Cancer therapy using live attenuated bacteria

Abstract

The present invention relates to the field of cancer therapy. In particular, the present invention relates to a first composition comprising a live attenuated bacterium for use in treating, preventing, reducing, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the same or different live attenuated bacterium as the live attenuated bacterium of the first composition, and a method thereof.

Description

[Technical field]

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

[0002] The field of cancer therapy is constantly evolving with new therapies as our understanding of the underlying mechanisms associated with cancer formation and progression improves.Currently, patients diagnosed with cancer have numerous treatment options, including surgery, radiation therapy, chemotherapy and immunotherapy.However, despite the variety of treatment options available to cancer patients, the majority of patients relapse, remain refractory to treatment, or experience toxic side effects that result in the discontinuation of treatment.Therefore, there is a continuing need for new, effective and safe cancer treatment strategies.

[0003] One therapeutic approach that has been adopted in the field of oncology is that of using compositions containing live attenuated bacteria. For example, one therapeutic strategy that has been at least partially successful in the field of bladder cancer is that of intravesical immunotherapy using Bacillus Calmette-Guerin (BCG). However, while BCG therapy has had some clinical success, it is estimated that nearly 90% of patients experience some type of adverse side effect, ranging from cystitis to sepsis and death, and that patient compliance is low in some cases due to the demanding dosing schedule (six consecutive weekly intravesical instillations). Additionally, BCG is used in combination with other therapies in an effort to enhance efficacy, but these combination therapies do not improve the side effects disclosed above.

[0004] There have been separate attempts to develop compositions comprising attenuated recombinant bacteria and / or attenuated tumor-targeting bacteria, including attenuated Salmonella Typhimurium, for inhibiting the growth of solid tumor cancer or reducing its volume, such as WO 03 / 063593 (Vion Pharmaceuticals); US Patent Publication No. 2007 / 0298012 (I. King & LM Zheng); WO 2009 / 098246 (Aeterna Zentaris GmbH); WO 2006 / 076678 (Univ. John Hopkins). Salmonella Typhi strains have also been proposed for use in the oncology field, for example for the treatment of bladder cancer (see WO 2014 / 180929). Zhou et al., (Nature Reviews Cancer, 18:12:727-743, 2018) have disclosed engineered tumor-targeting bacteria to fight cancer, and Chorobik et al., (Acta Biochimica Polonica, 60:3:285-297, 2013) have disclosed Salmonella as a possible therapeutic agent in the context of cancer. However, numerous challenges remain with this approach, including suboptimal levels of anticancer activity.

[0005] Thus, there remains a significant need in the field of cancer for new therapeutic strategies. In particular, there remains a need for methods that have improved efficacy of said treatment in individuals who show a positive response. At the same time, there remains a significant need for methods that show sustained efficacy in individuals who are otherwise refractory to said treatment. Finally, there remains a need for therapeutic strategies that improve patient compliance. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides an effective method for treating, preventing and / or preventing recurrence of neoplastic disease in a subject by administering a first and a second composition comprising both live attenuated bacteria.The inventors have surprisingly found that this combination results in a more effective therapy than when the subject is treated with a single composition comprising live attenuated bacteria, i.e., a synergistic or additive effect is achieved.It is believed that the systemic modification observed from the administration of the live attenuated bacteria of the first composition, in combination with the systemic immune memory generated against said live attenuated bacteria, can enhance the antitumor activity of the live attenuated bacteria of the second composition when the second composition is administered locally to the site of neoplastic disease through the bystander effect of enhancing innate immune response and recruiting effector memory cells.

[0007] Thus, in a first aspect, the present invention provides a first composition comprising a live attenuated bacterium for use in treating, preventing, reducing, inhibiting, preventing recurrence or controlling a neoplastic disease in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the same or different live attenuated bacterium as the live attenuated bacterium of the first composition, the first composition being formulated for oral, intravenous, intranasal, intradermal or subcutaneous delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, and the second composition is for local administration to the site of the neoplastic disease.

[0008] In a second aspect, the present invention provides a method of treating, preventing, reducing, inhibiting, preventing recurrence or controlling a neoplastic disease in a subject, the method comprising simultaneously, separately or sequentially administering to the subject (i) a first composition comprising a live attenuated bacterium and (ii) a second composition comprising a live attenuated bacterium that is the same or different from the live attenuated bacterium of the first composition, wherein the first composition is formulated for oral, intravenous, intranasal, intradermal or subcutaneous delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, and the second composition is for local administration to the site of the neoplastic disease. [Brief description of the drawings]

[0009] [Figure 1A] Figure 1A shows a schematic illustrating the timeline of subcutaneous systemic and local administration of Salmonella typhi in an orthotopic murine MB49 bladder cancer model. Sc, subcutaneous; IVES, intravesical. [Figure 1B] Figure 1B shows the survival percentage of mice treated with subcutaneous systemic and topical administration of Salmonella typhi. C57BL / 6 mice were treated or not with subcutaneous ZH9, inoculated with MB49 tumor cells in the bladder, and treated with intravesical ZH9 or PBS control. Survival was monitored over 100 days. Graphs are combined results from 5 independent studies. Statistics are log-rank (Mantel-Cox) tests. **p<0.01. ****p<0.0001. [Diagram 2] Figure 2 shows a schematic illustrating the timeline of flow cytometry analysis following subcutaneous systemic and local administration of Salmonella typhi in healthy mice. Sc, subcutaneous; IVES, intravesical. [Figure 3A]Figures 3A-3C show the changes 24 hours and 7 days after subcutaneous systemic and local administration (ives) of Salmonella Typhi in healthy mice in the numbers of neutrophils (Figure 3A), CD4+ T cells (Figure 3A), monocytes (Figure 3B), CD8+ T cells (Figure 3B), cross-presenting dendritic cells (Figure 3C) and natural killer cells (Figure 3C). Individual data points represent pools of cells from n=2 (treated with ZH9 in ives) or n=3 (treated with PBS in ives) mice and two independent experiments combined with n=4 pools each. Lines indicate group means. Dendritic cell populations shown are defined as CD11b+ Ly6G- CD11c+ Ly6C+ CD103+. Statistics indicated are one-way ANOVA with Sidak's post-hoc test. [Figure 3B] Figures 3A-3C show the changes 24 hours and 7 days after subcutaneous systemic and local administration (ives) of Salmonella Typhi in healthy mice in the numbers of neutrophils (Figure 3A), CD4+ T cells (Figure 3A), monocytes (Figure 3B), CD8+ T cells (Figure 3B), cross-presenting dendritic cells (Figure 3C) and natural killer cells (Figure 3C). Individual data points represent pools of cells from n=2 (treated with ZH9 in ives) or n=3 (treated with PBS in ives) mice and two independent experiments combined with n=4 pools each. Lines indicate group means. Dendritic cell populations shown are defined as CD11b+ Ly6G- CD11c+ Ly6C+ CD103+. Statistics indicated are one-way ANOVA with Sidak's post-hoc test. [Figure 3C]Figures 3A-3C show the changes 24 hours and 7 days after subcutaneous systemic and local administration (ives) of Salmonella Typhi in healthy mice in the numbers of neutrophils (Figure 3A), CD4+ T cells (Figure 3A), monocytes (Figure 3B), CD8+ T cells (Figure 3B), cross-presenting dendritic cells (Figure 3C) and natural killer cells (Figure 3C). Individual data points represent pools of cells from n=2 (treated with ZH9 in ives) or n=3 (treated with PBS in ives) mice and two independent experiments combined with n=4 pools each. Lines indicate group means. Dendritic cell populations shown are defined as CD11b+ Ly6G- CD11c+ Ly6C+ CD103+. Statistics indicated are one-way ANOVA with Sidak's post-hoc test. [Figure 4] Figure 4 shows a schematic illustrating the timeline of oral systemic administration of Salmonella Typhimurium followed by local administration (intratumoral) of Salmonella Typhi in a syngeneic subcutaneous MC38 mouse colon cancer model. IT, intratumoral. [Figure 5A] Figure 5A and Figure 5B show the numbers of tumor-infiltrating CD8 T cells, CD4 T cells and regulatory T cells (Tregs) after 1 day (Figure 5A) and 7 days (Figure 5B) of systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone. N=3 or 4 mice / group per time point. Bars are mean ± SEM. [Figure 5B] Figure 5A and Figure 5B show the numbers of tumor-infiltrating CD8 T cells, CD4 T cells and regulatory T cells (Tregs) after 1 day (Figure 5A) and 7 days (Figure 5B) of systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone. N=3 or 4 mice / group per time point. Bars are mean ± SEM. [Figure 6]Figure 6 shows CD8 T cell proliferation in the tumor and periphery 1 and 7 days after systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone. N=4 mice / group per time point. Bars are mean ± SEM. [Figure 7] Figure 7 shows CD4 T cell proliferation in the tumor and periphery 1 and 7 days after systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone. N=4 mice / group per time point. Bars are mean ± SEM. [Figure 8] Figure 8 shows the functional potential of CD8 T cells in mice treated with systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone. N=4 mice / group per time point. Bars are mean ± SEM. [Figure 9] Figure 9 shows the functional potential of CD4 T cells in mice treated with systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone. N=4 mice / group per time point. Bars are mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description So that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0011] As used herein, the term "attenuated" refers in the context of the present invention to the modification of a microorganism to reduce its pathogenicity while maintaining its viability, rendering it harmless to the host. This method is commonly used in vaccine development due to its ability to induce a highly specific immune response while maintaining an acceptable safety profile. The development of such vaccines can involve a number of methods, examples of which include, but are not limited to, passaging the pathogen under in vitro conditions until pathogenicity is lost, chemical mutagenesis, and genetic engineering techniques. Such attenuated microorganisms are preferably live attenuated microorganisms, although non-live attenuated microorganisms are also disclosed.

[0012] As used herein, the term "inactivating mutation" refers to a modification of the natural genetic code of a particular gene or the gene promoter associated with that gene, for example by changing the nucleotide code or 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 as an inactive protein, so that the natural function of the gene is lost or reduced to such an extent that it is not measurable. Thus, a mutation in a gene inactivates the function of the gene or the function of the protein that it codes for.

