily identified and separated from gram-positive bacteria via Gram's differential staining techniques, in which gram-negative bacteria do not retain crystal violet dye.

[0040] Preferably, the live attenuated Gram-negative bacterium of the present invention may be a Salmonella bacterium. Examples of Salmonella bacterium for use in the present invention include Salmonella enterica and Salmonella bongori. Salmonella enterica may be further subdivided into serotypes or serotypes. Examples of said serotypes or serotypes for use in the present invention are Salmonella enterica typhi, Salmonella enterica paratyphi A, Salmonella enterica paratyphi B, Salmonella enterica paratyphi C, Salmonella enterica typhimurium, and Salmonella enterica enteritidis, or any combination thereof. In a preferred embodiment, the live attenuated gram-negative bacterium may be Salmonella enterica serotype typhi and / or Salmonella enterica typhimurium. In a most preferred embodiment, the live attenuated gram-negative bacterium is Salmonella enterica serotype typhi.

[0041] The live attenuated Gram-negative bacteria of the present invention may include genetically modified non-natural bacteria. As will be appreciated by those skilled in the art, genes may be mutated by a number of methods well known in the art, such as homologous recombination with a recombinant plasmid targeting the gene of interest, where an engineered gene that is homologous to the target gene is incorporated into a suitable nucleic acid vector (such as a plasmid or bacteriophage) and transfected into a target cell. The engineered homologous gene is then recombined with the native gene to replace or mutate the native gene to achieve the desired inactivation mutation. Such modifications may be in the coding portion of the gene, such as the promoter region, or any regulatory portion. As will be appreciated by those skilled in the art, any suitable genetic modification technique, such as the CRISPR / Cas system, e.g., CRISPR / Cas9, may be used to mutate the gene of interest.

[0042] Numerous methods and techniques for genetically engineering strains in this way will be known to those skilled in the art. These techniques include those required to introduce heterologous genes into bacteria via chromosomal integration or through the introduction of stable autosomal self-replicating genetic elements. Exemplary methods for genetically modifying (also referred to as "transforming" or "engineering") bacterial cells include bacteriophage infection, transduction, conjugation, lipofection, or electroporation. General discussions of these and other methods in molecular and cellular biochemistry can be found in standard textbooks such as Friedrichsson, et al., Molecular and Cellular Biochemistry, vol. 14, no. 13; ...

[0043] Thus, the live attenuated Gram-negative bacteria may have a genetic structure that has been modified in some form, for example, via genetic engineering or via chemical mutagenesis, to introduce changes, such as mutations, additions, or deletions. The live attenuated Gram-negative bacteria may be genetically modified such that the live attenuated Gram-negative bacteria is a recombinant strain that may or may not further comprise a heterologous polynucleotide encoding a polypeptide. The polypeptide may be a therapeutic molecule in itself or a molecule to support / enhance the effect of the Gram-negative bacteria. Alternatively, the live attenuated Gram-negative bacteria may be a non-recombinant strain of bacteria.

[0044] It is anticipated that any live attenuated Gram-negative bacterium capable of inducing a systemic immune response in a subject according to the present invention may be used. In a preferred embodiment, any attenuated non-pathogenic Salmonella enterica serovar Typhi or Typhimurium strain may be used. In a further preferred embodiment, the live attenuated Gram-negative bacterium may be selected from the group including Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09 (used interchangeably with "ZH9"), ZH9PA, x9633, x639, x9640, x8444, DTY88, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R, or any combination thereof. In a preferred embodiment, the live attenuated bacterium is M01ZH09 or MD58.

[0045] Thus, when the live attenuated Gram-negative bacterium is a genetically modified non-naturally occurring bacterium, it is preferred that said genetically modified non-naturally occurring bacterium is derived from a bacterium of the genus Salmonella. It is further preferred that the live attenuated Salmonella microorganism may comprise an attenuating mutation in the Salmonella pathogenicity island 2 (SPI-2) gene and / or an attenuating mutation in a second gene. Preferably, the genetically modified non-naturally occurring bacterium is derived from Salmonella and comprises both an attenuating mutation in the SPI-2 gene and an attenuating mutation in a second gene. Suitable genes and details of such live attenuated Salmonella microorganisms are as described in U.S. Patent No. 5,399,623, which is incorporated herein by reference in its entirety.

[0046] The SPI-2 gene may be a ssa gene. For example, the invention includes attenuating mutations in one or more of ssaV, ssaJ, ssaU, ssaK, ssaL, ssaM, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaD, ssaE, ssaG, ssaI, ssaC, and ssaH. Preferably, the attenuating mutation is in the ssaV or ssaJ gene. More preferably, the attenuating mutation is in the ssaV gene.

[0047] The genetically engineered Salmonella microorganism may also include an attenuating mutation in a second gene, which may or may not be present in the SPI-2 region. The mutation may be outside the SPI-2 region and involved in the biosynthesis of aromatic compounds. For example, the present invention may include an attenuating mutation in the aro gene. In a preferred embodiment, the aro gene is aroA or aroC. More preferably, the aro gene is aroC.

[0048] When the genetically engineered Salmonella microorganism contains double attenuating mutations, both mutations may be in the SPI-2 gene or both mutations may be in a second gene that may or may not be in the SPI-2 region. Preferably, the genetically engineered Salmonella microorganism contains attenuating mutations in the ssaV gene and the aro gene, more preferably the aro gene is aroC.

