Cancer treatment

Abstract

The present invention relates to the field of cancer therapy. In particular, the present invention relates to a live attenuated Gram-negative bacterium for use in treating, reducing, suppressing or controlling neoplastic disease in a subject receiving or intended to receive checkpoint inhibitor therapy, adoptive T cell therapy and / or allogeneic or autologous CAR-T therapy, either simultaneously, separately or sequentially with the administration of the live attenuated Gram-negative bacterium.

Description

[Technical Field]

[0001] This invention relates to the field of cancer treatment. In particular, this invention relates to a method for preventing, treating, or suppressing the development of neoplastic diseases in a subject. [Background technology]

[0002] The field of cancer treatment is constantly evolving, with new therapies emerging as our understanding of the underlying mechanisms related to cancer formation and progression progresses.

[0003] One of the most promising treatment strategies for cancer patients today is immunotherapy. This type of therapy aims to enhance the body's natural defenses against cancer or tumors. Immunotherapy utilizes substances that the body produces or that are artificially created in a laboratory to improve or restore immune system function. Types of immunotherapy include monoclonal antibodies, cross-organ therapy, nonspecific immunotherapy, oncolytic virus therapy, adoptive cell transfer, such as CAR-T cell therapy, and cancer vaccines. Nonspecific immunotherapy includes treatment with interferon or interleukin, molecules that help the immune system fight cancer, slow the growth of cancer cells, or, in some cases, destroy cancer. Immunotherapy may be administered as an alternative to or in combination with conventional cancer treatments such as chemotherapy or radiation therapy.

[0004] While immunotherapy is successful to some extent, significant challenges remain to be overcome. These challenges primarily involve side effects. The goal of immunotherapy is to stimulate the immune system, thereby making cancer or tumors more susceptible to attack by circulating immune cells. However, by blocking the regulatory mechanisms of immune activation, some immunotherapies cause side effects due to autoimmune toxicity. Furthermore, some immunotherapies fail to distinguish between cancerous and healthy cells. As a result, an individual's quality of life can be severely affected.

[0005] Furthermore, while immunotherapy has proven effective, a large proportion of individuals still do not respond to cancer treatment. There are many reasons why such treatments are not as effective as initially expected for some individuals. These include the complexity of the cancer being treated, the health status of the individual receiving treatment (elderly individuals and those with underlying medical conditions are known to have weakened immune systems), and the challenges posed by cancer cells, such as their ability to "hide" from the immune system and weaken the immune response.

[0006] Therefore, in the field of cancer, there remains a considerable need for ways to improve the toxicity profile of currently used immunotherapies while maintaining the effectiveness of these treatments in individuals who respond positively. At the same time, there is a considerable need for ways to improve the effectiveness of immunotherapy in individuals who do not respond to this treatment unless the effectiveness of immunotherapy can be improved. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2000 / 68261 [Patent Document 2] International Publication No. 2019 / 110819 [Patent Document 3] International Publication No. 2020 / 157203 [Patent Document 4] International Publication No. 2012 / 001352 [Patent Document 5] International Publication No. 2005 / 052167 [Patent Document 6] International Publication No. 2020 / 157203 [Patent Document 7] European Patent No. 109942 [Patent Document 8] European Patent No. 180564 [Patent Document 9] European Patent No. 231039 [Non-patent literature]

[0008] [Non-licensed Document 1] Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001) [Non-licensed Document 2] Short Protocols in Molecular Biology,4th Ed. (Ausubel, John Wiley & Sons 1999) [Non-licensed Document 3] Protein Methods(Bollagら, John Wiley & Sons 1996) [Non-licensed Document 4] ORT-VAC; Garmoryら, 2005, Infect.Immun., Volume 73: Pages 2005~2011 [Non-licensed Document 5] Degryse, 1991, Mol.Gen.Genet., Volume 227: Pages 49~51 [Non-licensed Document 6] Adoptive cellular therapies:the current landscape,Rohaanら、2019,Virchows Arch.474(4):449-461 [Non-licensed Document 7] Rohaanら、2019, Virchows Arch.474(4):449-461 [Non-licensed Document 8] Remington's Pharmaceutical Sciences,18th Ed.Mack Printing Company,1990 [Non-licensed Document 9] Remington's Pharmaceutical Sciences,18th Ed.Mack Printing Company,1990,pp.1289-1329 [Non-licensed Document 10] Duan and Luo,Targeting macrophages in cancer immunotherapy,Signal Transduction and Targeted Therapy,2021(6:127) [Non-Patent Document 11] Quaranta and Schmid,Macrophage-Mediated Subversion of Anti-Tumour Immunity,Cells,2019 (8(7):747 [Overview of the project]

[0009] This invention provides an effective method for treating and / or preventing neoplastic diseases in subjects by administering attenuated live Gram-negative bacteria in combination with checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy. Surprisingly, the inventors found that this combination resulted in a higher therapeutic effect than treating the subject with any of the elements alone, i.e., achieving an additive or synergistic effect. The systemic modifications observed from the administration of attenuated live Gram-negative bacteria, combined with the intestinal uptake of these bacteria, are thought to enhance the antitumor activity of cancer immunotherapy.

[0010] In a first aspect of the present invention, there are attenuated live Gram-negative bacteria used for the treatment, reduction, suppression or control of neoplastic diseases in subjects receiving or intended to receive checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy, either concurrently with, separately, or over time with the administration of attenuated live Gram-negative bacteria, wherein the attenuated live Gram-negative bacteria are administered orally, subcutaneously, or intramuscularly.

[0011] A second aspect of the present invention provides attenuated live Gram-negative bacteria for use in the treatment, reduction, suppression or control of neoplastic diseases in subjects receiving or intended to receive checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy, wherein the attenuated live Gram-negative bacteria are to be administered in the first phase of treatment, and the checkpoint inhibitor therapy, adoptive T cell therapy and / or allogeneic or autologous CAR-T therapy are to be administered in the second phase of treatment.

[0012] In a third aspect of the present invention, there are attenuated live Gram-negative bacteria for use in the treatment, reduction, suppression or control of neoplastic diseases in subjects receiving or intended to receive checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy, either concurrently with, separately, or over time, said attenuated live Gram-negative bacteria, wherein the said attenuated live Gram-negative bacteria are non-recombinant or do not contain heterologous eukaryotic DNA encoding a therapeutic protein.

[0013] A fourth aspect of the present invention provides a method for treating, suppressing or controlling a neoplastic disease in a subject, the method comprising (i) administering attenuated live Gram-negative bacteria, which are administered orally, subcutaneously, or intramuscularly, and (ii) administering checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy to the subject simultaneously, separately, or over time, the method being characterized by enhancing the therapeutic effect compared to administering the attenuated live Gram-negative bacteria or checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy alone.

[0014] A fifth aspect of the present invention is a method for treating, suppressing or controlling a neoplastic disease in a subject, the method comprising simultaneously, separately, or over timely administration to the subject (i) attenuated live Gram-negative bacteria, the attenuated live Gram-negative bacteria being administered in the first phase of treatment, and (ii) checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy being administered in the second phase of treatment, the method being enhanced compared to the administration of the attenuated live Gram-negative bacteria or checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy alone.

[0015] A sixth aspect of the present invention provides a method for treating, suppressing or controlling a target neoplastic disease, the method comprising (i) an attenuated live Gram-negative bacterium, wherein the attenuated live Gram-negative bacterium is non-recombinant or does not contain heterologous eukaryotic DNA encoding a therapeutic protein, and (ii) administering checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy to the target simultaneously, separately, or over time, the method providing enhanced therapeutic effects compared to the administration of the attenuated live Gram-negative bacterium or checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy alone. [Brief explanation of the drawing]