[0013] As used herein, the term "immunotherapy" refers to any therapy that aims to modulate immune system response, for example, by antibodies or immune cells, or by drugs or other agents that stimulate, inhibit, or modulate the immune system. These include immunomodulators, drugs, or substances that affect the immune system; this includes, but is not limited to, antibodies (e.g., antitumor antigen antibodies), epitope-binding portions of antibodies, antibody-drug conjugates, including cytotoxic conjugates, radiological agents, other tumor targeting agents, oncolytic viruses, cytokines, chemokines, interferons, interleukins, colony-stimulating factors, drugs or other agents that modulate immune response, immune cells, or engineered cells that interact with, recognize, or bind to target cells.

[0014] As used herein, the term "cellular components of the immune system" refers to immune cells, such as lymphocytes, such as T and B lymphocytes, gamma-delta T cells, and NK cells, that can recognize specific antigens, such as prions, viruses, bacteria, yeast, fungi, parasites, tumor-associated or tumor-specific antigens, or other antigens associated with a particular disease, disorder, or condition. Other immune cells that we refer to include white blood cells, which may be granulocytes or agranulocytes. Examples of immune cells include neutrophils, eosinophils, basophils, lymphocytes, monocytes, and macrophages. Dendritic cells, microglia, and other antigen-presenting cells are also included in this definition.

[0015] As used herein, the term "non-naturally occurring bacteria" refers to bacterial (prokaryotic) cells that have been genetically modified or "engineered" to be altered with respect to naturally occurring cells. Such genetic modification may be, for example, the incorporation of additional genetic information into the cell, the modification of existing genetic information, or the actual deletion of existing genetic information. This may be accomplished, for example, by transfection of a recombinant plasmid into the cell or modifications made directly to the bacterial genome. Additionally, bacterial cells may be modified by chemical modifications, for example to achieve attenuation. They may be genetically modified by mutagenesis, methods of which are well known to those of skill in the art, and thus the term "non-naturally occurring bacteria" can refer to both recombinantly and non-recombinantly modified bacterial strains.

[0016] As used herein, the terms "recombinant", "recombinant strain" or "recombinant bacterium" are used interchangeably and refer to a strain of bacteria that has undergone genetic engineering, in the context of the present invention, such that the bacterial DNA has been modified by the introduction of new DNA. Recombinant DNA methods generally 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 can confer advantageous properties to the bacterial strain, such as prolonged activity, eliciting a stronger immune response in a subject, or introducing a desired molecule.

[0017] As used herein, the term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which results in selective damage to, destruction of, or elimination of cancerous cells from the human body.

[0018] As used herein, the terms "systemic immune response" and "systemic immunity" are used interchangeably and refer to a widespread immune response throughout the subject's body directed against an elicitor, as well as widespread non-specific immune activation, as opposed to a local, spatially restricted response. Such a response involves complex interactions between different cells of the immune response, such as lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver, and is believed to prime the subject's immune system in the context of the present invention so that the subject is more responsive to the second composition, when the second composition containing live attenuated bacteria is administered locally to the site of neoplastic disease. A "systemic immune response" thus refers to the activation of neutrophils, monocytes, dendritic cells, T cells (e.g., CD4+, CD8 ... + and / or CD8 +The "systemic immune response" may be measured and quantified through the analysis of a variety of different immune cell types, including, but not limited to, immune cells (e.g., T cells) and natural killer cells. Methods by which these effects can be measured are well known to those skilled in the art, such as flow cytometry. The "systemic immune response" may also be measured and quantified by the presence of antibodies, including, but not limited to, IgG and IgA isotype antibodies. Methods by which these antibodies can be measured are well known to those skilled in the art, such as ELISA. Thus, the first composition may act to prime (used interchangeably with "conditioning", "boost", "amplify", "enhance", "improve", "augment" or "promote") the immune response of the subject after administration of the second composition.

[0019] As used herein, the terms "locally" and "administered locally" are used interchangeably and refer to the manner in which the second composition is administered to a subject in the context of the present invention. Thus, the second composition comprising live attenuated bacteria may be administered in / on / around the neoplastic disease. For example, the second composition may be administered directly to the neoplastic disease, for example, via intratumoral injection, or may be administered such that the second composition contacts the tissue / surface of the tumor, for example, intraperitoneally, intrapleurally, intravesically, peritumorally. The term "locally" may therefore refer to both direct and indirect contact with the neoplastic disease in question, for example, local instillation, intratumoral injection, peritumoral injection, intrapleural, intravesical and / or intraperitoneal injection. Thus, the resulting immune response generated in the subject is also said to be local to the site of the neoplastic disease, i.e., it is not a widespread systemic immune response, as is generated using the first composition of the present invention.

[0020] The terms "tumor," "cancer," "malignancy," and "neoplasm" Neoplasia) is used interchangeably and refers to a cell or population of cells whose growth, proliferation or survival is greater than that of a normal counterpart cell, e.g., a cell proliferative or differentiative disorder. Typically, the growth is uncontrolled. The term "malignant tumor" refers to the invasion of nearby tissues. The term "metastasis" refers to the spread or dissemination of a tumor, cancer or neoplasm to other sites, locations or regions within a subject, where the site, location or region is distinct from the primary tumor or cancer.

[0021] 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 can be reduction, amelioration, alleviation, reduction, delay, and / or relief of one or more of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. With respect to cancer, an effective amount may include an amount sufficient to cause tumor shrinkage and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay progression or prolong survival, or induce stabilization of a cancer or tumor.

[0022] In some embodiments, therapeutically effective amount is sufficient to prevent or delay recurrence.Therapeutically effective amount can be administered in one or more doses.Therapeutically effective amount of drug or combination can result in one or more of the following: (i) reduction in the number of cancer cells; (ii) reduction in tumor size; (iii) inhibition, delay, slowing to some extent and preferably stopping cancer cell invasion into peripheral organs; (iv) inhibition (i.e. slowing to some extent and preferably stopping) of tumor metastasis; (v) inhibition of tumor growth; (vi) prevention or delay of tumor occurrence and / or recurrence; and / or (vii) alleviation to some extent of one or more of symptoms associated with cancer.

[0023] For example, for the treatment of tumors, a "therapeutically effective dosage" may induce tumor shrinkage of at least about 5%, such as at least about 10%, or about 20%, or about 60% or greater compared to a baseline measurement. The baseline measurement may be from an untreated subject.

[0024] A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art can 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.

[0025] The terms "treatment" or "therapy" refer to the administration of active agents with the purpose of curing, curing, alleviating, mitigating, altering, treating, ameliorating, improving, or affecting a condition (e.g., a disease), the symptoms of a condition, or preventing or delaying the onset of symptoms, complications, biochemical signs of a disease, or otherwise halting or inhibiting further progression of a disease, condition, or disorder in a statistically significant manner.

[0026] As used herein, the term "subject" is intended to include humans and non-human animals. Preferred subjects include human patients who need an enhanced immune response. The method is particularly suitable for treating human patients with disorders that can be treated by increasing the immune response. In certain embodiments, the method is particularly suitable for treating cancer in vivo.

[0027] As used herein, the terms "concurrent administration" or "concurrently" or "simultaneously" mean that administration occurs on the same day. "Subsequently" or "separately" means that the administration occurs on different days.

[0028] As defined herein, "concurrent" administration includes administration of a first composition and a second composition within about 2 hours or about 1 hour or less of each other, and even more preferably at the same time.

[0029] As defined herein, "separate" administration includes administration of a first composition and a second composition separated by more than about 12 hours, or about 8 hours, or about 6 hours, or about 4 hours, or about 2 hours.

[0030] As defined herein, "sequential" administration includes administering a first composition and a second composition in multiple aliquots and / or doses, respectively, and / or on separate occasions. The first composition may be administered to the patient before and / or after administration of the second composition.

[0031] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any described or listed components.

[0032] 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 depends in part on how the value 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 according to practice in the art. Alternatively, "about" can mean within a range of up to 20%. When a particular value is provided in this application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

[0033] The present invention provides an effective method by which neoplastic disease in a subject can be prevented and / or treated by the use of multiple doses (i.e., at least two doses) of a live attenuated bacterium, where the doses are administered via separate routes of administration to effectively activate the subject's immune system against the neoplastic disease.

[0034] Thus, in a first aspect, the present invention provides a first composition comprising a live attenuated bacterium for use in treating, preventing, reducing, inhibiting, preventing recurrence or controlling a neoplastic disease in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the same or different live attenuated bacterium as the live attenuated bacterium of the first composition, the first composition being formulated for oral, intravenous, intranasal or subcutaneous delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, and the second composition is for local administration to the site of the neoplastic disease.

[0035] It is therefore envisioned that the live attenuated bacteria of the first composition act as a "priming" agent for the subject's immune system, resulting in a systemic immune response and, subsequently, the ability of the subject's immune system to mount an effective adaptive response in addition to a more effective innate immune response against neoplastic disease when the live attenuated bacteria of the second composition are further administered locally.

[0036] The first composition comprising the live attenuated bacteria is prepared for oral, intravenous, intranasal, intradermal or subcutaneous delivery. The first composition is formulated for administration to the patient. Suitable formulations for these routes of delivery will be apparent to those skilled in the art. The first composition may preferentially be a liquid frozen formulation or may be lyophilized by methods such as freeze-drying and appropriately stored, for example in a sachet, for later rehydration and administration. Alternatively, the first composition may be distributed in an enteric coated capsule. For encapsulated formulations, the lyophilized first composition is preferably mixed with a bile-adsorbing resin, such as cholestyramine, to enhance survival when released from the capsule into the small intestine (see WO2010 / 079343 for further details). The specific formulation of the first composition may vary depending on various factors, such as the route of administration or the target patient population, i.e., young children, adolescents, or adults. The first composition may also be formulated to include any other suitable adjuvants, diluents, or excipients. Suitable adjuvants, diluents or excipients include, but are not limited to, disodium hydrogen phosphate, soy peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, calcium chloride, sucrose, sterile saline, and sterile water.

