Compositions containing bacteria for cancer immunotherapy

Abstract

The present invention provides an immunogenic composition comprising: (a) a bacterial population comprising at least three pathogenic bacterial species selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis, wherein the bacteria are inactivated bacteria comprising the entire bacterial cell wall; and (b) one or more tumor antigenic determinants. Pharmaceutical compositions comprising the immunogenic composition and uses thereof for preventing or treating tumors in subjects in need thereof are also provided.

Description

Technical Field

[0001] The present invention relates to the field of cancer immunotherapy. In particular, the present invention relates to an immunogenic composition comprising a combination of bacterial species for cancer treatment.

Background Art

[0002] Malignant tumors are one of the major challenges in modern medicine. It is widely accepted that conventional treatment methods such as surgery, chemotherapy and radiation have limited effectiveness in many common types and stages of cancer.

[0003] Immunotherapy is an evolving field of research and an additional option for the treatment of certain types of cancer. Despite the presence of antigen presentation by cancer cells and immune cells that can potentially react against tumor cells, in many cases, the immune system has been found to be either not sufficiently activated against the tumor or is effectively suppressed. The key to this phenomenon is the ability of tumors to develop several immunoregulatory mechanisms to avoid anti-tumor immune responses. Immunotherapy approaches are based on the rationale that the immune system can be stimulated to identify tumor cells and target them for destruction.

[0004] In this sense, one of the conventional approaches was to use inactivated bacteria as therapeutic vaccines for treating malignant tumors. For example, Coley's vaccine (a combination of Streptococcus pyogenes and Serratia marcescens) has long been known to be useful for the treatment of sarcoma and lymphoma, and BCG (Mycobacterium bovis) vaccine treatment has been reported to be effective for the treatment of some types of cancer.

[0005] Recently, other types of immunotherapy have been explored. For example, the use of monoclonal antibodies (mAbs) that block immune checkpoint molecules such as programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) has achieved some success, highlighting the potential of immunotherapy in the field of oncology. Furthermore, strategies that directly utilize immune cell effectors such as activated natural killer cells, chimeric antigen receptor (CAR) T cells, tumor-infiltrating lymphocytes, and tumor antigen-loaded dendritic cells have been used to enhance anti-tumor immunity. However, despite high expectations and sometimes promising results, clinical outcomes are often inconsistent, and tumors can find escape routes that can even metastasize over the long term.

[0006] From the above, it is clear that alternative immunotherapy approaches are needed to enhance host anti-tumor immunity and target tumor cells for destruction. [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors have found that a combination of inactive pathogenic bacteria can induce a specific anti-tumor immune response when in contact with antigens derived from tumor cells. This application demonstrates that this immune response effectively slows the growth rate of specific tumors from which the antigens originate. This application also demonstrates that the immunogenic compositions of the present invention, containing the bacterial population and antigens derived from tumor cells, can even achieve tumor disappearance and result in complete remission and memory responses to new tumor challenges / recurrences. [Means for solving the problem]

[0008] Therefore, a first aspect of the present invention is a) Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, Moraxella catarrhalis, Bordetella pertussis, Neisseria flava, Escherichia coli, and Proteus mirabilis. The present invention provides an immunogenic composition comprising (b) a bacterial population comprising at least three pathogenic bacterial species selected from the group consisting of (mirabilis), wherein the bacteria are inactivated bacteria, and (b) a tumor antigen determinant.

[0009] While we do not wish to be bound by theory, we hypothesize that tumor antigens act by directing a nonspecific immune response produced by a bacterial population. Presumably, activation of leukocytes mediating innate immunity, triggered by pattern recognition receptors located within bacterial cells, enhances the uptake of antigens from the tumor, transforming the initially occurring innate immune response into a specific adaptive immune response to that tumor. In this sense, we have also found that the immune response is enhanced when the bacteria in the population maintain their structural integrity, i.e., when the bacteria are whole bacteria. The immune response to tumors is also enhanced by a specific population of bacterial species contained in the immunogenic compositions of the present invention.

[0010] Furthermore, the inventors have also found that the immunogenic compositions of the present invention generate a memory immune response against tumors, effectively preventing recurrence and / or metastasis (see examples below).

[0011] A second aspect of the present invention provides a method for preparing an immunogenic composition, comprising contacting one or more tumor antigen determinants with a bacterial population comprising at least three pathogenic bacterial species selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, and Moraxella catarrhalis, wherein the bacteria are inactivated bacteria.