[0049] In yet another embodiment, the genetically engineered microorganism may be derived from a Salmonella microorganism and may contain inactivating mutations in one or more genes selected from pltA, pltB, cdtB and ttsA, and further contains attenuating mutations in one or more genes selected from aroA and / or aroC and / or ssaV. Preferably, the attenuating mutations are in aroC and ssaV. Details of the genes and mutations are as described in US Pat. No. 6,399,623, which is incorporated herein by reference in its entirety.

[0050] The live attenuated Gram-negative bacteria disclosed herein may comprise a heterologous polynucleotide encoding a target protein or peptide. The heterologous polynucleotide may encode a target protein or polypeptide that has anti-cancer properties by itself and / or the heterologous polynucleotide may encode a protein or peptide that supports and / or enhances the properties of the live attenuated Gram-negative bacteria and / or chemotherapeutic properties. Preferably, the target protein or peptide is a cytokine and / or a chemokine. More preferably, the target protein or peptide is selected from the list comprising IL-15, IL-21, IFNα, CXCL9, CXCL10, IL-18, IL-27, or any combination thereof. One skilled in the art will immediately appreciate that the specific target protein or peptide encoded by the heterologous polynucleotide will depend on a variety of factors, including but not limited to the type of cancer, the severity of the cancer, and the demographics of the patient.

[0051] The chemotherapeutic agent of the present invention may be any chemotherapeutic agent that prevents cancer cells from growing and dividing, including, but not limited to, alkylating agents, plant alkaloids, antitumor antibiotics, metabolic inhibitors and / or topoisomerase inhibitors, or any combination thereof.

[0052] Alkylating agents act by attaching alkyl groups to DNA nucleotides, causing cross-linking of DNA strands, abnormal base pairing, or DNA strand breaks, preventing DNA replication. Examples of alkylating agents include, but are not limited to, altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, and trabectedin.

[0053] Plant alkaloids act by interfering with DNA topoisomerase, inhibiting DNA replication. Plant alkaloids can also bind to microtubule proteins at the metaphase of the cell cycle, thereby causing mitotic arrest and cell death. Examples of plant alkaloids include, but are not limited to, actinomycin D, doxorubicin, and mitomycin.

[0054] Antitumor antibiotics (also called antineoplastic antibiotics) are usually derived from Streptomyces bacteria and have broad-spectrum antitumor effects on both solid and hematological tumors. Antitumor antibiotics' mechanisms include free radical DNA damage, topoisomerase II inhibition, DNA intercalation, alteration of ion transport, alteration of cell membrane fluidity and DNA binding. Examples of antitumor antibiotics include, but are not limited to, bleomycin, dactinomycin, and anthracycline.

[0055] Metabolic inhibitors act by mimicking the nucleotide bases required during DNA and RNA synthesis, thus interfering with the mechanism for cell proliferation and leading to cell death.Metabolic inhibitors are primarily effective in rapidly dividing cells, such as tumor cells.Examples of metabolic inhibitors include, but are not limited to, fludarabine, 5-fluorouracil, gemcitabine, cytarabine, and pemetrexed.

[0056] Topoisomerase inhibitors inhibit cell proliferation by preventing DNA replication, stimulating DNA damage, and inducing cell cycle arrest. There are two main classes of topoisomerases: type I topoisomerases and type II topoisomerases. These enzymes play important roles in cell division and DNA formation.

[0057] For example, topoisomerase mediates DNA strand breaks to relax DNA supercoils and allow DNA replication. Examples of topoisomerase inhibitors include, but are not limited to, irinotecan, topotecan, etoposide, and teniposide.

[0058] Preferably, the chemotherapeutic agent is selected from the group comprising cyclophosphamide, gemcitabine, methotrexate, vinorelbine, docetaxel, bleomycin, vinblastine, dacarbazine, mustine, viscristine, procarbazine, prednisoline, etoposide, epirubicin, capecitabine, folinic acid, doxorubicin, carboplatin, cisplatin, daunorubicin, oxaliplatin, 5-fluorouracil, paclitaxel, mitomycin C, mitoxantrone, irinotecan, bleomycin, pemetrexed, trifluridine / tipiracil (TAS-102), anthracyclines, topoisomerase II inhibitors, or any combination thereof. More preferably, the chemotherapeutic agent is selected from the group comprising cyclophosphamide, gemcitabine, cisplatin, carboplatin, methotrexate, vinblastine, doxorubicin, paclitaxel, oxaliplatin, and mitomycin C, or any combination thereof. One of skill in the art will immediately appreciate that subjects receiving live attenuated gram-negative bacteria and chemotherapy may also be receiving other therapies or medical interventions to enhance the effectiveness of the treatments described herein.

[0059] Chemotherapy drugs may be administered in various doses. For example, chemotherapy drugs may be administered in maximum effective doses. Maximum effective doses are the highest doses at which chemotherapy drugs are effective and have tolerable side effects. It is therefore understood that maximum effective doses are specific to the chemotherapy drugs used and the specific subjects. Thus, even when the maximum effective dose of the drug itself is achieved, the present invention can increase the efficacy of chemotherapy drugs. In addition, in some cases, subjects can benefit from improved efficacy of chemotherapy drugs to the extent that fewer cycles of chemotherapy are required, thus reducing the chances that subjects will experience adverse side effects from chemotherapy drugs.