[0016] [Figure 1A] Figure 1A shows that orally administered Salmonella induces significant long-term phenotypic changes in systemic myeloid cells. A) The graph shows the median fluorescence intensities of the markers CD80, CD86, and PD-L1 for viable CD11chigh, HLA-DR+, CD11b+ / -, PDCA-1-normal dendritic cells and viable CD11c- / low, PDCA1+, HLA-DR- / Int, CD11b-plasmacytoid dendritic cells. n=4 or 5 mice / group. [Figure 1B]Figure 1B shows that orally administered Salmonella induces significant long-term phenotypic changes in systemic myeloid cells. B) The graph shows the median fluorescence intensities of the markers CD80, CD86, and PD-L1 for viable CD11chigh, HLA-DR+, CD11b+ / -, PDCA-1-normal dendritic cells and viable CD11c- / low, PDCA1+, HLA-DR- / Int, CD11b-plasmacytoid dendritic cells. n=4 or 5 mice / group. [Figure 1C] Figure 1C shows that orally administered Salmonella induces significant long-term phenotypic changes in systemic myeloid cells. C) The graph shows the median fluorescence intensities of the markers PD-L1, CD80, and HLA-DR in viable CD11c-, CD11b+, Ly6C+, F4 / 80- monocytes and viable CD11c-, CD11b+, Ly6C-, F4 / 80+ macrophages. n=4 or 5 mice / group. [Figure 1D] Figure 1D shows that orally administered Salmonella induces significant long-term phenotypic changes in systemic myeloid cells. D) The graph shows the median fluorescence intensities of the markers PD-L1, CD80, and HLA-DR in viable CD11c-, CD11b+, Ly6C+, F4 / 80- monocytes and viable CD11c-, CD11b+, Ly6C-, F4 / 80+ macrophages. n=4 or 5 mice / group. [Figure 2A] Figure 2A shows the time course of phenotypic changes induced by Salmonella. A) This is a schematic diagram of the experiment detailing the experimental schedule. [Figure 2B] Figure 2B shows the time course of phenotypic changes induced by Salmonella. B) The graph shows the median fluorescence intensities of markers CD80 and CD86 as the mean proportion of the PBS control group for viable CD11chigh, HLA-DR+, CD11b+ / -, PDCA-1-normal dendritic cells (cDCs) and viable CD11c- / low, PDCA1+, HLA-DR- / Int, CD11b plasmacytoid dendritic cells (pDCs). The mean is shown for n=4-5 mice / group. [Figure 2C]Figure 2C shows the time course of phenotypic changes induced by Salmonella. C) The graph shows the median fluorescence intensities of markers CD80, PD-L1, and HLA-DR as the mean proportion of viable CD11c-, CD11b+, Ly6C+, F4 / 80- monocytes and CD11c-, CD11b+, Ly6C-, F4 / 80+ macrophages in the PBS control group. The mean values ​​are for n=4-5 mice / group. [Figure 3A] Figure 3A shows that oral administration of Salmonella enhances bone marrow hematopoiesis. A) The graph shows the percentage of lineage-negative (CD5-, CD11b-, B220-, GR-1-, Terr-119-, Ly-6B.2-) viable cells expressing both c-Kit and Sca-1, referred to as LKS cells, in the total bone marrow cells. n=4-5 mice / group. [Figure 3B] Figure 3B shows that oral administration of Salmonella enhances bone marrow hematopoiesis. B) This is a representative flow cytometry plot showing the increase in viable LKS cells in the bone marrow of animals orally treated with Salmonella. [Figure 3C] Figure 3C shows that oral administration of Salmonella enhances bone marrow hematopoiesis. C) The proportion of viable LKS cells to total bone marrow cells 14 days after Salmonella treatment correlated with the proportion of monocytes (viable CD11c-, CD11b+, Ly6C+, F4 / 80- cells) at the same point in time, using Spearman's rank correlation. [Figure 4] Figure 4 shows that orally administered Salmonella induces a state of heightened responsiveness in systemic dendritic cells that lasts for at least 14 days. Single-group studies involved n=5 mice per group; bars are mean + / - SEM; statistics shown are Mann-Whitney tests. [Figure 5A]Figure 5A shows that oral administration of Salmonella, when combined with immune checkpoint inhibitors, slows tumor growth and enhances efficacy. A) The graph shows MC38 tumor volume over time and illustrates tumor growth. The table summarizes the mean suppression rates of tumor growth by various treatments from day 14 to day 30 in the control group (solvent + isotype). Data are mean + / - SEM for 10 mice / group. [Figure 5B] Figure 5B shows that oral administration of Salmonella, when combined with immune checkpoint inhibitors, slows tumor growth and enhances efficacy. B) The graph shows tumor volume over time and illustrates tumor growth. The table summarizes the mean suppression of tumor growth by various treatments from day 16 to day 18 in the control group (solvent + isotype). Data are mean + / - SEM from 9-10 mice / group. [Figure 6] Figure 6 shows that in vitro conditioning of macrophages with Salmonella further polarizes M1 cells into an inflammatory phenotype and further induces M2 cells into M1-like phenotypic macrophages. The data represent monocyte-derived macrophages from three separate donors. [Figure 7A] Figure 7A shows that conditioning human monocytes in vitro overcomes M2 polarization and reduces their ability to suppress M2. A) The graph shows the replication index, indicating a doubling of the proportion of responsive T cells and T cells that undergo more than two divisions. [Figure 7B] Figure 7B shows that conditioning human monocytes in vitro overcomes M2 polarization and reduces M2 suppression ability. B) Positive control data showing effective stimulation of T cells by anti-CD3 / 28 antibody. [Figure 7C] Figure 7C shows that conditioning human monocytes in vitro overcomes M2 polarization and reduces their ability to suppress M2. C) This is a representative flow cytometry plot showing increased T cell proliferation in the presence of monocyte-derived macrophages conditioned with Salmonella. [Figure 8A]Figure 8A shows that in vitro conditioning of human monocytes overcomes M2 polarization and enhances anti-PD-L1 Ab activity. Human monocyte-derived macrophages conditioned with Salmonella enhance PD-L1 blocking activity. A) The graph shows the replication index, indicating a doubling of the proportion of responsive T cells and T cells that undergo more than two divisions. [Figure 8B] Figure 8B shows that in vitro conditioning of human monocytes overcomes M2 polarization and enhances anti-PD-L1 Ab activity. Human monocyte-derived macrophages conditioned with Salmonella enhance PD-L1 blocking activity. B) This is positive control data showing that anti-CD3 / 28 antibody effectively stimulates T cells. [Figure 8C] Figure 8C shows that in vitro conditioning of human monocytes overcomes M2 polarization and enhances anti-PD-L1 Ab activity. Human monocyte-derived macrophages conditioned with Salmonella enhance PD-L1 blocking activity. C) This is a representative flow cytometry plot of CellTrace dilution showing that combining PD-L1 blocking with Salmonella-conditioned monocyte-derived macrophages increases T cell proliferation. [Modes for carrying out the invention]

[0017] To facilitate understanding of this invention, certain terms are defined first. Additional definitions will be explained throughout the detailed description.

[0018] As used herein, the term "attenuated" refers to bacteria that have been genetically modified to not cause disease in humans or animal subjects / models.

[0019] "Immunotherapy" refers to a specific treatment method using drugs or other agents that aim to regulate immune system components such as antibodies or immune cells, or that stimulate, suppress, or regulate the immune system. In the context of the present invention, the term "immunotherapy" refers to checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T cell therapy.

[0020] The term "cellular components of the immune system" refers to immune cells such as T and B lymphocytes, γδT cells, and NK cells, which can recognize specific antigens such as prions, viruses, bacteria, yeast, fungi, parasites, tumor-related or tumor-specific antigens, or other antigens associated with specific diseases, disorders, or conditions. Other immune cells mentioned include leukocytes, 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.

[0021] The term "immunotherapy composition" refers to any composition containing an immunotherapy agent. Examples may include pharmaceutically acceptable carriers, bio-response modifiers that enhance the immune response, and / or adjuvants / additives or diluents.

[0022] As used herein, the term “attenuated” in the context of the present invention refers to a microbial alteration that reduces the pathogenicity of a microbial organism while maintaining its viability, thereby 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. Many methods may be involved in such vaccine development, including, but are not limited to, the passage of pathogens under in vitro conditions until pathogenicity is lost, chemical mutagenesis, and genetic engineering techniques. Such attenuated microorganisms are preferably live attenuated microorganisms, but non-live attenuated microorganisms are also disclosed.

[0023] This term refers to a bacterial cell (prokaryote) whose genes have been modified or "manipulated" in a manner similar to those altered in naturally occurring cells by "non-natural bacteria or multiple non-natural bacteria." Such genetic modification may, for example, involve the incorporation of additional genetic information into the cell, modification of existing genetic information, or, in fact, the deletion of existing genetic information. This may be achieved, for example, by introducing recombinant plasmids into the cell or by directly modifying the bacterial genome.

[0024] An "inactivating mutation" refers to an alteration of the natural genetic code of a specific gene or its associated gene promoter, such as a change in the nucleotide code, deletion of a nucleotide section, or addition of non-coding nucleotides or non-native nucleotides, which results in the loss or reduction of the gene's original function to an immeasurable degree, causing the gene to not be properly transcribed or translated, or to be expressed as an inactive protein. Therefore, a gene mutation inactivates the function of that gene or the protein it encodes.

[0025] The terms "tumor," "cancer," and "neoplasia" are used interchangeably and refer to cells or cell populations whose growth, proliferation, or survival rate is greater than that of normal corresponding cells, e.g., cell proliferation or differentiation disorders. Typically, growth is uncontrolled. The term "malignancy" refers to invasion of nearby tissue. The term "metastasis" refers to the spread or dissemination of a tumor, cancer, or neoplasm to other sites, locations, or areas within the subject, distinct from the primary tumor or cancer.

[0026] A “checkpoint inhibitor” is an agonist that acts on surface proteins considered to be checkpoints in the immune response, and the surface proteins are of any type, such as TNF receptors, the B7 superfamily, or others, and include agonists that bind to negative costimulatory molecules. Examples of such checkpoint inhibitors include, but are not limited to, agonists that bind to the respective ligands of CTLA-4, PD-1, TIM-3, BTLA, TIGIT, VISTA, LAG-3, and / or PD-L1. In the context of the present invention, the term “agonist” may refer to an antibody, small molecule, antibody fragment, or any other conjugate and / or blocker.

[0027] 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 common epitope with PD-1. The complete PD-1 sequence can be found at GenBank accession number NP005009.2.

[0028] The terms "PD-L1," "PDL1," "programmed death ligand 1," CD274, and "programmed cell death 1" are used interchangeably and are thought to include variants, isoforms, and species homologs of human PD-L1, as well as analogs that share at least one common epitope with PD-L1. The complete PD-L1 sequence can be found at GenBank accession number NP054862.1.

[0029] The terms "cytotoxic T lymphocyte 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 common epitope with CTLA-4. The complete CTLA-4 sequence can be found at GenBank accession number NP_005205.2.

[0030] The terms "LAG-3," "LAG3," CD223, and "lymphocyte activation gene 3" are used interchangeably and are thought to include variants, isoforms, and species homologs of human LAG-3, as well as analogs that share at least one common epitope with LAG-3. The complete LAG-3 sequence can be found at GenBank accession number NP_002277.4.

[0031] The terms "TIM-3," "TIM3," "HAVCR2," "Hepatitis A virus receptor 2," CD366, and "T cell immunoglobulin domain and mucin domain 3" are used interchangeably and are thought to include variants, isoforms, and species homologs of human TIM-3, as well as analogs that share at least one common epitope with TIM-3. The complete TIM-3 sequence can be found at GenBank accession number NP_116171.3.

[0032] The terms "BTLA," "B and T lymphocyte attenuator," and "CD272" are used interchangeably and are thought to include variants, isoforms, and species homologs of human BTLA, as well as analogs that share at least one common epitope with BTLA. The complete BTLA sequence can be found at GenBank accession number NP_861445.4.

[0033] The terms "VISTA," "V-set immunomodulatory receptor," "B7H5," "B7-H5," "PD-1H," and "V-domain Ig suppressor of T cell activation" are used interchangeably and are thought to include variants, isoforms, and species homologs of human VISTA, as well as analogs that share at least one common epitope with VISTA. The complete VISTA sequence can be found at GenBank accession number NP_071436.1.