[0012] The immunogenic compositions of the present invention can be prepared in situ by contacting a bacterial population with antigenic determinants obtained from / derived from a tumor. In this case, it is advantageous that the bacterial population is readily available, for example, as a suspension or lyophilized preparation. Accordingly, a third aspect of the present invention provides a bacterial population comprising Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis, wherein the bacteria are inactivated bacteria and include the entire bacterial cell wall.

[0013] The immunogenic compositions of the present invention can also be provided as pharmaceutical compositions ready for administration to subjects requiring administration. Accordingly, a fourth aspect of the present invention refers to a pharmaceutical composition comprising a therapeutically effective amount of the immunogenic composition defined in the first aspect or a bacterial population according to the third aspect of the present invention, together with pharmaceutically acceptable excipients and / or carriers.

[0014] A fifth aspect of the present invention provides a kit of parts comprising (a) a bacterial population as defined in the third aspect, and optionally (b) instructions for its use in the preparation of an immunogenic composition of the first aspect.

[0015] A sixth aspect provides the use of a bacterial population according to a third aspect of the present invention or a kit of parts according to a fifth aspect of the present invention for preparing an immunogenic composition as defined in a first aspect of the present invention.

[0016] A seventh aspect provides a pharmaceutical composition for use as a pharmaceutical, comprising an immunogenic composition as defined in a first aspect of the present invention or said immunogenic composition as defined in a fourth aspect of the present invention. This can be rephrased as the use of a pharmaceutical composition comprising an immunogenic composition as defined in a first aspect of the present invention or said immunogenic composition as defined in a fourth aspect of the present invention for the preparation of a pharmaceutical. The present invention also discloses a method for preventing and / or treating a subject in need of prevention and / or treatment, comprising administering a pharmaceutical composition comprising an immunogenic composition as defined in a first aspect of the present invention or said immunogenic composition as defined in a fourth aspect of the present invention.

[0017] An eighth aspect provides a pharmaceutical composition comprising an immunogenic composition as defined in the first aspect of the present invention or as defined in the fourth aspect of the present invention, for use in a method for preventing and / or treating tumors. This can be rephrased as the use of a pharmaceutical composition comprising an immunogenic composition as defined in the first aspect of the present invention or as defined in the fourth aspect of the present invention, for preparing a pharmaceutical for preventing and / or treating tumors. The present invention also discloses a method for preventing and / or treating tumors, comprising administering a pharmaceutical composition comprising an immunogenic composition as defined in the first aspect of the present invention or as defined in the fourth aspect of the present invention to a subject in need of prevention and / or treatment.

[0018] In a ninth aspect, the present invention provides a pharmaceutical composition comprising an immunogenic composition as defined in a first aspect of the present invention or as defined in a fourth aspect of the present invention for use in combination therapy with surgery, radiation or an antitumor agent for the prevention and / or treatment of tumors. This aspect can also be formulated as a use of the pharmaceutical composition comprising an immunogenic composition as defined in a first aspect of the present invention or as defined in a fourth aspect of the present invention for the manufacture of a pharmaceutical for the prevention and / or treatment of tumors in combination therapy with surgery, radiation or an antitumor agent. This aspect can also be formulated as a method for the prevention and / or treatment of tumors, the method comprising administering the pharmaceutical composition comprising an immunogenic composition as defined in a first aspect of the present invention or as defined in a fourth aspect of the present invention in combination with surgery, radiation or an antitumor agent to a subject in need of prevention and / or treatment.

[0019] Furthermore, the inventors have surprisingly discovered that a bacterial community consisting of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis, in which the bacteria are inactivated, is effective in treating colon tumors. Accordingly, a tenth aspect of the present invention provides a bacterial community comprising Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis, for use in the prevention and / or treatment of tumors of the gastrointestinal tract, nervous system, hematopoietic system, reproductive system (e.g., cervix, ovaries, uterus, vagina and vulva), urinary tract, endocrine system, skin, heart, brain, eye, testes, muscle, bone or breast, wherein the bacteria are inactivated bacteria. This embodiment can also be formulated as the use of a bacterial community comprising Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis for the manufacture of pharmaceuticals for the prevention and / or treatment of tumors of the gastrointestinal tract, nervous system, hematopoietic system, reproductive system (e.g., cervix, ovaries, uterus, vagina and vulva), urinary tract, endocrine system, skin, heart, brain, muscle, eye, testes, bone or breast, wherein the bacteria are inactivated bacteria.This embodiment can also be formulated as a method for preventing and / or treating tumors of the gastrointestinal tract, nervous system, hematopoietic system, reproductive system (e.g., cervix, ovaries, uterus, vagina and vulva), urinary tract, endocrine system, skin, heart, brain, eye, testes, muscle, bone or breast, and the method comprises administering a bacterial community consisting of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis, wherein the bacteria are inactivated, to a subject in need of prevention and / or treatment.