[0060] In other embodiments, the chemotherapeutic agent may be administered to a subject at a dose lower than the maximally effective dose, i.e., a "sub-maximal dose," and the efficacy is increased through administration of a live attenuated Gram-negative bacterium as described herein. Without being bound by theory, administration of a sub-maximal dose of the chemotherapeutic agent is expected to provide a particularly effective treatment when used in combination with the Gram-negative bacterium described herein, with the dose damaging the cancer cells without killing the Gram-negative bacterium (Non-Patent Document 5). Damaging the cancer cells in the presence of a Salmonella-conditioned immune system is expected to increase antigen presentation due to increased Salmonella-conditioned induced uptake of damaged cancer cells by conditioned antigen-presenting cells. Such changes are expected to result in a greater degree, magnitude, and duration of specific adaptive anti-cancer immune responses. Thus, in these instances, the desired efficacy of the selected chemotherapy can be produced while chemotherapy-induced toxicity is minimized or at least reduced from the start of the treatment regimen.

[0061] The live attenuated Gram-negative bacteria and chemotherapy of the present invention are administered to a subject in need of protective or curative cancer treatment in a well-defined time regimen, i.e., the Gram-negative bacteria are administered in a first treatment phase and the chemotherapy is administered in a second treatment phase, i.e., the Gram-negative bacteria are administered to the patient or subject before the chemotherapy, so that the immune system of the treated subject has the necessary time to be conditioned in response to the live attenuated Gram-negative bacteria, as described above. The present invention described herein is therefore in stark contrast to the disclosure of Friedrichsson et al., J. Immunol. 1999, 123:1311-1323, who found that the bacteria must be administered 12 days after the administration of chemotherapy to avoid lethal toxicity. Therefore, the good safety profile of the treatment regimen combined with the effective results described herein is a surprising finding of the present invention.

[0062] The present invention discloses that a live attenuated Gram-negative bacterium is administered to a subject in a first treatment phase, and a chemotherapeutic agent is administered to a subject in a second treatment phase. In a preferred embodiment, the first treatment phase is initiated at least one week before the second treatment phase. More preferably, the first treatment phase is initiated at least 10 days before the second treatment phase. For example, the first treatment phase may be initiated 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 month, 2 months, or 3 months before the second treatment phase, i.e., the start of administration of the chemotherapeutic agent. Each of the treatment phases may include repeated administration of either the Gram-negative bacterium or the chemotherapeutic agent. In a preferred embodiment, the live attenuated Gram-negative bacterium is administered before a chemotherapy cycle, and administration of the Gram-negative bacterium may be repeated before future additional chemotherapy cycles as long as required by the subject being treated. It is further understood that the terms "first treatment phase" and "second treatment phase" refer to a single "cycle" of the prophylactic / curative treatment disclosed herein, and that multiple cycles of said prophylactic / curative treatment are possible depending on the requirements of the relevant subject.

[0063] The present invention provides live attenuated Gram-negative bacteria that, when used in combination with chemotherapy, can be used in the prevention and / or treatment of neoplastic disease, and / or a second disease associated with a neoplastic disease. In one embodiment, the neoplastic disease may be a solid cancer and / or a hematological malignancy. Neoplasms, tumors and cancers include benign, malignant, metastatic and non-metastatic types, and include neoplasms, tumors or cancers of any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.), or neoplasms, tumors, cancers or metastases that are progressing, worsening, stabilized or in remission.

[0064] It is anticipated that live attenuated Gram-negative bacteria for use in the prevention or treatment of neoplastic disease in subjects undergoing or to undergo chemotherapy with chemotherapeutic agents will reduce metastasis of primary tumors. In particular, protective Salmonella treatment in combination with chemotherapeutic agents will reduce metastasis to the lungs.

[0065] A metastatic cancer is a cancer that has spread from its original primary site to a secondary site within a subject's body. The newly formed secondary pathological site is called a metastasis. The process of metastasis includes five steps: invasion, intravasation, circulation, extravasation, and colonization.

[0066] Tumor cells may undergo a transdifferentiation process of epithelial-mesenchymal transition, where they may develop the ability to penetrate and invade adjacent tissues. Such tumor cells, able to cross the basement membrane and extracellular matrix (at the primary site), may invade lymphatic or vascular circulation (intravasation). Once in the circulation, tumor cells may proceed to a process of extravasation, where the vascular basement membrane and extracellular matrix are invaded at secondary sites, allowing the cells to attach and colonize the secondary sites. In addition to metastasis through the vasculature, tumor cells may directly invade the adjacent tissues surrounding the primary site.

[0067] Common metastatic sites include lungs, lymph nodes, liver, and bone. Certain types of cancer may also be associated with some common metastatic sites. For example, breast cancer may metastasize to lungs, liver, bone, and / or brain. Bladder cancer may metastasize to lungs, liver, and bone. Lung cancer may metastasize to the adrenal glands, bone, brain, liver, and / or other lungs.

[0068] Metastatic cancer may be classified as stage 3 cancer, where the cancer has spread to secondary sites in surrounding tissues or lymph nodes. Metastatic cancer may also be classified as stage 4 cancer, where the cancer has spread to secondary sites in another organ, which may be far from the primary site. Metastatic cancer may be staged using the TNM system, where T describes the size of the tumor on a scale of 1 to 4; N describes the degree of spread to lymph nodes on a scale of 0 to 3, and M describes the degree of metastasis on a scale of 0 to 1.