[0034] The terms "TIGIT," "T cell immunoreceptor with Ig and ITIM domains," "WUCAM," and "Vstm3" are used interchangeably and are thought to include variants, isoforms, and species homologs of human TIGIT, as well as analogs that share at least one common epitope with TIGIT. The complete TIGIT sequence can be found at GenBank accession number NP_776160.2.

[0035] As used herein, the term “therapeutic antibody” includes the entire antibody that produces a therapeutic effect and any antigen-binding fragment (i.e., “antigen-binding portion”) or a single chain of antigen-binding fragment.

[0036] Such therapeutic antibodies may be directed towards the checkpoint inhibitors indicated above and include agonist antibodies directed towards costimulatory molecules 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 the subject to receive both types of therapeutic antibodies.

[0037] As used herein, the term "monoclonal antibody" refers to a formulation of an antibody molecule in a single-molecule composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope. This monoclonal antibody may be a human antibody or a humanized antibody.

[0038] The term “adoptive cell therapy” is intended to refer to any treatment involving the transfer / administration of cells to a subject, preferably a human. The cells may be allogeneic or autologous. Preferably, the cells are usually derived from the immune system, with the aim of improving immune function. Examples of adoptive cell therapies include, but are not limited to, CAR-T cell therapy (chimeric antigen receptor T cells), TIL therapy (tumor-infiltrating lymphocytes), TCR therapy (modified T cell receptor therapy), NK therapy (natural killer cells), and / or iPSC-derived therapy (induced pluripotent stem cells).

[0039] The term “effective dose” or “pharmaceutical effective dose” refers to the amount of an agonist sufficient to produce a desired biological or therapeutic outcome. This outcome may be a reduction, recovery, remission, lessening, delay, and / or alleviation of one or more signs, symptoms, or causes of disease, or any other desired change in a living system. In the case of cancer, the effective dose may contain an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (such as inhibiting tumor growth) or to inhibit or delay other unwanted cell growth. In some embodiments, the effective dose is sufficient to delay the onset of cancer or tumor, or to extend survival or induce stabilization.

[0040] In some embodiments, the therapeutically effective dose is sufficient to prevent or delay recurrence. The therapeutically effective dose may be administered in one or more doses. The therapeutically effective dose of a drug or combination may cause one or more of the following: (i) a reduction in the number of cancer cells; (ii) a reduction in tumor size; (iii) some degree of inhibition, retardation, slowing, or preferably cessation of the invasion of cancer cells into peripheral organs; (iv) inhibition of tumor metastasis (i.e., some degree of slowing, preferably cessation); (v) inhibition of tumor growth; (vi) prevention or delay of tumor development and / or recurrence; and / or (vii) some degree of relief of one or more cancer-related symptoms.

[0041] For example, in relation to tumor treatment, a “therapeutic dose” may include at least about 5% tumor reduction compared to baseline measurements, such as at least about 10%, about 20%, about 60%, or more. Baseline measurements may be derived from untreated subjects.

[0042] A therapeutically effective amount of a therapeutic compound can reduce tumor size or restore the symptoms of the target. A person skilled in the art would be able to determine such an amount based on factors such as the size of the target, the severity of the symptoms of the target, and the specific composition or route of administration selected.

[0043] The term "immune response" refers to the action of lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, for example, causing selective damage, destruction, or removal of cancer cells in the human body.

[0044] The term "treatment" or "therapy" refers to a state (example). This refers to administering an agonist with the aim of curing, alleviating, relieving, altering, treating, recovering from, improving, or influencing the symptoms of a disease or condition, or preventing or delaying the onset of symptoms, complications, or biochemical signs of a disease, or statistically significantly stopping or suppressing further deterioration of a disease, condition, or disorder.

[0045] As used herein, the term “subject” is intended to include human and non-human animals. Preferred subjects include human patients requiring enhancement of the immune response. The method is particularly suitable for treating human patients with disorders that can be treated by enhancing the immune response. In certain embodiments, the method is particularly suitable for in vivo treatment of cancer.

[0046] As defined herein, "simultaneous" administration includes administering attenuated live bacteria and an immunotherapy composition to each other within about two hours or about one hour, and more preferably simultaneously.

[0047] As defined herein, “individual” administration includes administering attenuated live bacteria and immunotherapy compositions at intervals of more than about 12 hours, or about 8 hours, or about 6 hours, or about 4 hours, or about 2 hours.

[0048] As defined herein, “sequential” administration includes administering attenuated live bacteria and immunotherapy compositions in multiple aliquots and / or doses and / or at different times. The immunotherapy composition may be administered to the patient before or after administration of attenuated live bacteria, or the immunotherapy composition may be continued after treatment with attenuated live bacteria.

[0049] The use of substitutes (e.g., "or") should be understood to represent any one, both, or any combination of these substitutes. Where used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any listed or enumerated components.

[0050] Where used herein, “approximately” means within the tolerance range of a particular value as determined by those skilled in the art, and this range depends in part on how the value is measured or determined, i.e., the limits of the measuring system. For example, “approximately” may mean a standard deviation of 1 or more than 1 per run in the art. Alternatively, “approximately” may mean a range of 20% or less. Where a particular value is provided in this application and claims, unless otherwise specified, the meaning of “approximately” should be presumed to be within the tolerance range for that particular value.

[0051] As used herein, the terms “non-recombinant” and “non-recombinant strain” are interchangeable and, in the context of the present invention, refer to the fact that these strains do not contain eukaryotic genes or gene fragments. Thus, the strains disclosed herein do not function as “carrier strains” intended to deliver therapeutic molecules to subjects / patients. Accordingly, the strains disclosed herein do not encode eukaryotic heterologous DNA, or eukaryotic heterologous DNA encoding therapeutic molecules, or eukaryotic DNA encoding proteins or protein fragments intended to be antigens.

[0052] As used herein, the terms “systemic” and “systemically activated” are interchangeable and, in the context of the present invention, refer to a broad immune response throughout the subject’s body, as opposed to a localized, spatially limited response. Preferably, a systemic immune response involves the activation and / or maturation of myeloid cells, such as dendritic cells, monocytes, and / or macrophages, and, in the context of the present invention, is considered to help condition the subject’s immune system so that it responds to immunotherapies such as checkpoint inhibitors, adoptive cell therapy, and / or CAR-T cell therapy. Thus, Gram-negative bacteria after administration of the second composition The target immune response may be stimulated, boosted, amplified, enhanced, improved, increased, pre-activated, or promoted. The aforementioned terms are used interchangeably with the term "conditioned."

[0053] As used herein, the terms "tumor-agnostic therapy" or "agnostic to tumor type" are interchangeable and refer to a type of treatment that uses drugs or other substances to treat cancer based on genetic and / or molecular characteristics, regardless of the type of cancer or where the cancer originated in the body.

[0054] In a first aspect of the present invention, there are attenuated live Gram-negative bacteria used for the treatment, reduction, suppression or control of neoplastic diseases in subjects receiving or intended to receive checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy, either concurrently with, separately, or over time with the administration of attenuated live Gram-negative bacteria, wherein the attenuated live Gram-negative bacteria are administered orally, subcutaneously, or intramuscularly.

[0055] A second aspect of the present invention provides attenuated live Gram-negative bacteria for use in the treatment, reduction, suppression or control of neoplastic diseases in subjects receiving or intended to receive checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy, wherein the attenuated live Gram-negative bacteria are to be administered in the first phase of treatment, and the checkpoint inhibitor therapy, adoptive T cell therapy and / or allogeneic or autologous CAR-T therapy are to be administered in the second phase of treatment.

[0056] In a third aspect of the present invention, there are attenuated live Gram-negative bacteria for use in the treatment, reduction, suppression or control of neoplastic diseases in subjects receiving or intended to receive checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy, either concurrently with, separately, or over time, said attenuated live Gram-negative bacteria, wherein the said attenuated live Gram-negative bacteria are non-recombinant or do not contain heterologous eukaryotic DNA encoding a therapeutic protein.

[0057] The aforementioned attenuated live Gram-negative bacteria may therefore act as a modulator of the target immune system, and it is assumed that, when treated with checkpoint inhibitors, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy, the target immune system can initiate an effective immune response against neoplastic diseases.

[0058] The attenuated live bacteria of the present invention are Gram-negative bacteria. Examples of Gram-negative bacteria used in the present invention include, but are not limited to, Escherichia coli, Salmonella, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia, and Yersinia.

[0059] Preferably, the attenuated gram-negative bacteria are Salmonella species. Examples of Salmonella species used in the present invention are Salmonella Enterica and Salmonella bongori. Salmonella Enterica can be further subdivided into different serotypes or serovars. Examples of these serotypes or serovars used in the present invention are Salmonella enterica Typhi, Salm These are Salmonella enterica Paratyphi A, Salmonella enterica Paratyphi B, Salmonella enterica Paratyphi C, Salmonella enterica Typhimurium, and Salmonella enterica Enteritidis. In a preferred embodiment, the attenuated gram-negative bacterium is Salmonella enterica Typhi or Salmonella enterica Typhimurium.

[0060] In another embodiment of the present invention, the attenuated live Gram-negative bacteria are genetically modified non-natural bacteria. In a preferred embodiment, the attenuated live Gram-negative bacteria are non-recombinant bacteria. In another preferred embodiment, the Gram-negative bacteria disclosed herein do not contain eukaryotic genes or gene fragments.

[0061] As will be understood by those skilled in the art, genes may be mutated by many methods well known in the art, such as homologous recombination using recombinant plasmids that target the gene of interest. In homologous recombination, a genetically modified gene homologous to the target gene is incorporated into a suitable nucleic acid vector (such as a plasmid or bacteriophage), and the incorporated nucleic acid vector is introduced into target cells. The homologous genetically modified gene is recombined with the native gene to achieve a desired inactivating mutation by either substituting or introducing a mutation into the native gene. Such modifications may be in any regulatory region of the gene, such as the translational region or promoter region. As will be understood by those skilled in the art, any suitable gene modification technique may be used to introduce mutations into the gene of interest, such as a CRISPR / Cas system, such as CRISPR / Cas9.