[0020] An eleventh aspect of the present invention refers to a pharmaceutical composition comprising a bacterial population as defined in the third aspect or as defined in the fourth aspect for use in the prevention and / or treatment of a tumor, wherein the prevention and / or treatment comprises the simultaneous, sequential, or separate administration of the bacterial population and one or more tumor antigenic determinants within a therapeutic interval. This can be rephrased as the use of a pharmaceutical composition comprising a bacterial population as defined in the third aspect or as defined in the fourth aspect for preparing a pharmaceutical for the prevention and / or treatment of a tumor, wherein the pharmaceutical is administered simultaneously, sequentially, or separately within a therapeutic interval together with one or more tumor antigenic determinants. The present invention also discloses a method for the prevention and / or treatment of a tumor, comprising administering to a subject in need of prevention and / or treatment a therapeutically effective amount of a pharmaceutical composition comprising a bacterial population as defined in the third aspect or as defined in the fourth aspect, in combination with one or more tumor antigenic determinants, wherein the bacterial population and one or more tumor antigenic determinants are administered simultaneously, sequentially, or separately within a therapeutic interval. [Brief explanation of the drawing]

[0021] [Figure 1A]This figure shows the volume measurements of CT-26 tumors subcutaneously implanted in 12-week-old Balb / c mice. The four experimental groups tested (n=5 mice / group) are shown as follows: Group 1 - control, Group 2 - bacterial population, Group 3 - immunogenic composition, and Group 4 - immunogenic composition containing glucose. [Figure 1B] This figure shows the details of the antitumor activity observed in experimental groups 3 and 4 (n=5 mice / group). The X-axis represents the variable "time," indicating the number of days elapsed since the start of treatment. The Y-axis represents the variable "tumor size," indicating the volume (cm3) gained by the tumor since the start of treatment. Detailed description of the invention

[0022] All terms used herein are to be understood in the ordinary sense known in the art unless otherwise specified. Other, more specific definitions of any particular terms used herein are given below and are intended to apply uniformly throughout the specification and claims unless a more explicit definition is provided.

[0023] As used herein, “cancer” refers to any undesirable proliferation of cells that does not perform physiological function. Generally, cancer cells are cells that are freed from their normal cell division controls, i.e., their proliferation is not regulated by the normal biochemical and physical influences in the cellular environment. Thus, “cancer” is a general term for diseases characterized by abnormal, uncontrolled cell proliferation. In most cases, cancer cells proliferate to form benign or malignant cloned cells. The resulting mass or cell mass, or “tumor,” can generally invade and destroy surrounding normal tissue. “Malignant tumor” means any abnormal proliferation of any cell type or tissue that has harmful effects in an organism having abnormal proliferation. The terms “malignant” or “cancer” include cell proliferation that is technically benign but has the risk of becoming malignant. Tumor cells can spread from their original site to other parts of the body via the lymphatic system or bloodstream in a process known as “metastasis,” potentially causing “secondary tumors” that result in “metastatic cancer” disease.

[0024] An "immunogenic composition" is a composition that, when administered to a subject, directly or indirectly induces, or is capable of inducing, an immune response in the subject.

[0025] "Trained immunity" is a new concept that defines the ability of the innate immune system to generate immune memory and provide long-term protection against pathogens. It is a mechanism involving epigenetic changes in cells involved in the innate immune system, but it means the adaptation of innate immune processes in true innate immune memory (Saeed et al., Science, 2014). Trained immunity is thought to provide protection by (i) increasing the non-specific effector responses of innate immune cells (e.g., monocytes / macrophages) and (ii) enhancing the adaptive T cell responses to both specific antigens and unrelated (bystander) antigens by exploiting the activated state of dendritic cells.

[0026] In the context of the present invention, the composition of the present invention containing both pathogen-associated molecular patterns from selected bacterial species and antigenic determinants of specific tumors induces such a trained immune response so as to target specific types of tumors.