[0069] Cancers that may be treated by the present invention include, but are not limited to, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus cells or neoplasms. In addition, the cancer may be of the following specific histological types, but is not limited to: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant cell and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; hairy cell carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; mixed hepatomas; trabecular adenocarcinomas; adenoid cystic carcinomas; adenocarcinomas in adenomatous polyps; familial adenocarcinomatous polyposis coli; solid tumors; malignant carcinoid tumors; bronchioloalveolar adenocarcinomas; papillary adenocarcinomas; chromophobe carcinomas; Eosinophilic carcinoma;Eosinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Follicle-type papillary adenocarcinoma;Non-encapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrial carcinoma;Skin adnexal carcinoma;Apocrine gland carcinoma;Sebaceous gland carcinoma;Earwax gland carcinoma;Mucoepidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Serous papillary cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease of the breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Malignant thymoma;Malignant ovarian stromal tumor;Malignant thecoma; Malignant granulosa cell tumor;Malignant androblastoma;Sertoli cell carcinoma;Malignant Leydig cell tumor;Malignant lipid cell tumour, malignant;Malignant paraganglioma;Malignant extramammary paraganglioma;Pheochromocytoma;Glomus tumor;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma of giant pigmented nevus;Epithelioid cell melanoma;Malignant blue nevus;Sarcoma;Fibrosarcoma;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stroma sarcoma;Mixed tumor;Müllerian mixed tumor;Nephroblastoma;Hepa toblastoma;carcinosarcoma;malignant mesenchymoma;malignant Brenner tumor;malignant phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonal carcinoma;malignant teratoma;malignant ovarian goiter;choriocarcinoma;malignant mesonephric carcinoma;angiosarcoma;malignant hemangioendothelioma;Kaposi's sarcoma;malignant hemangiopericytoma;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;malignant chondroblastoma;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;malignant odontogenic tumor;ameloblastoma;malignant ameloblastoma;ena Melanoblastoma;Malignant pinealoma;Chordoma;Malignant glioma;Epicenteric tumor;Astrocytoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Anaplastic neuroectodermal;Cerebellar tumor;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neuroendocrine tumor;Malignant meningioma;Neurofibrosarcoma;Malignant neurilemmoma;Malignant granular cell tumor;Malignant lymphoma;Hodgkin's disease;Hodgkin's disease 's);lateral granuloma;malignant small lymphocytic lymphoma;malignant diffuse large cell lymphoma;malignant follicular lymphoma;mycosis fungoides;other specific types of non-Hodgkin's lymphoma;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphoblastic leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia.

[0070] Preferably, the solid cancer and / or hematological malignancy may be a cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colon cancer, bladder cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, or sarcoma.

[0071] In a preferred embodiment, the treatment regimen of the invention will be administered to a patient with breast cancer with metastasis, said metastasis being within the lungs.

[0072] Administration of live attenuated Gram-negative bacteria in combination with a chemotherapeutic agent may reduce tumor burden, tumor growth, tumor progression, and / or tumor metastasis. The term "tumor burden" refers to the number of cancer cells, the size of a tumor, or the amount of tumor metastases in a subject. Reduced tumor burden is associated with improved treatment response and survival outcomes. Reduced tumor burden may be indicated, for example, by a reduction in tumor volume, size, or mass in a subject, a reduction in tumor growth rate, a reduction in tumor progression rate, a reduction in tumor metastasis rate, a reduction in the number of tumor metastases, a reduction in the number of tumors, or an overall reduction in the amount of cancer in a subject. Thus, tumor burden may be quantified, for example, by measuring tumor volume, tumor mass, tumor size (diameter, circumference, etc.). Methods for quantifying tumor burden are known in the art, including, but not limited to, clinical imaging (MRI, PET-CT, etc.).

[0073] The amount of live attenuated Gram-negative bacteria administered to a subject is sufficient to elicit a systemic immune response in the subject, such that the subject's immune system is effectively conditioned prior to receiving the chemotherapy agent. The immune response initiated by administration of the Gram-negative bacteria may itself be therapeutic, or may be subtherapeutic, requiring a subsequent administration of the chemotherapy agent for the desired response. The live attenuated Gram-negative bacteria may be administered in an amount of 10 4 ~10 12 CFU, where CFU is colony forming units. For example, a suitable dose may be between 10 4 ~10 5 Between CFU, 10 4 ~10 6 Between CFU, 104 ~10 7 Between CFU, 10 4 ~10 8 Between CFU, 10 4 ~10 9 Between CFU, 10 4 ~10 10 Between CFU, 10 4 ~10 11 Between CFU, 10 4 ~10 12 Between CFU, 10 5 ~10 6 Between CFU, 10 5 ~10 7 Between CFU, 1 0 5 ~10 8 Between CFU, 10 5 ~10 9 Between CFU, 10 5 ~10 10 Between CFU, 10 5 ~10 11 Between CFU, 10 5 ~10 12 Between CFU, 10 6 ~10 7 Between CFU, 10 6 ~10 8 Between CFU, 10 6 ~10 9 Between CFU, 10 6 ~10 10 Between CFU, 10 6 ~10 11 Between CFU, 10 6 ~10 12 Between CFU, 10 7 ~10 8 Between CFU, 10 7 ~10 9 Between CFU, 10 7 ~10 10 Between CFU, 10 7 ~10 11 Between CFU, 10 7 ~10 12 Between CFU, 10 8 ~10 9 Between CFU, 10 8 ~10 10 Between CFU, 10 8 ~10 11Between CFU, 10 8 ~10 12 Between CFU, 10 9 ~10 10 Between CFU, 10 9 ~10 11 Between CFU, 10 9 ~10 12 Between CFU, 10 10 ~10 11 Between CFU, 10 10 ~10 12 Between CFU or 10 11 ~10 12 It may be between CFU.