[0062] Accordingly, numerous methods and techniques for genetically modifying bacterial species are well known to those skilled in the art. These techniques include those required to introduce heterologous genes into bacteria by chromosome integration or the introduction of stable autosomal self-replicating genetic elements. Examples of methods for genetically modifying (also referred to as "transforming" or "genetically modifying") 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 Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3, which are incorporated herein by reference.

[0063] Accordingly, the present invention discloses attenuated biogram-negative bacteria for use, which may be selected from the group including Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, x9633, x9640, x8444, DTY88 ZH9PA, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, or A1-R. Preferably, the bacteria disclosed herein are non-recombinant strains such as Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, x9633, x9640, x8444, DTY88, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, or A1-R. In another embodiment, the bacteria disclosed herein are strains modified to contain heterologous prokaryotic DNA, such as ZH9PA. In addition to the aforementioned strains, any attenuated, non-pathogenic Salmonella enterica serovar Typhi or Salmonella enterica serovar Typhimurium strains may be used, as disclosed herein, i.e., as modifiers, to initiate a more effective immune response after immunotherapy administration to a subject.

[0064] In one embodiment, the genetically modified non-natural bacterium is Salmonella Pa The attenuated bacteria may be derived from a Salmonella species that contains attenuated mutations in the thogenicity Island 2 (SPI-2) gene and a second gene. Suitable genes and details of such attenuated bacteria are described in Patent Document 1, which is incorporated herein by reference in its entirety.

[0065] In one embodiment, the SPI-2 gene is the ssa gene. For example, the present invention includes one or more attenuating mutations 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. Much more preferably, the attenuating mutation is in the ssaV gene.

[0066] The genetically modified non-natural bacteria may also contain an attenuating mutation in a second gene, which may or may not be in the SPI-2 region. This mutation may be outside the SPI-2 region and may be involved in the biosynthesis of aromatic compounds. For example, the present invention contains an attenuating mutation in the aro gene. In a preferred embodiment, the aro gene is aroA or aroC. Much more preferably, the aro gene is aroC.

[0067] Genetically modified non-natural bacteria may further contain one or more gene cassettes. Such gene cassettes may be used to maintain the function of the genetically modified non-natural bacteria for the purpose of conditioning the immune system, or to deliver additional prokaryotic molecules to maintain the activity of related immunotherapies, namely checkpoint inhibitor therapy, adoptive cell therapy, and / or CAR-T cell therapy. Those skilled in the art will recognize that the maintenance molecules thus delivered may depend on the immunotherapy to be administered.

[0068] In yet another embodiment, the genetically modified non-natural bacteria may be derived from Salmonella species and may contain inactivating mutations in one or more genes selected from pltA, pltB, cdtB, and ttsA, and may further contain attenuating mutations in one or more genes selected from aroA and / or aroC and / or ssaV. Details of these genes and mutations are described in Patent Document 2, which is incorporated herein by reference in whole.

[0069] It is hypothesized that inactivating mutations (e.g., deletions) in the genes pltA, pltB, and cdtB would prevent Salmonella species from producing typhoid toxin, and that inactivating mutations (e.g., deletions) in ttsA would prevent Salmonella species from secreting typhoid toxin. Non-natural bacteria may originate particularly from Salmonella Enterica.

[0070] In one embodiment, the genetically modified microorganism may be derived from Salmonella enterica serovar Typhi and may include a modification that expresses the lipopolysaccharide O2O antigen of Salmonella enterica serovar Paratyphi A. In yet another preferred embodiment, the genetically modified microorganism may be derived from Salmonella enterica serovar Typhi, and this strain may include a modification that expresses the flagellar protein of Salmonella enterica serovar Paratyphi A. In several examples, the genetically modified microorganism may be derived from Salmonella enterica serovar Typhi and may include a modification that expresses both the lipopolysaccharide O2O antigen and the flagellar protein of Salmonella enterica serovar Paratyphi A. Details of such modifications can be found in Patent Document 3. Such strains are considered non-recombinant in the context of the present invention, since the term "non-recombinant" refers to bacteria that do not contain eukaryotic genes or gene fragments, or bacteria that act as "carrier" strains for the purpose of delivering therapeutic molecules or heterologous eukaryotic DNA encoding therapeutic molecules.

[0071] When the methods described herein involve the use of a plasmid, the plasmid ideally has an origin of replication selected from pMB1, ColEI, p15A, pSC101, and RK2. The plasmid may also contain an antibiotic resistance gene selected from β-lactamase (bla), kanamycin phosphotransferase (kan), tetracycline efflux protein (tetA), or chloramphenicol acetyltransferase (cat). Ideally, the antibiotic resistance gene is excised before or immediately after transformation into a viable vector strain by a mechanism such as, for example, "X-mark" (Cranenburgh and Leckenby, 2012, Patent Document 4). Plasmid retention systems may be required to prevent plasmid loss. These may include mechanisms for placing native chromosome genes under heterologous promoters, such as, for example, the "Operator-Repressor Titration for Vaccines" system (Non-Patent Document 4) or the "oriSELECT" system (Cranenburgh, 2005, Patent Document 5), neither of which requires the presence of additional selection marker genes on the plasmid. Alternatively, one could use a selection marker gene that is not an antibiotic resistance gene, such as a gene for complementing host cell mutations (Non-Patent Document 5).

[0072] The present invention also relates to the above-mentioned attenuated viable bacterial strain, wherein the strain's natural fliC gene is replaced with the fliC gene of Salmonella enterica serover Paratyphi A so as to change the resulting serotype from Hd serotype to Ha serotype, and it is assumed that this also includes strains in which "serotype" refers to a distinct variant within a bacterial species. Details of such modifications can be found in Patent Document 6.

[0073] A further embodiment of the present invention is the attenuated live strain described above, which strain may be further modified to contain a functional fepE gene such that a long-chain O antigen chain is produced, preferably where the O antigen chain is a trisaccharide backbone 100 repeat units in length. Details of such modifications can be found in Patent Document 6.

[0074] The fepE gene encodes a factor that controls the length of the ultra-long-chain O antigen chain, and "ultra-long-chain" is considered to represent a trisaccharide backbone of more than 100 repeat units. Salmonella enterica serovar Typhi does not have these long-chain O antigen chains due to a mutation that introduces a stop codon into this gene. By many methods, Salmonella enterica serovar Typhi may be engineered to express these long-chain O antigen chains, such as replacing the native promoter of fepE with another promoter such as P araBAD , repairing the chromosomal mutation of fepE in Salmonella enterica serovar Typhi, or inserting a functional copy of fepE somewhere in the Salmonella enterica serovar Typhi chromosome. An in vivo-inducible promoter or a constitutive promoter may be utilized, and examples of such promoters include P pagC , P nirB , P ssaG , P sifA , P sifB , P sseA , P sseG , P sseJ , P lac , P tac , P trc , and λP L / P R . Similar modified sequences include P pagC , P nirB , P ssaG , P sifA , P sifB , P sseA , P sseG , P sseJ , and λP L / P RExamples include sequences that have at least approximately 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the wild-type sequences.

[0075] Preferably, the introduction of these long O antigen chains induces an LPS-specific immune response. It may be beneficial. If LPS is naturally very long-chain due to fepE expression, etc., there may be further advantages.

[0076] It is further conceivable that the aforementioned attenuated viable bacterial strains may be modified to constitutively express gtrC or to express gtrC in the trans form. Details of such modifications can be found in Patent Document 6.

[0077] It is further conceivable that the aforementioned attenuated viable bacterial strains may be further modified to include additional tviA gene copies under the control of a phagosome-inducible promoter. Details of such modifications can be found in Patent Document 6.

[0078] The amount of attenuated live Gram-negative bacteria administered to the subject, whether natural or unnatural, is sufficient to induce an immune response in the subject, thereby effectively conditioning the subject's immune system to receive checkpoint inhibitor therapy, adoptive cell therapy, and / or CAR-T cell therapy, and when treated with this therapy, the subject's immune system can initiate an effective immune response against cancer or tumor. The immune response initiated by the administration of attenuated live Gram-negative bacteria may be at a therapeutic level, or it may be below the therapeutic level that requires the administration of the aforementioned immunotherapies to exert a therapeutic effect.

[0079] Attenuated live Gram-negative bacteria may be administered at least once or twice, with a 2-week interval between doses. Attenuated live Gram-negative bacteria may be administered before, during, or after checkpoint inhibitor therapy, adoptive cell transfer therapy, and / or CAR-T cell therapy. Preferably, at least one administration of attenuated live Gram-negative bacteria occurs before immunotherapy treatment, and such administration may occur at least one week before immunotherapy treatment. Administration of attenuated live Gram-negative bacteria may be repeated depending on the immunotherapy treatment plan. 5 ~10 12 It may be administered in doses equivalent to CFUs, where CFU is a colony-forming unit. For example, a suitable dose is 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 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 11CFU, 10 8 ~10 12 CFU, 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's fine to be a CFU.

[0080] In one embodiment, the immunotherapy is a checkpoint inhibitor, and the term “checkpoint inhibitor” as used herein refers to an antagonist formulated against a checkpoint molecule. The antagonist may be an antagonist, an inhibitor, or a blocking antibody. Thus, the antagonist may be a small molecule or a biological agent, and in certain examples, a monoclonal antibody. In a preferred embodiment, the checkpoint inhibitor is formulated against CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, BTLA, TIGIT, VISTA, or any combination thereof. For example, the checkpoint inhibitor may be formulated against PD-1 and PD-L1, PD-1 and CTLA-4, or PD-L1 and CTLA-4.

[0081] Even more preferably, checkpoint inhibitors are formulated to target CTLA-4, PD-1, or PD-L1. In some examples, the blockers are ipilimumab (Yervoy®, targeting CTLA-4), nivolumab (Opdivo®, targeting PD-1), and pembrolizumab (Keytruda®, targeting PD-1). ), atezolizumab (Tecentriq® targeting PD-L1), semiprimab (Libtayo® targeting PD-1), or durvalumab (Imfinzi® targeting PD-L1).