[0037] It is envisioned that any live attenuated bacterium, or combination of live attenuated bacteria, capable of generating the required immune response in a subject may be used in the present invention. In a preferred embodiment, the live attenuated bacteria of the first and / or second composition may be a Gram-negative bacterium. 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. Gram-negative bacteria can be easily identified and differentiated from Gram-positive bacteria via Gram's differential staining technique, in which Gram-negative bacteria do not retain crystal violet stain.

[0038] Preferably, the live attenuated bacteria of the first and / or second composition may be of the genus Salmonella. Examples of Salmonella species for use in the present invention are Salmonella enterica and Salmonella bongori. Salmonella enterica may be further divided into different serotypes or serovars. Examples of said 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. In a preferred embodiment, the live attenuated bacterium may be Salmonella enterica serotype Typhi and / or Salmonella enterica Typhimurium.

[0039] The live attenuated bacteria of the first and second compositions may be the same live attenuated bacteria or different live attenuated bacteria. Thus, various different combinations of live attenuated bacteria are disclosed herein according to the present invention. In a preferred embodiment, the live attenuated bacteria of the first and second compositions are of the same species. In a most preferred embodiment, the live attenuated bacteria of the first and second compositions are both of the Salmonella enterica species.

[0040] The live attenuated bacteria of the first composition and the live attenuated bacteria of the second composition are genetically modified It may include non-natural bacteria. As will be understood by those skilled in the art, genes may be mutated by a number of well-known methods in the art, such as by homologous recombination using recombinant plasmids targeted to the gene of interest, in which case an engineered gene with homology to the target gene is incorporated into a suitable nucleic acid vector (e.g., a plasmid or bacteriophage) and transfected into the target cell. The homologous engineered 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 part of the gene or any regulatory part, such as the promoter region. As will be understood by those skilled in the art, any suitable genetic modification technique may be used to mutate the gene of interest, for example, CRISPR / Cas system, such as CRISPR / Cas 9, may be used.

[0041] Thus, many methods and techniques for genetically engineering bacterial strains are well known to those skilled in the art. These techniques include those required for introducing 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 "transformation" 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 Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996), which are incorporated herein by reference.

[0042] Thus, both the first composition and the second composition may comprise a bacterium whose genetic makeup has been altered in some form to induce changes, such as mutations, additions or deletions, for example, via genetic engineering or via chemical mutagenesis. The first composition and / or the second composition may be genetically modified such that the first composition and the second composition comprise a recombinant strain of bacteria. Alternatively, the first composition and / or the second composition may comprise a non-recombinant strain of bacteria. The first composition and the second composition may comprise the same live attenuated bacteria that have been genetically modified as described above, or the first and second compositions may comprise two different bacterial strains (which may or may not be within the same genus) that have been genetically modified. Alternatively, the first composition may comprise a recombinant strain of bacteria and the second composition may comprise a non-recombinant strain of bacteria, and vice versa.

[0043] It is envisaged that any bacterium capable of inducing a systemic immune response in a subject may be used according to the present invention. Thus, any live attenuated bacterium capable of inducing a systemic immune response is disclosed herein as part of a first composition, and any live attenuated bacterium capable of inducing a local immune response is disclosed herein as part of a second composition. In a preferred embodiment, any attenuated, non-pathogenic, Salmonella enterica serovar Typhi or Typhimurium strain may be used. In further preferred embodiments, the live attenuated bacteria of the first or second composition may be selected from the group including Ty21a, CVD 908-htrA, CVD 909, Ty800, ZH9 (also referred to as "M01ZH09"), ZH9PA, x9633, x639, x9640, x8444, DTY88, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R, or any combination thereof. The live attenuated bacteria of the first and second compositions may be the same, for example, both the first and second compositions may contain Ty21a, CVD 908-htrA, CVD 909, Ty800, ZH9, ZH9PA, x9633, x639, x9640, x8444, DTY88, MD58, WT05, ZH26, SL7838, SL7207, VNP20009 or A1-R. Alternatively, the live attenuated bacteria of the first and second compositions may be different, for example, the first composition may be Ty21a, and the second composition may be CVD 908-htrA, CVD 909, Ty800, ZH9, ZH9PA, x9633, x639, x9640, x8444, DTY88, MD58, WT05, ZH26, SL7838, SL7207, VNP20009 or A1-R, etc. In a preferred embodiment, the live attenuated bacteria of at least one of the first or second compositions is Salmonella typhi ZH9. For example, the live attenuated bacteria of the first composition may be Salmonella typhimurium MD58, and the live attenuated bacteria of the second composition may be Salmonella typhi ZH9. In a further preferred embodiment, the live attenuated bacteria of the first and second compositions are the same.In a most preferred embodiment, the live attenuated bacteria of the first and second compositions are both Salmonella typhi ZH9.

[0044] Thus, when the first and / or second composition comprises a genetically modified non-naturally occurring bacterium, it is preferred that said genetically modified non-naturally occurring bacterium is derived from the genus Salmonella. It is further preferred that said Salmonella 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 the genus 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 WO 2000 / 68261, which is incorporated herein by reference in its entirety.

[0045] The SPI-2 gene may be a ssa gene. For example, the present 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. Even more preferably, the attenuating mutation is in the ssaV gene.

[0046] The genetically engineered Salmonella microorganism may also include an attenuating mutation in a second gene, which may or may not be in the SPI-2 region. The mutation may be outside the SPI-2 region and may be 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. Even more preferably, the aro gene is aroC.

[0047] 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, which 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, and even more preferably, the aro gene is aroC.

[0048] The genetically engineered microorganism may further comprise one or more gene cassettes, which may be used to provide additional molecules to support the function of the genetically modified non-native bacterium as an immune system primer and / or stimulator.

[0049] In yet another embodiment, the genetically engineered microorganism may be derived from a Salmonella microorganism and may comprise an inactivating mutation in one or more genes selected from pltA, pltB, cdtB and ttsA, and further comprises an attenuating mutation in one or more genes selected from aroA and / or aroC and / or ssaV. Preferably, the attenuating mutation is in aroC and ssaV. Details of said genes and mutations are as described in WO 2019 / 110819, which is incorporated herein by reference in its entirety.

[0050] The present invention provides a first and second composition comprising a live attenuated bacterium disclosed herein that can be used for the prevention and / or treatment of neoplastic disease and / or secondary disease associated with 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 remitted.

[0051] Cancers that may be treated according to 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 uterine cells or neoplasms. Additionally, the cancer may be of the following histological types, among others, but not limited to: neoplasm, malignant tumor; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumors; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; Papillary adenocarcinoma; Chromophobe carcinoma; Eosinophilic carcinoma; Eosinophilic adenocarcinoma; Basophilic carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary follicular adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous 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; Theca cell tumor;Malignant granulosa cell tumor;Malignant androblastoma;Sertoli cell carcinoma;Malignant Leydig cell tumor;Malignant lipid cell tumor;Malignant paraganglioma;Malignant extramammary paraganglioma;Pheochromocytoma;Hemangiocytic angiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial melanoma;Malignant melanoma in giant pigmented nevus;Epithelioid cell melanoma;Malignant blue nevus;Sarcoma;Fibrosarcoma;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonic rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stroma sarcoma;Mixed tumor;Müllerian mixed tumor;Nephroblastoma; Hepatoblastoma;Carcinosarcoma;Malignant mesenchymoma;Malignant Brenner tumor;Malignant phyllodes tumor;Synovial sarcoma;Malignant mesothelioma;Dysgerminoma;Embryonal carcinoma;Malignant teratoma;Malignant ovarian thyroid tumor;Choriocarcinoma;Malignant mesonephroma;Hemangiosarcoma;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;Ameloblastic odontosarcoma;Malignant ameloblastoma;Ameloblastic fibrosarcoma;Malignant pinealoma;Chordoma;Malignant glioma;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Anaplastic neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neurobiotics;Retinoblastoma;Olfactory nerve tumor;Malignant meningioma;Neurofibrosarcoma;Malignant schwannoma;Malignant granular cell tumor;Malignant lymphoma;Hodgkin's disease disease);Hodgkin's disease;paragranulomatous;small lymphocytic lymphoma;diffuse large cell lymphoma;follicular lymphoma;mycosis fungoides;other specified non-Hodgkin's lymphoma;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lympho-sarcoma cell leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia.;

[0052] Preferably, the solid cancer and / or hematological malignancy may be a cancer selected from prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colon cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, mesothelioma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma. Even more preferably, the neoplastic disease may be associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular carcinoma, melanoma, esophageal cancer, gastric cancer, ovarian cancer, colon cancer, head and neck cancer or breast cancer. In a preferred embodiment, the neoplastic disease is colon cancer or bladder cancer. In a most preferred embodiment, the neoplastic disease is bladder cancer.

[0053] When the neoplastic disease to be prevented and / or treated is bladder cancer, bladder cancer can be non-muscle invasive bladder cancer or muscle invasive bladder cancer.Non-muscle invasive bladder cancer is defined as any bladder cancer that consists of cancerous cells contained inside the inner wall of the bladder, and is the most common type.Muscle invasive bladder cancer is defined as any bladder cancer whose cancerous cells have spread beyond the inner wall of the bladder and thus invade the surrounding bladder muscle layer.The latter is less common, but has a higher possibility of spreading to other parts of the body.

[0054] When the bladder cancer is non-muscle invasive bladder cancer, said bladder cancer may be referred to as transitional cell (urothelial) carcinoma (TCC). TCC accounts for about 95% of bladder cancer, and therefore the live attenuated bacteria of the first and second compositions of the present invention may preferably be used for the prevention and / or treatment of TCC. TCC may be further divided into two subtypes of bladder cancer: papillary carcinoma and flat carcinoma. Thus, in yet a further embodiment, the live attenuated bacteria of the first and second compositions of the present invention may preferably be used for the prevention and / or treatment of papillary carcinoma and / or flat carcinoma. Alternative bladder cancers for which the present invention may be suitable include, but are not limited to, squamous cell carcinoma, adenocarcinoma, small cell carcinoma and / or sarcoma.