[0074] The live attenuated Gram-negative bacteria disclosed herein may generate a systemic immune response in a subject when used. Preferably, the live attenuated Gram-negative bacteria generate a systemic immune response that results in increased activation and / or maturation of myeloid cells. Examples of such myeloid cells include, but are not limited to, conventional dendritic cells, plasmacytoid dendritic cells, monocytes and / or macrophages. Thus, a systemic immune response in the context of the present invention may refer to long-term phenotypic changes in the circulating and / or systemic myeloid compartment that, when used in combination with chemotherapeutic agents, result in either additive or synergistic effects, thus improving patient outcomes. Other forms or readouts of the systemic immune response will be apparent to those skilled in the art and include Salmonella-specific antibody production and proliferation of Salmonella-specific T cells. Such measurements may be used to provide a measure of the effectiveness of the treatment.

[0075] In a second aspect of the invention, there is a method of preventing or treating a neoplastic disease in a subject, the method comprising administering to the subject (i) a live attenuated Gram-negative bacterium in a first treatment phase and (ii) a chemotherapeutic agent in a second treatment phase, the method resulting in increased therapeutic efficacy compared to administration of either the bacterium or the chemotherapeutic agent alone.

[0076] The methods of the invention may therefore be used to reduce or inhibit metastasis of a primary tumor or cancer to other sites, or the formation or establishment of metastatic tumors or cancer at other sites distal to the primary tumor or cancer, thereby inhibiting or reducing relapse of the tumor or cancer, or progression of the tumor or cancer.The invention thus provides a detectable or measurable improvement, i.e., a therapeutic benefit or beneficial effect, in a given subject's condition, such as reducing or reversing one or more adverse (physical) symptoms or consequences associated with a cell proliferative or cell hyperproliferative disorder, a neoplasm, a tumor or cancer, or the presence of metastases.

[0077] Thus, the method of the present invention, involving administration of a live attenuated Gram-negative bacterium in a first treatment phase and a chemotherapeutic agent in a second treatment phase, is a combination therapy that has the potential to elicit a strong and durable immune response with increased therapeutic benefit. The additivity or synergy of the therapeutic combinations disclosed herein may result in lower levels of chemotherapy being required, resulting in reduced adverse effects with a more favorable toxicity profile.

[0078] The therapeutic benefit or beneficial effect is any objective or subjective transient, temporary, or long-term improvement in a condition or pathology associated with or caused by a cell proliferation or cell hyperproliferative disorder, such as a neoplasm, tumor or cancer, or metastasis, or a reduction in the onset, severity, duration, or frequency of adverse symptoms. It may lead to improved survival. A sufficient clinical endpoint of the treatment method according to the invention is realized, for example, when there is an incremental or partial reduction in the severity, duration, or frequency of one or more associated pathologies, adverse symptoms, or complications, or an inhibition or amelioration of one or more physiological, biochemical, or cellular manifestations or characteristics of a cell proliferation or cell hyperproliferative disorder, such as a neoplasm, tumor or cancer, or metastasis.

[0079] Thus, the therapeutic benefit or improvement may be, but is not limited to, the destruction of the target proliferative cells (e.g., neoplasm, tumor or cancer, or metastasis) or the ablation of one or more, most or all pathologies, adverse symptoms, or complications associated with or caused by a cellular hyperproliferative disorder, such as a neoplasm, tumor or cancer, or metastasis. However, it is not necessary for the cure or complete destruction of all the target proliferative cells (e.g., neoplasm, tumor or cancer, or metastasis) or the ablation of all pathologies, adverse symptoms, or complications associated with or caused by the cellular proliferation or cellular hyperproliferative disorder, such as a neoplasm, tumor or cancer, or metastasis. For example, partial destruction of a tumor or cancer cell mass or stabilization of tumor or cancer mass, size, or cell number by inhibiting tumor or cancer progression or worsening can reduce mortality and extend lifespan, even if only a portion or a large amount of tumor or cancer mass, size, or cells remain, even if only for a few days, weeks, or months.

[0080] Specific non-limiting examples of therapeutic benefit include reducing the volume (size or cell mass) or cell number of a neoplasm, tumor or cancer, or metastasis; inhibiting or preventing (e.g., stabilizing) an increase in the volume of a neoplasm, tumor or cancer; slowing or inhibiting the progression, progression or metastasis of a neoplasm, tumor or cancer; or inhibiting the proliferation, growth or metastasis of a neoplasm, tumor or cancer.

[0081] The methods of the invention may not take effect immediately, for example, following treatment, the number or mass of neoplastic, tumor or cancer cells may increase, but may be followed by an eventual stabilization or reduction in size or number of tumor cell mass, cells in a given subject over time.

[0082] Additional adverse symptoms and complications associated with neoplasia, cancer, and metastasis that may be inhibited, reduced, diminished, delayed, or prevented include, for example, nausea, loss of appetite, lethargy, pain, and discomfort. Thus, partial or complete reduction or reduction in the severity, duration, or frequency of adverse symptoms or complications associated with or caused by a hyperproliferative disorder of cells, improvement in the subject's quality of life and / or well-being, such as energy, appetite, mental well-being, etc., are all specific, non-limiting examples of therapeutic benefit.