[0082] The PD-L1 / PD-1 signaling pathway is a major mechanism by which cancer evades the immune system for several reasons. Firstly, and most importantly, this pathway is involved in the negative regulation of the immune response of activated effector T cells found in peripheral tissues. Secondly, PD-L1 is upregulated in the tumor microenvironment, but PD-1 is also upregulated on activated tumor-infiltrating T cells, potentially exacerbating a vicious cycle of suppression. Thirdly, this pathway is intricately involved in both innate and adaptive immune regulation through bidirectional signaling. Due to these factors, the PD-1 / PD-L1 complex becomes a major issue for cancer, enabling it to manipulate the immune response and promote its own progression. As a result, tumors can activate suppressive pathways of immune checkpoint molecules, suppressing the immune system and allowing cancer cells to proliferate unimpeded. After T cell activation, CTLA-4 is transported to the surface of antigen-presenting cells (APCs) to compete with CD28 for the same ligand, suppressing CD28 and subsequently inhibiting T cell activation and proliferation. Targeting PD-1, PD-L1, and CTLA-4 aims to prevent these events from occurring.

[0083] The present invention, as disclosed herein, may be able to condition the innate immune system and is therefore assumed to provide superior, more complete, and / or more durable support for the development, activation, and / or maintenance of checkpoint inhibitor-induced antitumor adaptive responses.

[0084] In one embodiment, immunotherapy is adoptive cell therapy, in which immune cells are transferred to a patient / subject, most commonly for improved functionality and properties. The cells to be transferred may originate from this subject (autologous) or another subject (allogeneic). Examples of such adoptive cell therapies include, but are not limited to, engineered or unengineered macrophages, engineered or unengineered γδ T cells, and engineered or unengineered natural killer cells. Therefore, adoptive cell therapies in the context of the present invention include, but are not limited to, tumor-infiltrating lymphocyte (TIL) therapy, genetically engineered T cell receptor (TCR) therapy, and / or natural killer (NK) cell therapy, the details of which are well known to those skilled in the art (Non-Patent Literature 6).

[0085] In another embodiment, the immunotherapy is CAR-T cell therapy. CAR-T cell therapy may be allogeneic or autologous. In some examples, CAR-T cell therapy is produced against the antigen CD19 present in B-cell derived cancers. Therefore, such therapies may be particularly suitable for B-cell derived cancers such as acute lymphoblastic leukemia (ALL) and diffuse large cell lymphoma (DLBCL). In other examples, CAR-T cell therapy is produced against tumor-associated antigens (TAAs) and is therefore more suitable for the treatment of solid tumors. Examples of such antigens include, but are not limited to, CD133, CD138, BCMA, 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 readers are directed to the review article titled “Adoptive cellular therapies: the current landscape” (Non-Patent Literature 7) for further information.

[0086] Checkpoint inhibitors may be therapeutic antibodies targeting cancer or tumors. In certain embodiments, the therapeutic antibody may be a monoclonal antibody, and in a much more preferred embodiment, a humanized or human monoclonal antibody. Methods for obtaining such monoclonal antibodies are known to those skilled in the art. The therapeutic antibody blocks abnormal proteins in cancer cells, adheres to specific proteins on cancer cells, or acts as a cytotoxic agent such as an anticancer agent. They can bind to molecules. The latter inform the immune system of cancer cells, and as a result, abnormal cells can be subsequently targeted and destroyed by cellular components of the immune system. Examples of monoclonal antibodies that are checkpoint inhibitors include, but are not limited to, ipilimumab (Yervoy®), nivolumab (Opdivo®), and pembrolizumab (Keytruda®).

[0087] Due to the multifactorial nature of the etiology, the immunotherapies described herein, namely checkpoint inhibitor therapy, adoptive cell therapy, and / or CAR-T cell therapy, are understood to be adjunctive therapies for patients / subjects. For example, adoptive cell therapy and checkpoint inhibitors may be used together. Whether adjunctive therapy is necessary is understood to be determined on an individual case basis and depends on numerous factors, such as the complexity of the neoplastic disease to be treated and / or the medical history of the patient / subject to be treated.

[0088] Regardless of the type of immunotherapy administered to the subject (the immunotherapy used may depend on the form and complexity of the neoplastic disease), the efficacy of this immunotherapy after administration of attenuated live Gram-negative bacteria is greater than that of immunotherapy components administered alone. In some cases, the efficacy of immunotherapy after administration of attenuated live Gram-negative bacteria is greater than that of any of the components administered alone. Therefore, the present invention provides either an additive or synergistic method for treating neoplastic diseases. This additive or synergistic effect is particularly advantageous when treating more complex and / or solid tumors and is particularly advantageous for patients / subjects who are unresponsive to immunotherapy alone.

[0089] The attenuated live Gram-negative bacteria used in the present invention may be used to treat neoplastic diseases associated with solid tumors or hematopoietic malignancies. Such diseases include sarcomas, carcinomas, adenocarcinomas, melanomas, myelomas, blastomas, gliomas, lymphomas, or leukemias. In preferred embodiments, the neoplastic disease is associated with solid tumors. In specific embodiments, the neoplastic disease is associated with cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, or sarcomas.

[0090] 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, or cancers or metastases that are progressing, worsening, stable, or in remission. Cancers that can be treated by the present invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestines, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, testes, tongue, or uterus. Furthermore, cancer is not limited to, but particularly includes, the following histological types, neoplasms, malignant cancers, carcinomas, undifferentiated carcinomas, giant cell and spindle cell carcinomas, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, gastrin-producing tumors, malignant cancers, cholangiocarcinomas, hepatocellular carcinoma, compound hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma of adenomatous polyps, adenocarcinoma, familial adenomatous polyposis, solid tumors, carcinoid tumors, malignant cancers, bronchioloalveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, eosinophilic carcinoma, eosinophilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, unencapsulated sclerosing carcinoma, adrenocortical carcinoma, endometrioid Cancer, skin adnexal carcinoma, apocrine gland carcinoma, sebaceous gland carcinoma, ear canal gland carcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous carcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease, mammary gland, acinar cell carcinoma, adenosquamous cell carcinoma, adenocarcinoma with squamous metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant theca cell tumor, malignant granulosa cell tumor, malignant androblastoma, Sertoli cell carcinoma, malignant Leydig cell tumor, malignant lipid cell tumortumor), malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, glomangiosarcoma, malignant melanoma, achromatic melanoma, superficial spreading melanoma, malignant melanoma of giant pigmented nevus, epithelioid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, mixed tumor, Müllerian mixed tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymal tumor, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, undifferentiated germ cell tumor, embryonic carcinoma, malignant teratoma, malignant ovarian goiter, choriocarcinoma, malignant mesonephroma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangioectocytoma, lymphangiosarcoma, osteosarcoma, paraosteal osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, ameloblastic Odontosarcoma, malignant ameloblastic fibrosarcoma, malignant pineal gland tumor, chordoma, malignant glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroblastoma, primitive neuroectodermal tumor, cerebellar sarcoma, gangliblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, malignant meningioma, neurofibrosarcoma, malignant schwannoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease; Paragranuloma, malignant lymphoma, small lymphocytic lymphoma, malignant lymphoma, large cell type, diffuse malignant lymphoma, follicular lymphoma, mycosis fungoides, other specific non-Hodgkin lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative bowel disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemialeukemia) may be myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid leukemia, and hairy cell leukemia. Preferably, the neoplastic disease may be a tumor equivalent to cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, head and neck cancer, skin cancer, and soft tissue sarcoma and / or other forms of cell tumor. The tumor may be metastatic or malignant.

[0091] In a preferred embodiment, the neoplastic disease is related to a cancer selected from lung cancer, kidney cancer, bladder cancer, stomach cancer, ovarian cancer, colorectal cancer, melanoma, head and neck cancer, or breast cancer.

[0092] In preferred embodiments of the present invention, attenuated live Gram-negative bacteria are administered orally, subcutaneously, or intramuscularly, as this method of administration is compatible with systemic activation of the immune system. Where used herein, the terms “orally” or “administered orally” are interchangeable and refer to the administration of Gram-negative bacteria through the mouth of the patient / subject. The terms “subcutaneous” or “administered subcutaneously” are interchangeable and refer to the administration of Gram-negative bacteria subcutaneously to the patient / subject. The terms “intramuscular” or “administered intramuscularly” are interchangeable and refer to the administration of Gram-negative bacteria into the muscle of the patient / subject. In the most preferred embodiment, attenuated live Gram-negative bacteria are administered orally. However, other methods of administration may also be used. Accordingly, in certain cases, the attenuated live Gram-negative bacteria of the present invention may be administered by injection, infusion, continuous infusion, intravenous, intradermal, intraarterial, intrafocal, intravaginal, intrarectal, intramuscular, intraperitoneal, subcutaneous, subconjunctival, mucosa, intrapericardial, intraumbilical, intraocular, intracranial, intraarticular, intraprostatically, intrapleural, intratracheal, intranasal, inhalation (e.g., aerosol inhalation), via catheter, via lavage, or other methods, or any combination thereof (see, for example, Non-Patent Document 8). Preferably, the Gram-negative bacteria of the present invention may be administered via mucosa, vaginal, intravenous, intranasal, intrapleural, or intradermal. In preferred embodiments, the Gram-negative bacteria disclosed herein are not administered intratumorally.

[0093] In another embodiment of the present invention, attenuated live Gram-negative bacteria are administered to the subject in the first phase of treatment, and checkpoint inhibitor therapy, adoptive T-cell therapy and / or allogeneic or autologous CAR-T therapy are administered in the second phase of treatment.

[0094] The terms "Phase 1 of treatment" and "Phase 2 of treatment" indicate a treatment process in which Phase 1 and Phase 2 of treatment are separated in time, such that there is a gap in time between Phase 1 and Phase 2 in which the patient / subject is not receiving the Gram-negative bacteria or immunotherapy disclosed herein.

[0095] While not constrained by theory, it is thought that administering attenuated live Gram-negative bacteria before the immunotherapy in question (i.e., in the first phase of treatment) could effectively condition the immune system before immunotherapy is administered, allowing for more effective targeting of cancer or tumors before the administration of checkpoint inhibitor therapy, adoptive cell therapy, and / or CAR-T cell therapy.