[0055] In one embodiment, the invention provides a first composition comprising a live attenuated bacterium for use in treating, reducing, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the live attenuated bacterium, the first composition is formulated for oral delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, the second composition is for local administration to a site of the neoplastic disease, and the live attenuated bacteria of the first and second compositions is Salmonella serovar Typhi ZH9 strain.

[0056] In a further embodiment, the invention provides a first composition comprising a live attenuated bacterium for use in treating, reducing, inhibiting, preventing recurrence, or controlling bladder cancer in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the live attenuated bacterium, the first composition is formulated for oral delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, the second composition is for local administration to the bladder, and the live attenuated bacteria of the first and second compositions is Salmonella serovar Typhi ZH9 strain.

[0057] In another embodiment, the present invention provides a method for treating, reducing, inhibiting, or preventing recurrence of TCC in a subject. The present invention provides a first composition comprising a live attenuated bacterium for use in the prevention or control of infection, wherein a subject has been administered or is intended to be administered a second composition comprising the live attenuated bacterium, the first composition is formulated for oral delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, the second composition is for local administration to the bladder, and the live attenuated bacteria of the first and second compositions is Salmonella serovar Typhi ZH9 strain.

[0058] In another embodiment, the invention provides a first composition comprising a live attenuated bacterium for use in treating, reducing, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the live attenuated bacterium, the first composition is formulated for subcutaneous delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, the second composition is for intravesical administration to a site of the neoplastic disease, and the live attenuated bacteria of the first and second compositions is Salmonella serovar Typhi ZH9 strain.

[0059] In another embodiment, the invention provides a first composition comprising a live attenuated bacterium for use in treating, reducing, inhibiting, preventing recurrence, or controlling bladder cancer in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the live attenuated bacterium, the first composition is formulated for subcutaneous delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, the second composition is for intravesical administration to the bladder, and the live attenuated bacteria of the first and second compositions is Salmonella serovar Typhi ZH9 strain.

[0060] In another embodiment, the invention provides a first composition comprising a live attenuated bacterium for use in treating, reducing, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the live attenuated bacterium, the first composition being formulated for oral delivery to stimulate a systemic immune response in the subject and for simultaneous, separate or sequential administration with the second composition, the second composition being for intratumoral administration to a site of neoplastic disease, wherein the live attenuated bacterium of the first composition is Salmonella serovar Typhimurium MD58 strain and the live attenuated bacterium of the second composition is Salmonella serovar Typhimurium ZH9 strain.

[0061] In another embodiment, the invention provides a first composition comprising a live attenuated bacterium for use in treating, reducing, inhibiting, preventing recurrence, or controlling colon cancer in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the live attenuated bacterium, the first composition is formulated for oral delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, the second composition is for intratumoral administration to a site of colon cancer, and wherein the live attenuated bacterium of the first composition is Salmonella serovar Typhimurium MD58 strain and the live attenuated bacterium of the second composition is Salmonella serovar Typhimurium ZH9 strain.

[0062] It is envisioned that the different delivery routes of the first and second compositions, i.e., the first composition is administered orally, intravenously, intranasally, intrapleurally, intradermally or subcutaneously to achieve a systemic effect, and the second composition is administered locally at the site of the neoplastic disease, complement each other in such a way that the neoplastic disease in the subject can be effectively prevented and / or treated. In particular, such a combination activates the anti-tumor activity of the immune system in the subject in a more specific, targeted and effective manner than if each of the first or second compositions were given in isolation.

[0063] As mentioned above, the second composition is for local administration to the site of the neoplastic disease in the subject, for example, the bladder. Thus, the second composition may be administered in any manner that would allow the second composition to be in close proximity to the neoplastic disease. As used herein, the term "close proximity" is intended to refer to a section or area surrounding the neoplastic disease up to a certain distance. For example, in one embodiment, the term "close proximity" may refer to a section / area that extends up to 10 mm from the border of the neoplastic disease. In another embodiment, the term "close proximity" may refer to a section / area that extends up to 5 mm from the border of the neoplastic disease. In yet another embodiment, the term "close proximity" may refer to a section / area that extends up to 2.5 mm from the border of the neoplastic disease. The distance at which the second composition is administered from the site of the neoplastic disease allows the biological effects of the second composition, such as the recruitment and activation of various immune cell types, to be effective against the neoplastic disease in question, with minimal or no effect on tissues located in unrelated compartments of the body. Therefore, the second composition may be administered directly to the neoplastic tissue, or to surrounding tissues in close proximity to the neoplastic disease. Additionally, the second composition may be administered "in" or "on" the neoplastic tissue or surrounding tissue. Therefore, the term "local administration" refers to any context in which the live attenuated bacteria can be contacted with the neoplastic disease or the immediately surrounding tissue to have a desired effect.

[0064] Preferably, the second composition may be administered via local instillation, intraperitoneal, intrapleural, intravesical, peritumoral or intratumoral injection, with "instillation" referring to the introduction of the second composition into the relevant anatomical site and allowing it to remain there for a particular amount of time before being drained, emptied or removed, "intraperitoneal" referring to the injection of the second composition into the peritoneum of a subject, "intrapleural" referring to the injection of the second composition into the pleura or pleural cavity of a subject, "intravesical" referring to injection or instillation via a catheter into the bladder, "peritumoral injection" referring to the injection of the second composition around the site of the neoplastic disease, and "intratumoral injection" referring to the injection of the second composition directly into the neoplastic disease of a subject. It is understood that the particular method of administration of the second composition may depend on both the location and type of neoplastic disease to be treated, e.g., the neoplastic disease to be treated. For example, when it is desired to treat a body cavity with a large surface area, such as the pleural cavity of a subject, administration via instillation may be most appropriate.Alternatively, when the neoplastic disease is located in the peritoneal cavity, such as ovarian cancer, administration via intraperitoneal injection may be most appropriate.Moreover, it is noted that when the neoplastic disease to be prevented and / or treated is a hematological malignancy, intratumoral injection may not be the administration method of choice.

[0065] In the case where the neoplastic disease to be prevented and / or treated is bladder cancer, the second composition can preferably be administered via intravesical instillation, also referred to as bladder instillation, intravesical treatment or intravesical therapy.Such administration method refers to the method in which the second composition can be delivered to the bladder via catheter.As previously described, such method is considered to be "local" due to reduced systemic side effects compared to oral or parenteral delivery.

[0066] Thus, in one embodiment, the invention provides a first composition comprising a live attenuated bacterium for use in treating, reducing, inhibiting, preventing recurrence, or controlling bladder cancer in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the same or different live attenuated bacterium as the live attenuated bacterium of the first composition, the first composition being formulated for oral or subcutaneous delivery to stimulate a systemic immune response in the subject, and is for simultaneous, separate or sequential administration with the second composition, and the second composition is for administration via intravesical instillation.

[0067] In another embodiment, the invention provides a first composition comprising a live attenuated bacterium for use in treating, reducing, inhibiting, preventing recurrence, or controlling bladder cancer in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising the live attenuated bacterium, the first composition is formulated for oral or subcutaneous delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, the second composition is for administration via intravesical instillation, and the live attenuated bacteria of the first and second compositions is Salmonella serovar Typhi ZH9 strain.

[0068] The first composition of the present invention is intended to generate a systemic immune response in a subject. Thus, the first composition of the present invention may be a vaccine or a vaccine composition. Such terms are used interchangeably and refer to a biological preparation that allows a subject to generate an immune response against said biological preparation, thus providing active acquired immunity against a specific infectious disease, such as a disease caused by Salmonella. In the context of the present invention, a vaccine may contain an agent that resembles an infection-causing bacterium, or a "foreign" agent, which is a weakened or killed form of said bacterium, or any part or fragment of bacterial protein, capsule, DNA or RNA. Such a foreign agent is recognized by the immune system of the vaccine recipient, which then destroys said agent and develops a "memory" against the bacterium, inducing a level of permanent protection against future bacterial infections from the same or similar viruses. It is assumed that when a vaccinated subject again encounters the same bacterium or bacterial isolate that the subject was vaccinated against through the vaccination route, including the vaccine composition of the present invention, the individual's immune system will thereby recognize the bacterium or bacterial isolate and induce a more effective defense against infection. The active adaptive immunity induced in the subject as a result of the vaccine may be humoral and / or cellular in nature. Thus, in an embodiment in which the first and second compositions are the same, the first composition of the present invention may not only induce a systemic immune response that boosts the non-specific natural immune response against the second composition, but may also confer the additional benefit that the subject acquires active immunity against the live attenuated bacterium of the first composition.

[0069] The first composition may be administered to a subject in need thereof simultaneously with the second composition, separately or sequentially.Without being bound by theory, it is believed that administering the first composition prior to administering the second composition allows the subject's immune system to be effectively primed prior to administering the second composition, thus resulting in a more effective prophylactic or therapeutic strategy.Thus, although there may be some cases in which the first and second compositions are administered within rapid succession of each other or at the same time, it is preferred that the live attenuated bacteria of the first composition is administered to the subject prior to the live attenuated bacteria of the second composition.

[0070] The amount of live attenuated bacteria of the first composition administered to the subject is sufficient to elicit a systemic immune response in the subject, such that the subject's immune system is effectively primed to receive the second composition at a site local to the neoplastic disease, resulting in the subject's immune system being able to mount an effective immune response against the cancer or tumor when treated with the two compositions in combination. The immune response initiated by administration of the composition may be therapeutic in itself, or may be sub-therapeutic requiring subsequent administration of the second composition to exert a therapeutic effect.