[0083] Therapeutic benefit or improvement therefore can also include a subjective improvement in the quality of life of the treated subject. In additional embodiments, the method extends or increases the lifespan (survival rate) of the subject. In further embodiments, the method improves the quality of life of the subject.

[0084] It is anticipated that the present invention may be particularly suitable for individuals who have been refractory to previous treatment with chemotherapy. "Refractory" is intended to refer to any neoplastic disease that does not respond to treatment, e.g., chemotherapy drugs. It is also anticipated that the present invention may be particularly suitable for individuals who have been previously low-, medium-, or high-responders to previous chemotherapy treatments. The determination of whether a subject is a responder or non-responder may be based on past response to chemotherapy treatments, or via biomarker analysis of biological samples to identify markers that are indicative of subjects that have responded or have not responded to a particular treatment.

[0085] The inventors of the present invention have surprisingly found that administering a live attenuated Gram-negative bacterium to a subject prior to administration of a chemotherapeutic agent results in increased effectiveness of said chemotherapy.The invention is further described with reference to the following non-limiting examples. EXAMPLES

[0086] Example 1 - Conditioning a subject's immune system with gram-negative bacteria allows for increased response rates to chemotherapy The present invention provides a method in which Gram-negative bacteria can be used to effectively and systemically condition a subject's immune system to enhance the effectiveness of a subsequently administered chemotherapeutic agent. Without being bound by theory, a possible mechanism of action demonstrating how such an effect occurs is provided in FIG.

[0087] Example 2 – Induction of long-term phenotypic changes in systemic bone marrow cells Adult female BALB / c mice were cultured at 1 × 10 9 CFU of Salmonella enterica serovar Typhimurium strain MD58 (ΔaroC) were orally administered. After 21 days, spleens were harvested, single cell suspensions were made, and flow cytometry staining was performed. high , HLA-DR + , CD11b + / - , PDCA-1 - CD11c on conventional and surviving dendritic cells - / low , PDCA1 + , HLA-DR - / Int , CD11b - The median fluorescence intensity of markers CD80, CD86, and PD-L1 on plasmacytoid dendritic cells was measured (see Figures 2A and 2B). - , CD11b + , Ly6C + , F4 / 80 - CD11c on and in monocytes - , CD11b + , Ly6C - , F4 / 80 + The median fluorescence intensity of the markers PD-L1, CD80, and HLA-DR on macrophages was also measured (see Figures 2C and 2D).

[0088] As can be seen in FIG. 2, various cell markers of myeloid cells, including conventional dendritic cells, plasmacytoid dendritic cells, monocytes and macrophages, showed a significant increase at 21 days after treatment with Salmonella, suggesting that the effects on immune cell activation after treatment with Salmonella can be sustained for a significant period of time.

[0089] Example 3 – Time course of Salmonella-induced phenotypic changes To explore the kinetics of the activation / maturation phenotype changes of myeloid cells observed in Figure 2, we performed a time course study. Adult female BALB / c mice were treated with 1 × 10 9 Mice were orally treated with CFU of Salmonella enterica serovar Typhimurium strain MD58(ΔaroC). After 1, 14, 21, or 42 days, spleens and femurs were harvested, single-cell suspensions were made, and flow cytometry staining was performed (see experimental schematic in Figure 3A).

[0090] Activation markers on systemic conventional (cDC) and plasmacytoid (pDC) dendritic cells were shown to be upregulated after oral Salmonella challenge, peaking at 3 weeks post-challenge, and returning to near basal levels by 6 weeks post-challenge (Figure 3B). Activation markers on systemic monocytes and macrophages were also shown to be upregulated after oral Salmonella challenge, peaking at 3 weeks post-challenge, and returning to near basal levels by 6 weeks post-challenge (Figure 3C).

[0091] Example 4 – Oral administration of Salmonella increases bone marrow hematopoiesis Adult female BALB / c mice were cultured at 1 × 10 9 Mice were orally treated with CFU of Salmonella enterica serovar Typhimurium strain MD58(ΔaroC). Flow cytometric staining of isolated bone marrow cells was performed 1, 14, 21, or 42 days later.

[0092] The number of hematopoietic stem cells / multipotent progenitors in the bone marrow is shown to be significantly increased after 2 weeks of oral administration (see FIG. 4A). Representative flow cytometry plots also demonstrate an increase in viable LKS cells in the bone marrow of animals orally treated with Salmonella (see FIG. 4B). In addition, the % of viable LKS cells relative to total bone marrow cells at 14 days after Salmonella treatment was significantly higher than the % monocytes (viable CD11c) at the same time point using Spearman rank correlation. - , CD11b + , Ly6C + , F4 / 80 - cells) (see Figure 4C).

[0093] Thus, the data herein support the hypothesis that administration of Salmonella conditions the immune system, particularly the myeloid arm of the immune system. These data demonstrate a long-term effect of Salmonella conditioning on the immune system, as demonstrated by the Salmonella-induced conditioning changes over a 3- to 6-week period. These changes may be systemically / centrally mediated, as suggested by the positive correlation between bone marrow hematopoietic progenitor cell numbers and splenic monocyte numbers.