[0096] While it is preferable to administer attenuated live Gram-negative bacteria before the immunotherapy composition, there may be cases where it is necessary to administer attenuated live Gram-negative bacteria simultaneously or afterward. Therefore, attenuated live Gram-negative bacteria and immunotherapy may be present in the same or separate pharmaceutical preparations and administered simultaneously or at different time points.

[0097] Preferably, the first and second phases of treatment are administered at least one week apart, and preferably, the first and second phases of treatment are administered two weeks apart.

[0098] The attenuated live Gram-negative bacteria disclosed herein induce a systemic immune response in the target when used. Preferably, the attenuated live Gram-negative bacteria induce a systemic immune response that enhances the activation and / or maturation of myeloid cells. Examples of such myeloid cells include, but are not limited to, conventional dendritic cells, plasmacytoid dendritic cells, monocytes, and / or macrophages. Therefore, in the context of the present invention, a systemic immune response may refer to a long-term phenotypic change in the circulating / systemic bone marrow compartment.

[0099] A fourth aspect of the present invention provides a method for treating, suppressing or controlling a target neoplastic disease, the method comprising (i) administering attenuated live Gram-negative bacteria, which are administered orally, subcutaneously, or intramuscularly, and (ii) administering checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy to the target simultaneously, separately, or over time, the method being characterized by enhancing the therapeutic effect compared to the administration of attenuated live Gram-negative bacteria or checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy alone.

[0100] A fifth aspect of the present invention is a method for treating, suppressing or controlling a target neoplastic disease, the method comprising simultaneously, separately, or over timely administration to the target of (i) attenuated live Gram-negative bacteria, the attenuated live Gram-negative bacteria being administered in the first phase of treatment, and (ii) checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy being administered in the second phase of treatment, the method being enhanced compared to the administration of the attenuated live Gram-negative bacteria or checkpoint inhibitor therapy, adoptive cell therapy and / or allogeneic or autologous CAR-T therapy alone.

[0101] A sixth embodiment of the present invention provides a method for treating, suppressing or controlling a target neoplastic disease, wherein the method comprises (i) a virulent, non-recombinant, or a eukaryote encoding a therapeutic protein. (ii) administering to the subject simultaneously, separately, or over time, attenuated live Gram-negative bacteria that do not contain heterologous DNA, and (ii) checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy, wherein the method enhances the therapeutic effect compared to the administration of the attenuated live Gram-negative bacteria or checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy alone.

[0102] Therefore, the method of the present invention may be used to reduce or suppress the metastasis of a primary tumor or cancer to other sites, or the formation or establishment of metastatic tumors or cancers at other sites distal to the primary tumor or cancer, thereby suppressing or reducing the recurrence or progression of the tumor or cancer. Accordingly, the present invention provides a detectable or measurable improvement of a particular subject's condition, such as reducing or relieving one or more adverse (physical) symptoms or consequences associated with the presence of cytoproliferative or hyperproliferative disorders, neoplasms, tumors or cancers, or metastases, i.e., therapeutic or beneficial effects.

[0103] The methods of the present invention are therefore combination therapies comprising attenuated live Gram-negative bacteria and immunotherapy, i.e., checkpoint inhibitor therapy, adoptive cell therapy, and / or CAR-T cell therapy, which may induce a potent and sustained immune response with enhanced therapeutic effects. Due to the additive or synergistic nature of the combination therapies disclosed herein, lower levels of treatment or therapy may be required, and adverse effects are reduced due to the favorable toxicity profile.

[0104] A therapeutic effect or beneficial effect is any objective or subjective, transient, temporary or long-term improvement of a condition or pathology, or a reduction in the occurrence, severity, duration or frequency of adverse symptoms associated with or caused by cell proliferation or hyperproliferation disorders such as neoplasms, tumors or cancers, or metastases. Survival time may be improved by the therapeutic effect or beneficial effect. A satisfactory clinical endpoint of the treatment according to the present invention is achieved, for example, by an increased or partial reduction in the severity, duration or frequency of one or more associated pathologies, adverse symptoms or complications, or by the suppression or recovery of one or more physiological, biochemical or cellular signs or characteristics of cell proliferation or hyperproliferation disorders such as neoplasms, tumors or cancers, or metastases. The therapeutic effect or improvement may, however, be the destruction of target proliferative cells (e.g., neoplasms, tumors or cancers, or metastases) or the disappearance of most or all of one or more conditions, adverse symptoms or complications associated with or caused by cell proliferation or hyperproliferation disorders such as neoplasms, tumors or cancers, or metastases. However, the therapeutic effect or improvement does not necessarily have to be the cure or complete destruction of all target proliferative cells (e.g., neoplasms, tumors or cancers, or metastases) or the disappearance of all conditions, adverse symptoms or complications associated with or caused by cell proliferation or hyperproliferation disorders such as neoplasms, tumors or cancers, or metastases. For example, stabilizing the amount, size or number of tumors or cancers by partially destroying a population of tumors or cancers, or by suppressing the progression or worsening of tumors or cancers, can reduce mortality and extend life by even just a few days, weeks, or months, even if some or most of the tumors or cancers remain.

[0105] Specific non-limiting examples of therapeutic effects include reduction of neoplasm, tumor, or cancer volume (size or cell mass) or cell number, inhibition or prevention (e.g., stabilization) of the increase in neoplasm, tumor, or cancer volume, slowing or inhibiting the progression, worsening, or metastasis of neoplasm, tumor, or cancer, or inhibiting the proliferation, growth, or metastasis of neoplasm, tumor, or cancer.

[0106] The method of the invention does not need to be immediately effective. For example, the number or volume of neoplasms, tumors, or cancer cells may increase after treatment, but the stabilization or decrease of tumor cell volume, size, or number of cells in a particular target may occur gradually thereafter.

[0107] Additional adverse symptoms and complications associated with neoplasms, tumors, cancers, and metastases that can be suppressed, reduced, decreased, delayed, or prevented include, for example, nausea, loss of appetite, lethargy, pain, and discomfort. Therefore, partial or complete reduction or decrease in the severity, duration, or frequency of adverse symptoms or complications associated with or caused by cellular hyperproliferative disorders, as well as improvements in the subject's quality of life and / or well-being, such as increased vitality, appetite, and psychological well-being, are all specific, non-limiting examples of therapeutic effects.

[0108] The therapeutic effect or improvement may therefore include a subjective improvement in the quality of life of the treated subject. In a further embodiment, the method extends or prolongs the lifespan (survival time) of the subject. In a further embodiment, the method improves the quality of life of the subject.

[0109] The present invention may be particularly suitable for individuals who have not responded to prior treatment with checkpoint inhibitors, adoptive cell therapy, and / or CAR-T cell therapy. The term "not responded" is intended to refer to any neoplastic disease that does not respond to treatment. The present invention may also be particularly suitable for individuals who previously responded poorly, moderately, or highly to prior immunotherapy treatments.

[0110] The attenuated live Gram-negative bacteria and immunotherapies in question are typically administered to a subject in a composition containing an effective amount of attenuated live Gram-negative bacteria or immunotherapy, further comprising a pharmaceutically acceptable carrier / adjuvant / diluent or additive. The phrase "pharmaceutically or pharmacologically acceptable" means molecular entities and compositions that, where necessary, do not produce harmful, allergic, or other undesirable reactions when administered to animals, such as humans. Such formulations are known to those skilled in the art. Furthermore, for animal (e.g., human) administration, it is understood that the formulation should, where applicable, meet standards of sterility, pyrogenicity, general safety, and purity.

[0111] As used herein, “pharmaceutically acceptable carriers / adjuvants / diluents / additives” include, but are not limited to, any and all solvents, dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobials, antifungals), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, colorants, such substances and combinations thereof, as are known to those skilled in the art (see, for example, Non-Patent Document 9). Examples include, but are not limited to, disodium hydrogen phosphate, soy peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, calcium chloride, sucrose, borate buffer, sterile saline (0.9% NaCl), and sterile water.

[0112] The composition, the attenuated live Gram-negative bacterial composition, or the immunotherapy may also contain additional components intended to enhance the immune response. Examples of such additional components include aluminum salts such as aluminum hydroxide, aluminum oxide, and aluminum phosphate; oily adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant; mycolic acid adjuvants (e.g., trehalose dimycolate); bacterial lipopolysaccharides (LPS); peptidoglycans (e.g., glycoproteins such as murein, mucopeptide, or N-Opaca, muramyl dipeptide [MDP], or MDP analogues); proteoglycans (e.g., extracted from Klebsiella pneumoniae); streptococcal preparations (e.g., OK432); muramyl dipeptide; immunostimulatory complexes ("Iscoms" disclosed in Patent Documents 7, 8, and 9); saponins; DEAE-dextran; neutral oils (e.g., migliol); vegetable oils (e.g., peanut oil); liposomes; polio Examples include, but are not limited to, 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, and IL-17.

[0113] The inventors of this invention have surprisingly found that the combination of attenuated live Gram-negative bacteria and immunotherapy is more effective in preventing and / or treating neoplastic diseases than when either component is used alone. The present invention will be further described with reference to the following non-limiting examples. [Examples]

[0114] Example 1 - Induction of long-term phenotypic changes in systemic myeloid cells Adult female BALB / c mice 1 × 10 9Mice were orally treated with 1×10⁷ colony forming units (CFU) of Salmonella enterica serovar Typhimurium strain MD58 (ΔaroC). After 21 days, spleens were harvested, single cell suspensions were prepared, and flow cytometry staining was performed. Viable CD11c high and HLA-DR + and CD11b + / - and PDCA-1 conventional dendritic cells and - viable CD11c - / low and PDCA1 + and HLA-DR - / Int and CD11b - The median fluorescence intensities of the markers CD80, CD86, and PD-L1 of plasmacytoid dendritic cells were measured (see Figures 1A and 1B). Viable CD11c - and CD11b + and Ly6C + and F4 / 80 - The median fluorescence intensities of the markers PD-L1, CD80, and HLA-DR of monocytes and viable CD11c - and CD11b + and Ly6C - and F4 / 80 + of macrophages were also measured (see Figures 1C and 1D).