[0071] The first and second compositions containing the live attenuated bacteria are administered at least once or at least two weeks apart. It is contemplated that the first composition may be administered at least twice. The first composition may be administered before, during or after administration of the second composition. Preferably, at least one administration of the first composition is administered prior to administration of the second composition, and such administration may be administered at least one week prior to administration of the second composition. It is contemplated that administration of the first and second compositions may be repeated depending on the treatment regimen. The live attenuated bacteria of the first and / or second compositions may be administered at least once every 10 4 ~10 12 CFU, where CFU is colony forming unit. For example, a suitable dose is 10 4 ~10 5 CFU, 10 4~10 6 CFU、10 4 ~10 7 CFU、10 4 ~10 8 CFU、10 4 ~10 9 CFU、10 4 ~10 10 CFU、10 4 ~10 11 CFU、10 4 ~10 12 CFU、10 5 ~10 6 CFU、10 5 ~10 7 CFU、10 5 ~10 8 CFU、10 5 ~10 9 CFU、10 5 ~10 10 CFU、10 5 ~10 11 CFU、10 5 ~10 12 CFU、10 6 ~10 7 CFU、10 6 ~10 8 CFU、10 6 ~10 9 CFU、10 6 ~10 10 CFU、10 6 ~10 11 CFU、10 6 ~10 12 CFU、10 7 ~10 8 CFU、10 7 ~10 9 CFU、10 7 ~10 10 CFU、10 7 ~10 11 CFU、10 7 ~10 12 CFU、10 8 ~10 9 CFU、10 8 ~10 10 CFU、10 8 ~10 11 CFU、10 8 ~10 12CFU, 10 9 ~10 10 CFU, 10 9 ~10 11 CFU, 10 9 ~10 12 CFU, 10 10 ~10 11 CFU, 10 10 ~10 12 CFU, or 10 11 ~10 12 It may be a CFU.

[0072] It is widely known that the causes of neoplastic disease are multifaceted and diverse, often leading to prevention and treatment strategies that include multiple therapies to achieve optimal results. Therefore, the present invention may involve combining the live attenuated bacteria of the first and / or second composition with other known cancer therapies. Preferably, the live attenuated bacteria of the first and / or second composition may be administered in combination with immunotherapy, radiotherapy, chemotherapy or anti-cancer agents. The term "anti-cancer agent" as used herein refers to any agent that is effective in killing cancer cells, stopping the division of cancer cells, or helps prevent the recurrence of cancer cells, but is not considered to be immunotherapy, radiotherapy or chemotherapy.

[0073] In a preferred embodiment, the live attenuated bacteria of the first and / or second composition may be administered in combination with immunotherapy, preferably comprising checkpoint inhibitors, antigen-specific T cells, adoptive T cell therapy, therapeutic antibodies, cancer vaccines or any other engineered cellular immunotherapy.

[0074] "Checkpoint inhibitors" are agents that act on surface proteins that are TNF receptors or any member of the B7 superfamily, or otherwise, and include agents that bind to negative costimulatory molecules. Examples of such checkpoint inhibitors include, but are not limited to, CTLA-4, PD-1, TIM-3, BTLA, TIGIT, VISTA, LAG-3, and / or their respective ligands, including PD-L1.

[0075] When the immunotherapy includes a checkpoint inhibitor, the checkpoint inhibitor may be directed against CTLA-4, PD-1, PD-L1, LAG3, TIM3, BTLA, VISTA, or TIGIT. In a preferred embodiment, the checkpoint inhibitor may be directed against CTLA-4, PD-1, or PD-L1. In some cases, the blocking agent may be ipilimumab (Yervoy®; targets CTLA-4), nivolumab (Opdivo®; targets PD-1), pembrolizumab (Keytruda®; targets PD-1), atezolizumab (Tecentriq®; targets PD-L1), cemiplimab (Libtayo®; targets PD-1), or durvalumab (Imfinzi®; targets PD-L1).

[0076] The terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1", CD279 and "PD1" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one epitope in common with PD-1. The complete PD-1 sequence can be found under GenBank Accession No. NP 005009.2.

[0077] The terms "PD-L1", "PDL1", "programmed cell death ligand 1", CD274 and "programmed cell death 1" are used interchangeably and are intended to include variants, isoforms and species homologs of human PD-L1, as well as analogs that share at least one epitope in common with PD-L1. The complete PD-L1 sequence can be found under GenBank Accession No. NP 054862.1.

[0078] The terms "cytotoxic T lymphocyte-associated antigen-4", "CTLA-4", "CTLA4", CD152 and "CTLA-4 antigen" are used interchangeably and include variants, isoforms, species homologs, and analogs of human CTLA-4 that share at least one epitope in common with CTLA-4. The complete CTLA-4 sequence can be found under GenBank Accession No. NP_005205.2.

[0079] The terms "LAG-3", "LAG3", CD223 and "lymphocyte activation gene 3" are used interchangeably and are intended to include variants, isoforms and species homologs of human LAG-3, as well as analogs that share at least one epitope in common with LAG-3. The complete LAG-3 sequence can be found under GenBank Accession No. NP_002277.4.

[0080] The terms "TIM-3", "TIM3", "HAVCR2", "Hepatitis A Virus Cellular Receptor 2", CD366 and "T-cell immunoglobulin and mucin domains 3" are used interchangeably and are intended to include variants, isoforms and species homologs of human TIM-3, as well as analogs that share at least one epitope in common with TIM-3. The complete TIM-3 sequence can be found under GenBank Accession No. NP_116171.3.

[0081] The terms "BTLA", "B and T lymphocyte attenuator" and "CD272" are used interchangeably and are intended to include variants, isoforms and species homologs of human BTLA, as well as analogs that share at least one epitope in common with BTLA. The complete BTLA sequence can be found under GenBank Accession No. NP_861445.4.

[0082] The terms "VISTA", "V-set immunomodulatory receptor", "B7H5", "B7-H5", "PD-1H" and "V-domain Ig inhibitor of T-cell activation" are used interchangeably and are intended to include variants, isoforms and species homologs of human VISTA, as well as analogs that share at least one epitope in common with VISTA. The complete VISTA sequence can be found under GenBank Accession No. NP_071436.1.

[0083] The terms "TIGIT", "T cell immunoreceptor with Ig and ITIM domains", "WUCAM" and "Vstm3" are used interchangeably and are intended to include variants, isoforms and species homologs of human TIGIT and analogs that share at least one epitope in common with TIGIT. The complete TIGIT sequence can be found under GenBank accession number NP_776160.2.

[0084] The PD-L1 / PD-1 signaling pathway is a major mechanism of cancer immune escape for several reasons. First, and most importantly, this pathway is involved in the negative regulation of the immune response of activated effector T cells found in the periphery. Second, PD-L1 is upregulated in the cancer microenvironment, and PD-1 may also be upregulated on activated tumor-infiltrating T cells, thus promoting a vicious cycle of inhibition. Third, this pathway is intricately involved in both innate and adaptive immune regulation through bidirectional signaling. These factors make the PD-1 / PD-L1 complex a focal point through which cancer can manipulate the immune response to promote its own progression. As a result, tumors can activate inhibitory immune checkpoint molecular pathways, resulting in suppression of the immune system and continued unhindered proliferation of cancerous cells. Following T cell activation, CTLA-4 is transported to the surface where it competes with CD28 for the same ligand on antigen presenting cells (APCs), resulting in the suppression of CD28 and subsequent suppression of T cell activation and proliferation. Targeting PD-1, PD-L1 and CTLA-4 aims to prevent these events from occurring.

[0085] When the immunotherapy involves antigen-specific T cells, the antigen-specific T cells may be the result of adoptive T cell therapy. The term "adoptive cell therapy" is intended to refer to any therapy involving the transfer / administration of cells into a subject, preferably a human. The cells may be autologous or allogeneic. Preferably, the cells are generally derived from the immune system with the goal of improving immune functionality. The adoptive cell therapy may include, but is not limited to, CAR-T cell therapy (chimeric antigen receptor T cells), TIL therapy (tumor infiltrating lymphocytes) and iPSC-derived therapy (induced pluripotent stem cells). It is particularly envisioned that the adoptive T cell therapy may be CAR-T cell therapy. In some cases, the CAR-T cell therapy is directed against the antigen CD19 present in B cell-derived cancers. Thus, such therapy may be particularly suitable for B cell-derived cancers, such as acute lymphoblastic leukemia (ALL) and diffuse large B cell lymphoma (DLBCL). In other cases, CAR-T cell therapy is directed against tumor-associated antigens (TAA), and thus more suitable for the treatment of solid tumors. Examples of such antigens include, but are not limited to, CD133, CD138, CEA, EGFR, EpCAM, GD2, GPC3, HER2, HerinCAR-PD1, MSLN, MG7, MUC1, LMP1, PSMA and PSCA. Such techniques are known to those skilled in the art, and the reader is directed to the review entitled "Adoptive cellular therapies: the current landscape" (Rohaan et al. 2019, Virchows Arch. 474(4): 449-461) for further information.

[0086] When immunotherapy includes therapeutic antibodies, said therapeutic antibodies may be directed to cancer or tumor. The term "therapeutic antibodies" as referred to herein includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof that result in a therapeutic effect. Such therapeutic antibodies may be directed to checkpoint inhibitor molecules directed to the above, or may include agonistic antibodies directed to costimulatory molecule targets, such as ICOS (inducible T cell costimulatory molecule / CD278), GITR (glucocorticoid-inducible TNF receptor / TNFRSF18 / CD357 / AITR), 4-1BB (CD137), CD27 and CD40. In some cases, it may be desirable for subjects to be given both types of therapeutic antibodies.

[0087] The therapeutic antibody may be a monoclonal antibody, more preferably a humanized or human monoclonal antibody. The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. The term "human antibody" as used herein refers to an antibody in which both the framework and CDR regions are derived from human germline clones. It is intended to include antibodies having variable regions derived from the germline of another mammalian species, such as a mouse, that have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0088] Methods for obtaining such monoclonal antibodies are known to those skilled in the art. Therapeutic antibodies may block abnormal proteins in cancer cells, attach to specific proteins on cancer cells, or be conjugated to cytotoxic molecules, such as anti-cancer drugs. The latter mark the cancer cells to the immune system, so that the abnormal cells can be subsequently targeted and destroyed by cellular components of the immune system. In some cases, monoclonal antibodies may also be checkpoint inhibitors. For example, ipilimumab (Yervoy®), nivolumab (Opdivo®) and pembrolizumab (Keytruda®) are all checkpoint inhibitors in addition to being monoclonal antibodies. In other cases, the monoclonal antibody may be an agonistic antibody directed against costimulatory molecule targets, such as ICOS (inducible T cell costimulatory molecule / CD278), GITR (glucocorticoid-inducible TNF receptor / TNFRSF18 / CD357 / AITR), 4-1BB (CD137), CD27, and CD40. Examples of non-checkpoint inhibitor monoclonal antibodies for the treatment of cancer include, but are not limited to, trastuzumab (Herceptin®), bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (Vectibix®), rituximab (Rituxan® and Mabthera®), alemtuzumab (Campath®), ofatumumab (Arzerra®), gemtuzumab ozogamicin (Mylotarg®), and brentuximab vedotin (Adcetris®).