[0027] A "pathogen-associated molecular pattern" is a small molecular motif on the surface of a microorganism that is recognized by pattern recognition receptors in the host and thereby activates the innate immune response in the host.

[0028] In the meaning of the present invention, a "bacterium containing the whole bacterial cell wall" is understood as a bacterium that is "whole" in the sense of retaining the integrity of the cell wall containing the pathogen-associated molecular pattern. Throughout the present disclosure, the expressions "bacterium containing the whole bacterial cell wall" and "whole bacterium" are used interchangeably. In contrast, "lysed bacteria" are those that do not retain the integrity of their cell walls, i.e., broken bacteria.

[0029] An "antigen" is a molecule or molecular structure, usually found on the surface of a cell, that can specifically interact with antigen-recognizing molecules of the immune system, triggering an immune response. The term "antigen determinant," also called an "epitope," refers to the part of the antigen that is recognized by the antigen-recognizing molecule.

[0030] An "adjuvant" is a drug that acts nonspecifically to increase the immune response.

[0031] The term "vaccine" refers to an immunogenic composition, accompanied by appropriate excipients and / or carriers, that, when administered to a target, directly or indirectly induces, or can induce, a protective immune response against the pathogen for which the vaccine is designed. As used herein, the terms “subject” or “host” refer to a target individual requiring administration, to which the immunogenic composition of the present invention is administered, in particular humans, mammals, livestock, or any other animal species readily treated with the composition of the present invention. In most embodiments of the present invention, the subject is human.

[0032] As used herein, the term “therapeutic dose” refers to the amount of the immunogenic composition of the present invention that, when administered, is sufficient to prevent or, to some extent, the onset of one or more symptoms of the disease being treated. Of course, the specific dose of the drug administered in accordance with the present invention will be determined by the specific circumstances surrounding the case, including the amount of the drug administered, the route of administration, the specific condition being treated, and similar considerations.

[0033] The term "pharmaceutical composition" encompasses both compositions intended for humans and non-human animals (i.e., veterinary compositions).

[0034] The expression "pharmaceutically acceptable carrier or excipient" refers to a pharmaceutically acceptable material, composition, or medium. Each component must be pharmaceutically acceptable in the sense that it is compatible with the other components of the pharmaceutical composition. It must also be free from excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications, and suitable for use in contact with human and non-human animal tissues or organs in a reasonable benefit / risk ratio.

[0035] Where percentages are given in this disclosure, they generally refer to percentage weight per unit weight (%w / w) unless the composition is liquid, in which case the percentage refers to percentage weight per unit volume. %w / w or %w / v for a component refers to the amount of a single component relative to the total weight or total volume of the composition.

[0036] As described above, the inventors have found that the immunogenic compositions disclosed in the first aspect of the present invention are effective in the prevention and treatment of tumors. When administered to cancer patients, a suitable combination of inactive bacteria selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, and Moraxella catarrhalis is hypothesized to generate a relevant innate immune response that is favorably directed towards tumor cells in the presence of tumor antigen determinants. This trained immunity counteracts immunomodulatory mechanisms that allow tumor cells to evade the immune system so that they become targets for destruction.

[0037] Bacterial colony The inventors have found the optimal combination of pathogenic bacteria to be included in an immunogenic composition. The selection of bacterial species is crucial for adequately stimulating the immune system in the sense of the present invention. Pathogens are known to contain pathogen-associated molecular patterns on their surface that are recognized by the host's pattern recognition receptors. However, arriving at the optimal combination of signals is not an easy task.

[0038] In some embodiments of the first aspect of the present invention, the bacterial community comprises at least four or at least five bacterial species selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, and Moraxella catarrhalis. In other embodiments, the bacterial community includes at least three or at least four species selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis.

[0039] In one particular embodiment, the bacterial population includes all five species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis. In another particular embodiment, the bacterial population consists of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis.

[0040] The formulation of the bacterial population is also relevant. Although it fluctuates to some extent, the proportions of each species in the bacterial population can affect the immune response, and the inventors have found the best formulation to achieve the desired effect. In some embodiments of the first aspect of the present invention, the bacterial population has the following composition: 5-25% Staphylococcus aureus, 1-20% Streptococcus pyogenes, 15-70% Streptococcus pneumoniae, 1-30% Haemophilus influenzae, 1-20% Klebsiella pneumoniae, and 1-25% Moraxella catarrhalis, where the total percentage of species in the bacterial population is 100.