[0115] As seen in Figure 1, various cell markers of myeloid cells, such as conventional dendritic cells, plasmacytoid dendritic cells, monocytes, and macrophages, showed a significant increase 21 days after treatment with Salmonella, suggesting that the effect on the activation of immune cells after treatment with Salmonella can be sustained for a considerable period of time.

[0116] Example 2 - Time course of phenotypic changes induced by Salmonella To investigate the kinetics of the activation / maturation phenotypic changes of myeloid cells observed in Figure 1, a time course study was performed. Adult female BALB / c mice were given 1×10 9CFUs were orally treated with Salmonella enterica serovar Typhimurium strain MD58 (ΔaroC). Spleens and femurs were collected at 1, 14, 21, or 42 days, single cell suspensions were prepared, and flow cytometry staining was performed (see schematic diagram of the experiment in Figure 2A).

[0117] Systemic conventional (cDC) and plasmacytoid (pDC) dendritic cell activation markers increased after oral administration of Salmonella, peaked at 3 weeks post-administration, and returned to near baseline levels by 6 weeks post-administration (see Figure 2B). Systemic monocyte and macrophage activation markers also increased after oral administration of Salmonella, peaked at 3 weeks post-administration, and returned to near baseline levels by 6 weeks post-administration (see Figure 2C).

[0118] Example 3 - Oral administration of Salmonella enhances bone marrow hematopoiesis. Adult female BALB / c mice 1 × 10 9 CFUs were orally treated with Salmonella enterica serovar Typhimurium strain MD58 (ΔaroC). Flow cytometry staining was performed on isolated bone marrow cells at 1, 14, 21, or 42 days.

[0119] The number of hematopoietic stem cells / progenitor cells in the bone marrow significantly increased two weeks after oral administration (see Figure 3A). Representative flow cytometry plots also demonstrate an increase in viable LKS cells in the bone marrow of animals orally treated with Salmonella (see Figure 3B). Furthermore, the proportion of viable LKS cells to total bone marrow cells 14 days after Salmonella treatment, when using Spearman's rank correlation, shows a significant increase in viable LKS cells compared to monocytes (viable CD11c cells) at the same point in time. - CD11b + Ly6C + F4 / 80 - This correlated with the proportion of cells (see Figure 3C).

[0120] Therefore, the data from this embodiment support the hypothesis that Salmonella administration conditions the immune system, particularly the bone marrow arm of the immune system. Furthermore, these data demonstrate a long-term effect of Salmonella conditioning the immune system, as the conditioned changes induced by Salmonella are observed over a period of 3–6 weeks. These changes appear to be systemically / centrally mediated, as suggested by the positive correlation between bone marrow hematopoietic progenitor cells and splenic monocytes.

[0121] Example 4 - Oral administration of Salmonella induces a state of heightened responsiveness in systemic dendritic cells that lasts for at least 14 days. Adult female BALB / c mice 1 × 10 9 The cells were orally treated with CFU Salmonella enterica serovar Typhimurium strain MD58 (ΔaroC). After 14 days, the spleen was harvested, a single cell suspension was prepared, and CD11c-expressing cells were concentrated by magnetic separation to obtain 1 × 10⁶ cells. 5 Cells / wells were incubated for 24 hours with the indicated stimulus (TLR9, TLR2 / 6, or TLR4 / 2 agonist or solvent). IL-6 in the supernatant was measured by the LegendPlex assay (see Figure 4).

[0122] As is clear from Figure 4, CD11c-expressing cells derived from animals treated with Gram-negative bacteria, such as Salmonella, showed increased IL-6 secretion (an indicator of the level of cellular immune responsiveness) in response to various stimuli compared to the solvent control group. This further supports the idea that administration of the Gram-negative bacteria of the present invention conditions immune cells, making them more responsive to subsequent stimuli.

[0123] As shown in Examples 1-4, the experimental data disclosed in these examples indicate that treatment with attenuated live Gram-negative bacteria, such as Salmonella, unexpectedly activates multiple immune cell types (e.g., dendritic cells, monocytes, and macrophages) over a long period, and also increases hematopoietic stem cells / progenitor cells in the bone marrow. While not theoretically constrained, it is thought that the systemic response induced by the administration of such attenuated live Gram-negative bacteria can condition the immune system of the subject or patient to enhance the antitumor activity of immunotherapies, such as checkpoint inhibitor therapy, adoptive cell therapy, or CAR-T cell therapy, when administered in combination with such immunotherapies. This is further demonstrated in Example 5 below. The systemic modifications observed from the administration of attenuated live Gram-negative bacteria, namely the activation of multiple immune cell types and the effect on bone marrow hematopoiesis (as shown herein), are thought to contribute to the enhanced antitumor effect when combined with the intestinal uptake of these attenuated live Gram-negative bacteria. Those skilled in the art will readily understand that the broad systemic effects induced by attenuated live Gram-negative bacteria can condition the patient's / subject's immune system so that the beneficial effects are observed across a multitude of checkpoint inhibitors and / or adoptive cell therapies, i.e., the beneficial effects are not limited to a single treatment but are applicable across the entire range of treatments. Furthermore, using Gram-negative bacteria to condition the subject's / patient's immune system may be particularly advantageous for patients / subjects who are known to be resistant to immunotherapy when immunotherapy is used alone.

[0124] Example 5 - Oral administration of Salmonella slows tumor growth, and its effectiveness is enhanced when administered in combination with an immune checkpoint inhibitor. Adult female C57BL / 6 mice, 1 x 10 6 MC38 (colon cancer) or B16-F10 (melanoma) tumor cells were subcutaneously disseminated.

[0125] In Figure 5A, animals with MC38 tumors were measured at 15 and 22 days after tumor dissemination, with 1 × 10⁶ measurements. 9CFU Salmonella enterica serovar Typhimurium strain MD58 (ΔaroC) or PBS control, and oral treatment with 10 mg / kg anti-PD-L1 twice weekly from day 17. The graphs show tumor volume over time and depict tumor growth, and the corresponding tables summarize the mean inhibition of tumor growth compared to the control solvent and isotype groups after treatment with Salmonella and isotype control, Salmonella and αPD-L1, or solvent and αPD-L1 from day 14 to day 30 (see Figure 5A).

[0126] For Figure 5B, animals with tumors B16-F10 were measured at 1 × 10⁶ on day 8 after tumor dissemination. 9 Tumors were treated orally with 10 mg / kg anti-PD-1 antibody on days 10, 13, and 17, with either CFU Salmonella enterica serovar Typhimurium strain MD58 (ΔaroC) or PBS control. The graphs show tumor volume over time and depict tumor growth, and the corresponding tables summarize the mean suppression rates of tumor growth by various treatments from days 14 to 30 compared to the control group (solvent + isotype) (see Figure 5B).

[0127] As discussed above, while immunotherapies such as checkpoint inhibitor therapy, adoptive cell therapy, and CAR-T therapy are effective for subpopulations of patients, it is known that a large majority of patients still show little to no effect from these treatments. Thus, finding a treatment to address this unresolved need in the field of oncology is of paramount importance. This issue is reflected in the tables shown in Figures 5A and 5B, where treatment with checkpoint inhibitors shows little to no effect on suppressing tumor growth, and is not significantly better than the effects produced by solvent and isotype controls. However, in stark contrast, when checkpoint inhibitors are administered in combination with attenuated live Gram-negative bacteria, such as Salmonella, the tumor growth inhibition rate is significantly improved (approximately 13 times greater inhibition than that seen in the checkpoint inhibitor alone group in Figure 5A). This effect is also considerably higher than that seen with Salmonella alone, highlighting the advantages of using the two components in combination. Therefore, the inventors of this invention have surprisingly found that the antitumor activity of immunotherapies such as checkpoint inhibitors, adoptive cell therapy, and CAR-T therapy can be significantly enhanced when administered in combination with attenuated live Gram-negative bacteria.

[0128] Furthermore, the inventors also demonstrated identical trends in additive and / or synergistic effects of two different checkpoint inhibitors in two unrelated tumor models, demonstrating and supporting the hypothesis that conditioning effects induced by attenuated bio-Gram-negative bacteria condition the patient / subject's immune system so that favorable effects are observed across numerous checkpoint inhibitors and / or adoptive cell therapies, and potentially favorable across a wide range of cancer signs. Thus, those skilled in the art will readily understand that the effects of the present invention are tumor type independent.

[0129] Example 6 - In vitro conditioning of macrophages with Salmonella further polarizes M1 cells into an inflammatory phenotype and further induces M2 cells into M1-like phenotypic macrophages. Macrophages are generally widely accepted to be classified into M1 macrophages (pro-inflammatory, classically activated) and M2 macrophages (anti-inflammatory, selectively activated / inhibitory) based on their phenotype and functionality. Macrophages traditionally play a role in angiogenesis, invasiveness, metastasis, control of the tumor microenvironment, and treatment resistance. Macrophages are known to be directly or indirectly involved in several key characteristics of malignant tumors. However, macrophages also have the ability to present tumor antigens, support the proliferation and expansion of tumor-specific T cells, phagocytose cancer cells, and reconstruct the tumor microenvironment, making tumors more susceptible to treatment. It is also known that repolarizing M2-phenotypic macrophages into M1-phenotypic macrophages is sufficient to produce an antitumor effect (Non-Patent Literature 10). Therefore, any treatment strategy that can initiate such a repolarization effect would be particularly advantageous for supporting and enhancing the antitumor effects of adoptive cell therapy and other immunotherapies such as checkpoint inhibitors.

[0130] As described herein, in vitro treatment of M2 macrophages with Gram-negative bacteria not only activates various immune cells and induces myelopoeia (see Examples 1-4), but also initiates a phenotypic shift from suppressive M2 macrophages to an M1-like phenotype (see Example 6). While not theoretically constrained, such repolarization from M2 macrophages to M1 macrophages is thought to further contribute to the antitumor effects described herein.