[0089] Immunotherapy may include cancer vaccine.Cancer vaccine may be a preventive vaccine or a therapeutic vaccine, preferably the vaccine is a therapeutic vaccine.The use of cancer vaccine teaches immune system to recognize and destroy the antigens presented by cancerous cells.Such vaccine may also include adjuvants to help boost response even further.

[0090] Immunotherapy may include any other engineered cellular immunotherapy.In the context of the present invention, "any other engineered cellular immunotherapy" refers to any cell type that is engineered in a manner designed to regulate immune system or immune response in a subject and provide favorable outcomes in terms of cancer prevention or treatment.Examples of such cells include, but are not limited to, natural killer cells, macrophages, lymphocytes, stem cells and dendritic cells.

[0091] In a second aspect, the present invention provides a method of treating, preventing, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, the method comprising simultaneously, separately or sequentially administering to the subject (i) a first composition comprising a live attenuated bacterium and (ii) a second composition comprising a live attenuated bacterium that is the same or different from the live attenuated bacterium of the first composition, wherein the first composition is formulated for oral, intravenous, intranasal, intradermal or subcutaneous delivery to stimulate a systemic immune response in the subject and is for simultaneous, separate or sequential administration with the second composition, and the second composition is for local administration to the site of the neoplastic disease.

[0092] The present invention therefore provides a method of treating, preventing, inhibiting, preventing the recurrence of, or controlling a neoplastic disease in a subject, comprising a live attenuated bacterium for use as described herein.

[0093] Thus, the methods of the invention may be used to reduce or inhibit metastasis of a primary tumor or cancer to other sites, or the formation or establishment of a metastatic tumor or cancer at other sites distal to the primary tumor or cancer, thereby inhibiting or reducing tumor or cancer recurrence or tumor or cancer progression.Thus, the invention provides a detectable or measurable improvement in a given subject's condition, such as the reduction or amelioration of one or more adverse (physical) symptoms or prognosis associated with a cell proliferative or hyperproliferative disorder, a neoplasm, a tumor or cancer, or the presence of a metastasis, i.e., a therapeutic benefit or beneficial effect.

[0094] The method of the present invention is therefore a combination therapy comprising the administration of two compositions whose characteristics are described above. Such a combination has the potential to induce a strong and durable immune response in a subject, resulting in an enhanced therapeutic benefit.

[0095] Therapeutic benefit or beneficial effect is any objective or subjective, transient, temporary or long-term improvement in a condition or pathology, or a reduction in the onset, severity, duration or frequency of adverse symptoms associated with or caused by a cell proliferation or cell hyperproliferative disorder, such as a neoplasm, tumor or cancer, or metastasis. It may also lead to improved survival. A satisfactory clinical endpoint of the therapeutic method according to the present invention is achieved, 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 reversal 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. Therapeutic benefit or improvement may therefore be, but is not limited to, the destruction of the target proliferative cell (e.g., a neoplasm, tumor or cancer, or metastasis) or the elimination of one or more, most or all of the pathologies, adverse symptoms or complications associated with or caused by a cell proliferation or cell hyperproliferative disorder, such as a neoplasm, tumor or cancer, or metastasis. However, a therapeutic benefit or improvement need not be a cure or complete destruction of all target proliferative cells (e.g., a neoplasm, tumor or cancer, or metastasis) or the elimination of all pathologies, adverse symptoms, or complications associated with or caused by a cell proliferation or cell hyperproliferative disorder, such as a neoplasm, tumor or cancer, or metastasis. For example, partial destruction of tumor or cancer cell mass, or stabilization of tumor or cancer mass, size, or cell number by inhibiting tumor or cancer progression or progression, can reduce mortality even if a portion or a majority of the tumor or cancer mass, size, or cells remain, and can prolong life, even if only for days, weeks, or months.

[0096] Specific non-limiting examples of therapeutic benefit include a reduction in the volume (size or cell mass) or number of cells of a neoplasm, tumor or cancer, or metastasis, inhibiting or preventing an increase in the volume of a neoplasm, tumor or cancer (e.g., stabilizing), 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.

[0097] The methods of the invention may not have an immediate effect, for example, treatment may be followed by an increase in neoplastic, tumor or cancer cell number or mass, but may be followed by an eventual stabilization or reduction in tumor cell mass, size or number of cells over time in a given subject.

[0098] Associated with neoplasms, tumors, cancers and metastases that can be inhibited, reduced, diminished, delayed or prevented Additional adverse symptoms and complications that may be observed include, for example, nausea, loss of appetite, lethargy, pain and discomfort. Thus, a partial or complete decrease or reduction in the severity, duration or frequency of adverse symptoms or complications associated with or caused by a cell hyperproliferative disorder, an improvement in the subject's quality of life and / or well-being, e.g., increased energy, appetite, psychological well-being, are all specific, non-limiting examples of therapeutic benefit.

[0099] The therapeutic benefit or improvement may therefore also include a subjective improvement in the quality of life of the treated subject. In additional embodiments, the method prolongs or extends the lifespan (survival) of the subject. In further embodiments, the method improves the quality of life of the subject.

[0100] Therapeutic benefit may also include prevention of recurrence of neoplasms, tumors, cancers and metastases, for example, where said neoplasms, tumors, cancers and metastases have been surgically or chemically resected.

[0101] The present invention may be suitable for individuals who have been refractory to previous preventive and / or therapeutic strategies. By "refractory" we mean to refer to any neoplastic disease that does not respond to treatment. It is also envisioned that the present invention may be suitable for individuals who have previously been low, intermediate or high responders to previous treatments.

[0102] The first and second compositions are typically administered to a subject in a composition that contains an effective amount of live attenuated bacteria, such as Salmonella serovar Typhi ZH9 strain, and further contains a pharma- ceutically acceptable carrier / adjuvant / diluent or excipient. The terms "pharmaceutical" and "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered appropriately to animals, such as humans. Such preparations are known to those skilled in the art. Furthermore, it is understood that for administration to animals (e.g., humans), preparations should meet sterility, pyrogenicity, general safety and purity standards, as applicable.

[0103] As used herein, "pharmaceutical acceptable carriers / adjuvants / diluents / excipients" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, the like and combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Examples include, but are not limited to, disodium hydrogen phosphate, soybean peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, calcium chloride, sucrose, borate buffer, sterile saline solution (0.9% NaCl) and sterile water.

[0104] The first and / or second composition may also comprise additional components intended to enhance the immune response. Examples of such additional components are aluminum salts, such as aluminum hydroxide, aluminum oxide and aluminum phosphate, oil-based adjuvants, such as Freund's complete and incomplete adjuvants, mycolic acid-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (e.g., murein, mucopeptides, or glycoproteins, such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), muramyl dipeptides, immune stimulating complexes (EP 109 942, EP 180 564, EP 231 563, EP 231 564, EP 231 565, EP 231 566, EP 231 567, EP 231 568, EP 231 569 ... 039), saponin, DEAE-dextran, neutral oils (e.g., miglyol), vegetable oils (e.g., peanut oil), liposomes, polyols, the Ribi adjuvant system (see, e.g., GB-A-2 189 141), vitamin E, carbopol, interferons (e.g., IFN-alpha, IFN-gamma, or IFN-beta), interleukins, particularly those that stimulate cell-mediated immunity (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21) or chemokines (e.g., CXCL9, CXCL10, CXCL11, CCL5, CCL2, CX3CL1).

[0105] The invention will now be further described with reference to the following non-limiting examples. EXAMPLES

[0106] Example 1 The interaction of ZH9 with urothelial cancer cells was investigated using in vitro invasion assays and flow cytometric staining for intracellular Salmonella common antigen (CSA-1) and propidium iodide for cell death. The therapeutic efficacy of ZH9 against bladder tumor growth was established in a mouse orthotopic, syngeneic MB49 bladder tumor model. Tumor-bearing animals were treated with a single intravesical dose of ZH9 or OncoTice BCG, and long-term survival comparisons were assessed by log-rank (Mantel-Cox) test. Local immune responses were analyzed by flow cytometric staining of disaggregated mouse bladders after treatment of healthy mice.

[0107] Mice treated with a single dose of ZH9 2 days after MB49 tumor inoculation demonstrated a significant survival benefit compared to both vehicle-treated (median survival 49.5 vs. 31 days, p=0.003) and BCG-treated animals (median survival 49.5 vs. 27.5 days, p<0.001). In a more rigorous model with treatment 4 days after tumor inoculation, ZH9 also demonstrated significant efficacy (median survival 30 vs. 20.5 (p=0.003) and 23.5 (p=0.025) days for vehicle and BCG, respectively). Surviving ZH9-treated animals, unlike naive controls, were resistant to further bladder inoculation with MB49 tumor cells, suggesting durable antitumor immunity resulting from ZH9 treatment (100% vs. 31.5% median survival day 45, p=0.01). In vitro, intracellular flow cytometry in human (UMUC3, T24, RT4, 5637) and murine (MB49) urothelial carcinoma cell lines showed that ZH9 infiltrated and induced cell death in all cell types 24 hours after a 1-hour exposure. In vivo, a single intravesical treatment with ZH9 resulted in a strong cellular immune response at day 7 characterized by robust recruitment of monocytes, NK cells, CD4+ and CD8+ T cells, and dendritic cells with an activated, cross-presenting (Ly6C+, CD103+) phenotype, which in all cases was greater in magnitude and duration than after a single treatment with a comparable dose of BCG.