[0094] Example 5 – Oral administration of Salmonella induces a systemic dendritic cell hyperresponsive state that persists for at least 14 days Adult female BALB / c mice were cultured at 1 × 10 9 Fourteen days later, spleens were harvested, single-cell suspensions were made, and CD11c-expressing cells were enriched by magnetic separation to yield a concentration of 1 × 10 5 Cells / well were incubated with the indicated stimuli (TLR9, TLR2 / 6 or TLR4 / 2 agonists, or controls) for 24 hours. IL-6 in the supernatants was measured by LegendPlex assay (see FIG. 5).

[0095] As can be seen from Figure 5, CD11c expressing cells from animals treated with Gram-negative bacteria, e.g., Salmonella, demonstrated increased IL-6 secretion (an indicator of the level of immune reactivity of the cells) in response to various stimuli compared to the vehicle control group, further supporting that administration of the Gram-negative bacteria of the present invention results in conditioning of immune cells, making them more responsive to the next stimulus, e.g., DAMP induced by a chemotherapeutic drug.

[0096] As demonstrated in Examples 1-5, the experimental data disclosed herein show that treatment with live attenuated Gram-negative bacteria, such as Salmonella, results in an unexpected long-term activation of multiple immune cell types (e.g., dendritic cells, monocytes, and macrophages) and an increase in hematopoietic stem cells / multipotent progenitors in the bone marrow. Without being bound by theory, it is believed that the systemic response induced by administration of such live attenuated Gram-negative bacteria can condition the immune system of a subject or patient, thereby enhancing the antitumor activity of said chemotherapy when administered in combination with a chemotherapeutic agent. It is believed that the systemic modifications observed by administering live attenuated Gram-negative bacteria, i.e., activation of multiple immune cell types and effect on the modeling system (as demonstrated herein) in conjunction with the intestinal uptake of said live attenuated Gram-negative bacteria, are responsible for the increased antitumor effect. One skilled in the art will immediately appreciate that the broad range of systemic effects induced by live attenuated Gram-negative bacteria can condition the immune system of a patient / subject in such a way that beneficial effects are observed regardless of the chemotherapeutic agent used. In addition, the use of Gram-negative bacteria to condition the immune system of a subject / patient may be particularly advantageous for those patients / subjects who have demonstrated resistance to chemotherapy when used alone.

[0097] Example 6 - In vitro conditioning of primary human monocytes with Gram-negative bacteria results in hyperresponsiveness to subsequent challenge with PAMPs and DAMPs Primary human monocytes were conditioned in vitro with Salmonella for 30 minutes, washed with antibiotics to remove all Salmonella, and then rested for 6 days in the presence of M-CSF. After the 6-day resting period, human monocytes were stimulated with PAMPs, DAMPs, or a media-only control, and the levels of TNF-α release were measured via ELISA.

[0098] The present inventors surprisingly found that cells conditioned with the Gram-negative bacteria disclosed herein exhibited hyperresponsiveness to both PAMPs and DAMPs, as reflected in the release levels of TNFα (FIG. 6). This is in contrast to the established conditioning agent β-glucan, which only induced hyperresponsiveness in response to challenge with PAMPs and showed no difference in TNFα release levels relative to media-treated cells (negative control). In contrast, cells conditioned with the Gram-negative bacteria disclosed herein and then challenged with HMGB1 (a known DAMP) showed an approximately 5-fold increase in TNFα release compared to the media-only control.

[0099] Example 6 – Treatment of Salmonella in Combination with Cyclophosphamide, Gemcitabine or Doxorubicin method Preparation of bacterial cells A dilution of attenuated Salmonella typhimurium strain (MD58) was administered at 1 × 10 9 Made up in PBS as needed to achieve CFU / 100 μl.

[0100] Orthotopic 4T1 mammary tumor model Six to seven week old female BALB / c mice were cultured at 1 × 10 9 Mice were pretreated orally with CFU Salmonella MD58 or PBS control. On day 0, mice were 5 4T1-Luc2-1A4 tumor cells were inoculated into the fourth mammary fat pad. On days 10 and 15 after tumor challenge, mice received 100 mg / kg cyclophosphamide intraperitoneally (IP) (Figure 7A). Primary tumor volumes were measured three times per week, lung tumor burden was measured by bioluminescence imaging (BLI), and survival was monitored until 52 days after tumor inoculation.

[0101] Experimental 4T1 lung metastasis model Six to seven week old female BALB / c mice were cultured at 1 × 10 9 Mice were pretreated orally with 1 × 10 CFU of Salmonella MD58 or PBS control. 5Mice were inoculated intravenously with 4T1-Luc2-1A4 tumor cells at 40 mg / kg for 3 h. Three days after tumor challenge, mice received 40 mg / kg cyclophosphamide (FIG. 8A) or 60 mg / kg gemcitabine (FIG. 9A) intraperitoneally (IP), or 10 mg / kg doxorubicin intravenously (IV) (FIG. 11A). Lung tumor growth was monitored by bioluminescence imaging (BLI) and survival was monitored.

[0102] LL / 2 lung tumor model Six to seven week old female albino C57BL / 6 mice were cultured at 1 × 10 9 Mice were pretreated orally with 1 × 10 CFU of Salmonella MD58 or PBS control. 5 Mice were inoculated intravenously with LL / 2-Luc-M38 tumor cells at 100 μg / mL. Three days after tumor challenge, mice received 40 mg / kg cyclophosphamide intraperitoneally (FIG. 10A). Lung tumor growth was monitored by bioluminescence imaging (BLI).