[0131] PBMCs from three different donors were separated by Ficoll-Paque gradient centrifugation using Buffycoat. CD14 +Monocytes were isolated using a CD14 microbead isolation kit (Miltenyi Biotec), and M1 cells were cultured in GM-CSF and M2 cells in M-CSF for 6 days. A cytokine cocktail was added on day 8 to polarize the cells into M1 and M2 cells. The resulting macrophages were collected, washed, and treated with Salmonella enterica Typhi strain ZH9 (ΔaroC, ΔssaV) at an infection efficiency of 1, or left untreated. After 1 hour, the cells were collected, washed, and cultured in medium for 48 hours. Before flow cytometry analysis, the cells were stained with antibodies against CD163 and CD86.

[0132] The results shown in Figure 6 demonstrate that Salmonella is effective against both M1 and M2 macrophages. When M1 macrophages are conditioned with Salmonella, almost all cells express CD86 and decrease CD163, and this combination of features is specific to pro-inflammatory antitumor macrophages. Similarly, this same CD86 is absent or present in small amounts in M2 suppressive macrophages. + CD163 - The population appears in this cell population after it has been conditioned with Salmonella. This suggests that the tumor microenvironment (TAM—often referred to as tumor-associated macrophages) and suppressive M2 macrophages, which are generally associated with poor prognosis, can be altered by Salmonella and conditioned into a more pro-inflammatory phenotype that better supports the antitumor response.

[0133] Example 7 - In vitro conditioning of human monocytes overcomes M2 polarization and reduces M2 inhibitory ability. M2-like macrophages, such as tumor-associated macrophages, are known to exert immunosuppressive effects on various immune cell types, including T cells, through several mechanisms (Non-Patent Literature 11). Due to the known cytotoxic capacity of T cells directed towards antigens, T cells are a primary target for the development of various immunotherapies. Therefore, we investigated the effect on T cell proliferation after in vitro conditioning of human monocytes with Gram-negative bacteria.

[0134] PBMC was separated by Ficoll-Paque gradient centrifugation using buffy coat, and CD14 + Monocytes were isolated using a CD14 microbead isolation kit (Miltenyi Biotec). Monocytes were treated with Salmonella enterica Typhi strain ZH9 (ΔaroC,ΔssaV) at an infection efficiency of 1 for 30 minutes (conditional cells), or incubated in culture medium alone (unconditional cells). Cells were washed twice with PBS to remove Salmonella. The cells were removed and cultured for 6 days in medium supplemented with gentamicin. IL-4, IL-10, and TGFβ were then added for 48 hours to polarize the cells into immunosuppressive macrophages. Autologous CD4 + T cells were isolated and labeled with CellTrace Far Red. The T cells were co-cultured with macrophages in the presence of anti-CD3 / CD28Ab. After 5 days, the cells were harvested and T cell proliferation was evaluated using flow cytometry.

[0135] Human monocyte-derived M2 macrophages conditioned with Salmonella did not suppress T cell proliferation as much as unconditioned macrophages, as demonstrated by the greater T cell proliferation in the presence of Salmonella-conditioned macrophages compared to unconditioned macrophages (see Figures 7A and 7C). As can be seen from Figure 7C, unconditioned (culture medium) myeloid cells behave like suppressive M2 cells, effectively suppressing T cell proliferation with only an average of 10% of them becoming activated T cells that undergo more than two divisions. In contrast, Salmonella-conditioned myeloid cells did not suppress T cell proliferation as much, with an average of 20% becoming T cells that undergo more than two divisions. Therefore, Salmonella-conditioned monocyte-derived macrophages are not significantly affected by M2 polarization.

[0136] These data support the hypothesis that conditioning myeloid cells with Gram-negative bacteria can enhance T cell proliferation under suppressive conditions such as the tumor microenvironment, and that conditioning by oral administration of Gram-negative bacteria enhances the proliferation of adoptively transferred and CAR-T cell therapies.

[0137] Example 8 - In vitro training of human monocytes overcomes M2 polarization and enhances anti-PD-L1 Ab activity. After discovering that conditioning myeloid cells with Gram-negative bacteria could enhance T cell proliferation under suppressive conditions, we continued to investigate whether this effect impacts the efficacy of immunotherapy.

[0138] PBMC was separated by Ficoll-Paque gradient centrifugation using buffy coat, and CD14 + Monocytes were isolated using a CD14 microbead isolation kit (Miltenyi Biotec). Monocytes were treated with Salmonella enterica Typhi strain ZH9 (ΔaroC,ΔssaV) at an infection efficiency of 1 for 30 minutes (conditional cells) or incubated in culture medium alone (unconditional cells). Cells were washed twice with PBS to remove Salmonella and cultured for 6 days in medium supplemented with gentamicin. To polarize the cells into immunosuppressive M2 macrophages, IL-4, IL-10, and TGFβ were then added for 48 hours. (Autologous CD4) + T cells were isolated and labeled with CellTrace Far Red. The cells were co-cultured with macrophages in the presence of either anti-CD3 / CD28 Ab or anti-PD-L1 Ab or associated isotype Ab control. After 5 days, the cells were harvested and T cell proliferation was evaluated using flow cytometry.

[0139] Human monocyte-derived macrophages conditioned with Salmonella showed enhanced PD-L1 blocking activity compared to human monocyte-derived macrophages not exposed to Salmonella (see Figures 8A and 8C). As can be seen from Figure 8C, unconditioned (culture medium) myeloid cells combined with PD-L1 blockade were not effective in reversing the suppression of T cell proliferation in this experimental setting that mimicked the tumor microenvironment. However, in myeloid cells conditioned with Salmonella combined with PD-L1 blockade, this suppression became sensitive to checkpoint inhibitors, and ~50% of cells underwent more than two divisions, reversing the suppression of T cell proliferation in the presence of PD-L1 blockade.

[0140] These data suggest that conditioning myeloid cells with Gram-negative bacteria can induce a more immunosuppressive environment, such as a tumor microenvironment, even in patients who are not responsive to checkpoint inhibitor therapy. In this context, checkpoint blockade enables and enhances the effectiveness of reversing the suppression of T cell proliferation. Therefore, this data supports the hypothesis that conditioning by administration of Gram-negative bacteria enhances the effectiveness of immunotherapies such as checkpoint inhibitors, adoptive cell therapy, and CAR-T therapy.

[0141] Therefore, the inventors of the present invention have shown, through numerous assays in both rodents and human cells, that combining Gram-negative bacteria with immunotherapies such as checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy can enhance the antitumor effects of these therapies compared to the use of immunotherapy alone. Thus, the present invention represents a significant improvement in the field of oncology and provides, at the very least, a novel treatment option for patients who are currently unresponsive to existing immunotherapies used in clinical practice for reasons that are currently unknown.

Claims

1. A pharmaceutical preparation containing attenuated live Gram-negative bacteria for use in inducing a systemic immune response in subjects with malignant neoplastic diseases, The subjects mentioned above are receiving, or intending to receive, checkpoint inhibitor therapy, adoptive cell therapy, and / or allogeneic or autologous CAR-T therapy simultaneously with, separately from, or over time with the administration of the attenuated live Gram-negative bacteria. The aforementioned attenuated live Gram-negative bacteria are non-recombinant or do not contain heterologous eukaryotic DNA encoding therapeutic molecules. The aforementioned attenuated live Gram-negative bacteria are administered orally, subcutaneously, intradermally, or intramuscularly. The aforementioned attenuated live Gram-negative bacteria is a Salmonella species. Pharmaceutical preparations.

2. The pharmaceutical preparation for use according to claim 1, wherein the attenuated live Gram-negative bacterium is Salmonella Enterica.

3. A pharmaceutical preparation for use according to claim 1 or 2, wherein the attenuated live gram-negative bacterium is Salmonella enterica serva Typhi, or the attenuated live gram-negative bacterium is Salmonella enterica serva Typhimurium.

4. The aforementioned attenuated live Gram-negative bacteria are genetically modified non-natural bacteria, as described in any one of claims 1 to 3, for use in a pharmaceutical preparation.

5. The attenuated live Gram-negative bacteria is selected from the group comprising Ty21a, CVD908-htrA, CVD909, Ty800, M01ZH09, x9633, x9640, x8444, DTY88, ZH9PA, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, and A1-R, and is a pharmaceutical formulation for use according to any one of claims 1 to 4.

6. The pharmaceutical formulation for use according to any one of claims 1 to 5, wherein the checkpoint inhibitor is a blocker formulated for CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, BTLA, TIGIT, VISTA, or any combination thereof.

7. The pharmaceutical formulation for use according to claim 6, wherein the checkpoint inhibitor is a blocker formulated for CTLA-4, PD-1, or PD-L1.

8. A pharmaceutical formulation for use according to any one of claims 1 to 7, wherein the adoptive cell therapy is tumor-infiltrating lymphocyte (TIL) therapy, modified T cell receptor (TCR) therapy, and / or natural killer (NK) cell therapy.

9. The aforementioned malignant neoplastic disease is related to a solid tumor or a hematopoietic tumor, and is a pharmaceutical preparation for use according to any one of claims 1 to 8.

10. The aforementioned malignant neoplastic disease relates to a solid tumor, and is a pharmaceutical formulation for use according to claim 9.

11. A pharmaceutical preparation for use according to any one of claims 1 to 10, wherein the malignant neoplastic disease relates to a cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, cell tumor, head and neck cancer, skin cancer, or sarcoma.

12. A pharmaceutical formulation for use according to claim 11, wherein the malignant neoplastic disease relates to a cancer selected from lung cancer, bladder cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer, melanoma, kidney cancer, or breast cancer.

13. The aforementioned attenuated live Gram-negative bacteria are administered orally in a pharmaceutical preparation for use according to any one of claims 1 to 12.

14. A pharmaceutical preparation for use according to any one of claims 1 to 13, wherein the attenuated live Gram-negative bacteria is administered in the first stage of treatment, and the checkpoint inhibitor therapy, the adoptive cell therapy and / or the allogeneic or autologous CAR-T therapy is administered in the second stage of treatment.

15. The pharmaceutical formulation for use according to claim 14, wherein the first and second stages of the treatment are administered at least one week apart.

16. The pharmaceutical preparation for use according to claim 15, wherein the first and second phases of the treatment are administered two weeks apart.

17. The systemic immune response increases the activation and / or maturation of myeloid cells, a pharmaceutical formulation for use according to any one of claims 1 to 16.

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