[0108] Thus, live-attenuated Salmonella strain ZH9 has been shown to demonstrate a clear survival benefit over standard of care in an orthotopic bladder cancer model, likely via both direct tumor cell killing and induction of a robust cellular immune response, thus indicating significant therapeutic potential of ZH9 in bladder cancer.

[0109] Example 2 Preceding systemic priming with subcutaneous Salmonella typhi enhances the efficacy of local bladder Salmonella typhi treatment The aim of the study described here was to evaluate the effect of subcutaneous (sc) priming with attenuated Salmonella Typhi strain ZH9 prior to intravesical (ives) ZH9 treatment on the level of mouse survival in a non-muscle invasive bladder cancer model (see Figure 1A).

[0110] As described in more detail below, the inventors of the present application have surprisingly shown that mice treated with both systemic and local administration of Salmonella typhi exhibit improved survival.

[0111] material and method Mouse cells The MB49 cell line (provided by Prof. A. Loskog, Uppsala University, Sweden) was derived from a carcinogen-induced urothelial carcinoma in male C57Bl / 6 mice (Summerhayes, Journal of the National Cancer Institute, 62(4):1017-1023, 1979). Luciferase expression (MB49-luc) was generated by transfection with a lentiviral vector encoding firefly luciferase (provided by Prof. D. Trono, EPFL, Lausanne, Switzerland).

[0112] MB49 orthotopic bladder tumor model Seven- to 10-week-old female C57Bl / 6 wild-type mice (Charles River) were used and all experiments were performed according to Swiss legislation and with the approval of the Cantonal Veterinary Office of Canton de Vaud, Switzerland. Bladder tumors were established in deeply anesthetized mice that were urethrally catheterized using an Introcan 24Gx3 / 4 catheter (Braun, Melsungen, Germany). A 15-min pretreatment with 100 μl of 22% ethanol was performed before the instillation of 500,000 MB49-luc cells in 50 μl. MB49-luc tumor growth was monitored by bioluminescence 15 min after intraperitoneal (IP) injection of D-luciferin (Promega, L8220, 150 μg per g body weight) in a Xenogen imaging system (Xenogen / IVIS Caliper Life Science; kindly provided by the Cellular Imaging Facility, CIF / UNIL, Lausanne, Switzerland). 100% of mice developed bladder tumors. Bioluminescence monitoring of MB49-luc tumors is highly efficient for assessing tumor establishment and growth during the first 3 weeks, however, growing tumors may then frequently exhibit uncontrollable loss of luminescence (Jurczok et al., BJU International, 101(1):120-124, 2008), requiring additional monitoring by palpation, hematuria, and general health of the mice. Mice were sacrificed when approved termination was reached.

[0113] Preparation of bacteria ives 3×10 for infusion 7 CFU / 50 μl and 1 × 10 for s.c. injection 7 Prepare dilutions of the attenuated Salmonella Typhi strain (ZH9) in PBS as required to achieve CFU / 100 μl.

[0114] Intravesical treatment As described above, 50 μl of bacterial suspension was instilled by urethral catheterization. The retention time in the bladder was 1 hour until the mouse spontaneously voided after waking from anesthesia. In this precise setting, a single intravesical therapeutic instillation was administered on day 5 (4 days after intravesical tumor cell instillation). Tumors were detectable by bioluminescence on day 5.

[0115] Survival was monitored over 100 days, and the results are representative of five separate studies, demonstrating the reproducibility of the results.

[0116] result Mice given local ZH9 to the bladder (ives) had improved survival at the end of the study compared to mice treated with PBS ives (p<0.01). A higher percentage of mice given ZH9 ives plus ZH9 sc survived (p<0.01) (see Figure 1B). Statistics are log-rank (Mantel-Cox) tests. Mice treated with both systemic and local bladder administration of Salmonella had a 77% survival rate at the end of the study compared to 43% for local bladder ZH9 alone and 26% for local bladder PBS treatment.

[0117] Example 3 Systemic priming with subcutaneous Salmonella Typhi enhances favorable bladder cellular immune responses to local Salmonella Typhi treatment The aim of the study described here was to assess by flow cytometry the level of immune cell infiltration in the bladder of mice that received subcutaneous systemic priming with Salmonella Typhi strain (ZH9) prior to local intravesical administration of Salmonella Typhi strain (ZH9) (see Figure 2).

[0118] As described in detail below, the present inventors have surprisingly shown that systemic priming increases the peak magnitude and duration of myeloid and lymphoid immune responses in the bladder, and that undesirable neutrophilic inflammation is not enhanced.

[0119] material and method 1 × 10 on days -35 and -14 7 Female C57BL / 6 mice were treated subcutaneously with 3 × 10 CFU of Salmonella typhi ZH9 or PBS control. 7 Mice were treated intravesically with CFU of Salmonella typhi ZH9 or PBS control. At the indicated time points (24 h and 7 days) after treatment, bladder tissue was harvested and digested with collagenase / dispase / DNase (see Figure 2). Single cell suspensions were generated and cells were stained for flow cytometry analysis.

[0120] Preparation of bacteria ives 3×10 for infusion 7 CFU / 50 μl and 1 × 10 for s.c. injection 7 Dilutions of the attenuated Salmonella Typhi strain (ZH9) were made in PBS as required to achieve CFU / 100 μl.

[0121] Intravesical treatment Seven- to ten-week-old female C57Bl / 6 wild-type mice (Charles River) were used, and all experiments were performed in accordance with Swiss legislation and with the approval of the Cantonal Veterinary Office of Canton de Vaud, Switzerland. Intravesical instillations were performed in deeply anesthetized mice that had been urethrally catheterized using an Introcan 24Gx3 / 4 catheter (Braun, Melsungen, Germany). 50 μl of bacterial suspension was instilled. Retention time in the bladder was approximately 1 h until the mice spontaneously urinated after waking from anesthesia.

[0122] 1. Preparation of the Bladder Mice were sacrificed by CO2 inhalation and bladders were collected. Single cell suspensions were obtained by mincing in DL-dithiothreitol (Sigma Merck KGaA) and digestion with 1 mg / mL collagenase / dispase (Roche, Basel, Switzerland) and 0.1 mg / ml DNAse I (Sigma Merck KGaA) with 20% fetal bovine serum (Gibco, MA, USA). Recovered cells were used for immunostaining.

[0123] Immunostaining and flow cytometry analysis The monoclonal anti-mouse antibodies used were Anti-CD3-PE(17A2), Anti-CD3-PerCP / Cy5.5(17A2), Anti-Ly6G-PE / Cy7(1A8), Anti-Ly6C-AF700(HK1.4), Anti-CD8-APC / Cy7(53-6.7), Anti-CD103-PcBlue(2E7), Anti-CD11b-FITC(M1 / 70), Anti-XCR1-APC(ZET)(Biolegend); Anti-CD4-APC(RM4-5), Anti-CD8-PE(53-6.7)(BD The antibodies were: Anti-CD11c-PE-eF610 (N418), Anti-CD45-FITC (30-F11), Anti-CD45-PerCP / Cy5.5 (30-F11), Anti-CD11b-AF700 (M1 / 70), Anti-CD335-eF450 (29A1.4) (eBioscience, Thermo Fisher Scientific, MA, USA). The following isotype controls were used: Rat IgG2a, kappa isotype control-APC-Cy7 (RTK2758), Rat IgG2a, kappa isotype control-APC (RTK2758) (Biolegend), Arm hamster IgG isotype control-PE-eF610 (eBio99Arm) (eBioscience).

[0124] Dead cells were excluded using a live / dead fixable aqua dead cell stain kit (Invitrogen Thermo Fisher Scientific, MA, USA). Cells were acquired and analyzed using a Gallios Flow Cytometer (Beckman Coulter, Nyon, Switzerland) and FlowJo software (Tree Star, Ashland, OR), respectively.

[0125] result Immune filtration is significantly increased by priming with ZH9 sc when administered prior to ZH9 ives (see Figures 3A, 3B and 3C). This is especially evident 7 days after instillation. Priming with ZH9 specifically increased T cells and NK cells 7 days after instillation ives, whereas myeloid cells were not significantly altered. Both CD4+ and CD8+ T cells are significantly increased by priming with ZH9 7 days after instillation ives (see Figures 3A and 3B). Neutrophils were not altered by priming with ZH9, whereas monocytes were significantly increased, especially 24 hours after ZH9 ives (see Figures 3A and 3B).

[0126] Example 4 Systemic priming with oral Salmonella Typhimurium enhances favorable cellular immune responses to local Salmonella Typhi treatment The aim of the study described here was to measure changes in T cell numbers, phenotype and function in tumor-bearing mice (a murine colon cancer model) after oral systemic priming with a Salmonella Typhimurium strain (MD58) combined with local intratumoral treatment with a Salmonella Typhimurium strain (ZH9).

[0127] material and method Preparation of bacterial cells 1 x 10 for oral gavage 9 Required to achieve CFU / 100 μl A dilution of an attenuated Salmonella Typhimurium strain (MD58) was made in PBS as described above. For intratumoral injection, 1 × 10 7 Dilutions of the attenuated Salmonella Typhi strain (ZH9) were made in PBS as required to achieve CFU / 40 μl.

[0128] MC38 colon cancer model Female C57BL / 6 mice aged 7-14 weeks were used for all experiments. MC38 colon cancer cell line was purchased from Kerafast, Inc. and maintained within exponential growth phase until injection. Cells were prepared by trypsinization, washed in simple medium, and viable cells were counted via an automated cell counter. Prior to tumor cell implantation, each mouse was anesthetized using 1-2% isoflurane, and the injection area was shaved and washed. 6 × 10 cells in 200 μL of serum-free medium were injected using a 27G syringe. 5 MC38 cells were injected subcutaneously in the rear flank. Tumor burden was monitored at regular intervals to ensure it did not exceed levels permitted by the Home Office licence for Prokarium. Mice were treated with oral Salmonella Typhimurium (7 days prior to MC38 inoculation) followed by intratumoral treatment with Salmonella Typhimurium (14 days after MC38 inoculation) or intratumoral treatment with Salmonella Typhimurium alone (14 days after MC38 inoculation) (see Figure 4). The effect of oral systemic prime with Salmonella Typhimurium on T cell infiltration, phenotype and functional status was assessed 1 and 7 days after intratumoral treatment with Salmonella Typhimurium.