[0103] In vivo bioluminescence imaging Lung metastases in all models were monitored by bioluminescence imaging (BLI). Ten minutes after intraperitoneal (IP) injection of D-luciferin (Promega, E1605, 3 mg / 20 g body weight), mice were imaged with an IVIS Spectrum (Perkin Elmer, Waltham, MA) using a single thoracic constant area ROI for each individual animal. Image analysis was performed with Living Image 4.7.1 (Perkin Elmer, Waltham, MA). Binning and exposure times were adjusted to obtain at least several hundred counts per image and to avoid saturation of the CCD chip.

[0104] result Systemic administration of cyclophosphamide in a syngeneic orthotopic 4T1 mouse breast cancer model following oral challenge with Salmonella typhimurium reduces primary orthotopic mammary tumor burden, lung metastases (ns), and improves survival (ns) compared to cyclophosphamide monotherapy (Figure 7A-D).

[0105] Oral systemic administration of Salmonella typhimurium followed by intravenous administration of 4T1 mouse mammary carcinoma cells and subsequent systemic cyclophosphamide inhibits experimental pulmonary metastases in 4T1 tumor-bearing mice. It reduced migration and reduced lung tumors on days 5 and 12 after tumor inoculation (Figures 8A-C).

[0106] Intravenous administration of 4T1 mouse breast cancer cells followed by oral systemic administration of Salmonella typhimurium, and subsequent intraperitoneal gemcitabine, reduced experimental 4T1 metastasis to the lungs (Figure 9A-B).

[0107] Oral administration of Salmonella Typhimurium followed by intravenous administration of LL / 2-Luc-M38 mouse lung cancer cells and subsequent systemic cyclophosphamide reduced the growth of LL / 2 lung tumors (Figure 10A-B). Oral administration of Salmonella Typhimurium followed by intravenous administration of 4T1 mouse breast cancer cells and subsequent intravenous doxorubicin reduced experimental 4T1 metastasis to the lung (Figure 11A-B).

[0108] Thus, the present inventors have shown that administration of a Gram-negative bacterium in a first treatment phase followed by administration of a chemotherapeutic agent in a second treatment phase allows for a more effective treatment than if either treatment was used alone, and thus the present invention provides a method by which outcomes for cancer patients may be improved.

Claims

1. 1. A composition comprising a live attenuated gram-negative bacterium for use in the prevention or treatment of neoplastic disease in a subject undergoing or to undergo chemotherapy with a chemotherapeutic agent, comprising: the live attenuated gram-negative bacterium is for administration in a first treatment phase and the chemotherapeutic agent is for administration in a second treatment phase; the live attenuated Gram-negative bacterium does not contain a heterologous polynucleotide encoding a polypeptide; the polypeptide is a therapeutic molecule; composition.

2. 10. The composition of claim 1, wherein the live attenuated gram-negative bacterium is formulated for oral administration.

3. 3. The composition of claim 1 or 2, wherein the live attenuated Gram-negative bacterium is a Salmonella bacterium.

4. 4. The composition of claim 3, wherein the live attenuated gram-negative bacterium is Salmonella enterica.

5. 5. The composition of claim 4, wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi and / or Salmonella enterica serovar Typhimurium.

6. 10. The composition of claim 1, wherein the live attenuated Gram-negative bacterium is a genetically modified, non-naturally occurring bacterium.

7. 7. The composition of claim 6, wherein the genetically modified non-naturally occurring bacterium comprises an attenuating mutation in a Salmonella pathogenicity island 2 (SPI-2) gene and in a second gene.

8. 8. The composition of claim 7, wherein the SPI-2 gene is the ssaV gene and the second gene is the aro gene.

9. The chemotherapeutic agent is methotrexate, vinorelbine, docetaxel, bleomycin , vinblastine, dacarbazine, mustine, vincristine, procarbazine, prednisolone, etoposide, epirubicin, capecitabine, folinic acid, doxorubicin, carboplatin, cisplatin, cyclophosphamide, daunorubicin, oxaliplatin, 5-fluorouracil, gemcitabine, paclitaxel, mitomycin C, mitoxantrone, irinotecan, pemetrexed, trifluridine / tipiracil (TAS-102), anthracyclines, topoisomerase II inhibitors, and any combination thereof.

10. 10. The composition of claim 9, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, gemcitabine, carboplatin, methotrexate, vinblastine, doxorubicin, paclitaxel, oxaliplatin, mitomycin C, and any combination thereof.

11. The composition described in claim 1, wherein the first treatment phase is initiated to allow sufficient time for a systemic immune response to occur in the subject before the second treatment phase.

12. 12. The method of claim 11, wherein the first treatment phase is initiated at least one week before the second treatment phase.

13. 13. The method of claim 12, wherein the first treatment phase is initiated at least 10 days before the second treatment phase.

14. The composition of claim 11, wherein the first treatment phase is initiated 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 month, 2 months, or 3 months before the second treatment phase.

15. The composition of claim 1 , wherein the neoplastic disease is associated with a solid tumor and / or a hematological malignancy.

16. 16. The composition of claim 15, wherein the neoplastic disease is associated with a cancer selected from the group consisting of prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colon cancer, bladder cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, and sarcoma.

17. The composition of claim 1 , wherein the subject has breast cancer with metastases in the lung.

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