[0129] Tissue isolation and flow cytometry Tumors and spleens were excised and mechanically dissociated using strainers and syringe plungers. The lysate was then filtered through a 70 μm cell strainer to lyse red blood cells. Tumors were additionally treated with a 35% Percoll solution to remove cellular debris. Cell pellets were resuspended in appropriate buffers for downstream analysis. For flow cytometry analysis, dead cells were excluded by live / dead fixable aqua dead cell stain kit (Thermo Fisher) and then stained with the following monoclonal antibodies: anti-CD90.2 (53-2.1; Biolegend), Anti-CD4 (RM4-5; Thermo Fisher), Anti-CD8α (53-6.7; Biolegend), Foxp3 (FJK-16s; Thermo Fisher), IFNγ (XMG1.2; Biolegend), and Ki-67 (16A8; Biolegend). Cell acquisition and analysis were performed using an ATTUNE NXT cytometer (Thermo Fisher) and FlowJo software (Tree Star), respectively.

[0130] Tumor-infiltrating CD8 + and CD4 + Assessment of T cell numbers Transfer 7- to 9-week-old female C57BL / 6 mice to 1 × 10 peritoneal sputum via oral administration (PO, "orally"). 9 CFU of Salmonella typhimurium MD58 or were left untreated. Seven days later, 6 × 10 5 All mice were inoculated subcutaneously with 1 × 10 MC38 (colon) tumor cells. After 14 days, all mice were inoculated with 1 × 10 7CFU of Salmonella typhi ZH9 were given intratumoral (IT) doses. Tumors and spleens were harvested 1 and 7 days after IT treatment, with n=3 or 4 mice / group per time point. Tumors were mechanically dissociated, filtered, and single cell suspensions were analyzed by flow cytometry on an Attune NXT cytometer. Cells were stained for surface markers, followed by fixation / permeabilization and then staining for intracellular molecules. Indicated T cell populations were gated on FSC / SSC, singlets, viability dye-, CD90+, followed by either CD4, CD8, or CD4 and Foxp3. Absolute cell counts were normalized to the weight of each tumor.

[0131] Assessment of T cell proliferation in tumors and the periphery Transfer 7- to 9-week-old female C57BL / 6 mice to 1 × 10 peritoneal sputum via oral administration (PO, "orally"). 9 C After 7 days, mice were immunized with 6 × 10 FU of Salmonella typhimurium MD58 or left untreated. 5 All mice were inoculated subcutaneously with 1 × 10 MC38 (colon) tumor cells. After 14 days, all mice were inoculated with 1 × 10 7 CFU of Salmonella typhi ZH9 were given in an intratumoral (IT) dose. Tumors and spleens were harvested 1 and 7 days after IT treatment, with n=4 mice / group per time point. Tumors were mechanically dissociated, filtered, and single cell suspensions were analyzed by flow cytometry on an Attune NXT cytometer. Cells were stained for surface markers, followed by fixation / permeabilization and then staining for intracellular molecules. CD8 T cells were gated on FSC / SSC, singlets, viability dye -, CD90+, and CD8+. CD4 T cells were gated on FSC / SSC, singlets, viability dye -, CD90+, CD4+, and Foxp3-. Proliferating T cells were defined as Ki-67+ and expressed as a percentage of total CD8 T cells.

[0132] CD8 + and CD4 +Assessment of T cell functional potential Transfer 7- to 9-week-old female C57BL / 6 mice to 1 × 10 peritoneal sputum via oral administration (PO, "orally"). 9 CFU of Salmonella typhimurium MD58 or were left untreated. Seven days later, 6 × 10 5 All mice were inoculated subcutaneously with 1 × 10 MC38 (colon) tumor cells. After 14 days, all mice were inoculated with 1 × 10 7 CFU of Salmonella typhi ZH9 were given in an intratumoral (IT) dose. Tumors and spleens were harvested 1 and 7 days after IT treatment, with n=4 mice / group per time point. Tumors were mechanically dissociated, filtered, and single cell suspensions were analyzed by flow cytometry on an Attune NXT cytometer. Cells were stained for surface markers, followed by fixation / permeabilization and then staining for intracellular molecules. CD8 T cells were gated on FSC / SSC, singlets, viability dye -, CD90+, and CD8+. CD4 T cells were gated on FSC / SSC, singlets, viability dye -, CD90+, CD4+, and Foxp3-. Proliferating T cells were defined as Ki-67+ and expressed as a percentage of total CD8 T cells. For assessment of cytokine production capacity, cells were stimulated for 4 hours at 37°C in the presence of Cell Activation Cocktail with Brefeldin A (Biolegend; 423303). After stimulation, cells were stained for surface markers, followed by fixation / permeabilization and then staining for intracellular molecules. CD8 T cells were gated on FSC / SSC, singlets, viability dye-, CD90+, and CD8+. IFNγ+ CD8 T cells are expressed as a percentage of total CD8 T cells. CD4 T cells were gated on FSC / SSC, singlets, viability dye-, CD90+, CD4+, and Foxp3-. IFNγ+ CD4 T cells are expressed as a percentage of total CD4 T cells. Functional potential is measured as % of T cells capable of producing IFNγ after stimulation with T cell activation cocktail (PMA+ionomycin+brefeldin A).

[0133] result The numbers of tumor-infiltrating CD8 and CD4 T cells, but not regulatory T cells (Tregs), were found to be increased after systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone (see Figures 5A and 5B). Additionally, T cell proliferation in the tumor and periphery was shown to be increased after systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone, suggesting activation of the adaptive immune system, both locally and systemically (see Figures 6 and 7). The functional potential of CD8 and CD4 cells was also shown to be increased in mice treated with systemic priming with oral Salmonella Typhimurium in combination with intratumoral Salmonella Typhi treatment compared to intratumoral treatment alone (see Figures 8 and 9). reference).

[0134] The examples disclosed herein demonstrate that systemic administration of Salmonella followed by local administration of Salmonella enhances the efficacy of local administration of Salmonella. The inventors have also demonstrated that the combination of systemic and local administration of Salmonella appears to induce a stronger immune response and drive anti-tumor activity than seen with local administration of Salmonella alone. Furthermore, the inventors have demonstrated herein that the effects exemplified herein are seen across multiple Salmonella strains, multiple modes of systemic and local administration, and in multiple cancer models.

Claims

1. A first composition comprising a live attenuated bacterium for use in treating, preventing, reducing, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the subject has been administered or is intended to be administered a second composition comprising a live attenuated bacterium that is the same or different from the live attenuated bacterium of the first composition; The first composition is formulated for oral, intravenous, intranasal, intradermal or subcutaneous delivery to stimulate a systemic immune response in the subject, and is for simultaneous, separate or sequential administration with the second composition; and A first composition, wherein said second composition is for local administration to the site of said neoplastic disease.

2. The first composition of claim 1 , wherein the live attenuated bacterium of the first and / or second composition is a gram-negative bacterium.

3. The first composition described in claim 2, wherein the live attenuated bacterium is a Salmonella bacterium.

4. The first composition according to any one of claims 1 to 3, wherein the live attenuated bacterium of the first and / or second composition is Salmonella enterica.

5. The first composition described in claim 4, wherein the live attenuated bacteria is Salmonella enterica serovar Typhi and / or Salmonella enterica serovar Typhimurium.

6. 2. The first composition of claim 1, wherein the live attenuated bacteria of the first composition and / or the live attenuated bacteria of the second composition comprise a genetically modified, non-naturally occurring bacterium.

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

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

9. 9. The first composition of claim 7, wherein the SPI-2 gene is a ssaV gene and the second gene is an aro gene.

10. The first composition of claim 1 , wherein the neoplastic disease is a solid cancer and / or a hematological malignancy.

11. The first composition of claim 10, wherein the solid cancer and / or the hematological malignancy is a cancer selected from the group consisting of prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colon cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer and sarcoma.

12. The first composition described in claim 10, wherein the neoplastic disease is associated with a cancer selected from the group consisting of bladder cancer, lung cancer, mesothelioma, hepatocellular carcinoma, melanoma, esophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer and breast cancer.

13. The first composition described in claim 10, wherein the neoplastic disease is associated with a cancer selected from bladder cancer or colorectal cancer.

14. The first composition of claim 10, wherein the neoplastic disease is non-muscle invasive bladder cancer or muscle invasive bladder cancer.

15. 2. The first composition of claim 1, wherein the second composition is administered via local instillation, intraperitoneal, intrapleural, intravesical, peritumoral or intratumoral injection.

16. The first composition described in claim 1, which is a vaccine.

17. 2. The first composition of claim 1, wherein the live attenuated bacteria of the first composition is administered to the subject prior to the live attenuated bacteria of the second composition.

18. The first composition of claim 1 , wherein the live attenuated bacteria of the first and / or second composition is administered in combination with immunotherapy, radiation therapy, chemotherapy or an anti-cancer drug.

19. The first composition described in claim 18, wherein the immunotherapy comprises a checkpoint inhibitor, antigen-specific T cells, adoptive T cell therapy, therapeutic antibody, cancer vaccine or any other engineered cellular immunotherapy.

20. 2. The first composition of claim 1, wherein the live attenuated bacterium of the first composition is Salmonella enterica serovar Typhi and the live attenuated bacterium of the second composition is Salmonella enterica serovar Typhi.

21. 21. The first composition of claim 20, wherein the live attenuated bacterium of the first composition is Salmonella enterica serovar Typhi ZH9, and the live attenuated bacterium of the second composition is Salmonella enterica serovar Typhi ZH9.

22. 2. The first composition of claim 1, wherein the live attenuated bacterium of the first composition is Salmonella enterica serovar Typhimurium and the live attenuated bacterium of the second composition is Salmonella enterica serovar Typhimurium.

23. 23. The first composition of claim 22, wherein the live attenuated bacterium of the first composition is Salmonella enterica serovar Typhimurium MD58 and the live attenuated bacterium of the second composition is Salmonella enterica serovar Typhimurium ZH9.

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