[0041] In some embodiments of the first aspect of the present invention, the bacterial population has the following composition: 5-25% Staphylococcus aureus, 1-20% Streptococcus pyogenes, 15-50% Streptococcus pneumoniae, 10-30% Haemophilus influenzae, 1-20% Klebsiella pneumoniae, and 5-25% Moraxella catarrhalis, where the total percentage of species in the bacterial population is 100.

[0042] In other embodiments, the bacterial population has the following composition: 10-20% Staphylococcus aureus, 5-15% Streptococcus pyogenes, 20-40% Streptococcus pneumoniae, 15-25% Haemophilus influenzae, 5-15% Klebsiella pneumoniae, and 10-20% Moraxella catarrhalis, where the total percentage of species in the bacterial population is 100. In certain embodiments, the bacterial community has the following composition: 15% Staphylococcus aureus, 10% Streptococcus pyogenes, 30% Streptococcus pneumoniae, 20% Haemophilus influenzae, 10% Klebsiella pneumoniae, and 15% Moraxella catarrhalis.

[0043] Furthermore, the inventors have found that the use of whole bacterial cells that maintain cellular structural integrity achieves better results in the context of the present invention than the use of lysed bacterial cells. Therefore, in certain embodiments, the inactivated bacteria include the entire bacterial cell wall. In other embodiments, the inactivated bacteria maintain the structural integrity of the bacterial cell wall. Methods for inactivating pathogenic bacteria while preserving the structural integrity of bacterial cells are known in the art. Non-limiting examples of these inactivation methods include phenolization, irradiation, and formalin and formaldehyde treatment (Fan et al., J Food Prot 2017; Levinson et al., JAMA, 1944; Hankaniemi et al., Antiviral Research 2019). In certain embodiments, bacterial cells present in the immunogenic composition of the present invention are inactivated by treatment with phenol. This can be achieved, for example, by treating bacteria with 0.5% (w / v) phenol at room temperature for 12 hours.

[0044] Some aspects of the present invention refer to the bacterial population itself, which is essential for preparing the immunogenic composition of the first aspect of the present invention, and is particularly designed for this purpose. In certain embodiments, the bacteria in the bacterial population are whole bacteria. In certain embodiments, the bacteria in the bacterial population are inactivated by phenolization. In certain embodiments, the bacteria in the combination maintain the structure and integrity of their cell walls.

[0045] Furthermore, in certain embodiments, the bacterial population includes whole inactive cells of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis. In another particular embodiment, the bacterial population consists of whole inactive cells of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis. In certain embodiments, the bacterial population has the following composition: 5-25% Staphylococcus aureus, 1-20% Streptococcus pyogenes, 15-50% Streptococcus pneumoniae, 10-30% Haemophilus influenzae, 1-20% Klebsiella pneumoniae, and 5-25% Moraxella catarrhalis, where the total percentage of species in the bacterial population is 100, and the bacterial cells are whole and inactive.In other specific embodiments, the bacterial population has the following composition: 10-20% Staphylococcus aureus, 5-15% Streptococcus pyogenes, 20-40% Streptococcus pneumoniae, 15-25% Haemophilus influenzae, 5-15% Klebsiella pneumoniae, and 10-20% Moraxella catarrhalis, where the total percentage of species in the bacterial population is 100, and the bacterial cells are whole and inactive. In a particular embodiment, the bacterial population has the following composition: 15% Staphylococcus aureus, 10% Streptococcus pyogenes, 30% Streptococcus pneumoniae, 20% Haemophilus influenzae, 10% Klebsiella pneumoniae, and 15% Moraxella catarrhalis, with the bacterial cells being whole and inactive.

[0046] The bacterial population can be provided, for example, as a suspension or a dehydrated preparation. Dehydration can be carried out by a freeze-drying process. In some embodiments, the bacterial population is a freeze-dried preparation. In other embodiments, the bacterial population is provided as a suspension.

[0047] Furthermore, additives or other additional compounds can be added to the bacterial population to protect the bacterial cells from damage. Therefore, the present invention also refers to bacterial compositions comprising the above-mentioned bacterial population. Suitable additives for bacterial compositions include preservatives, adjuvants, processing aids, cryoprotectants, and antioxidants. In this case as well, the bacterial composition can be provided as a dehydrated (e.g., lyophilized) composition or as a suspension. In one embodiment, the bacterial composition is lyophilized and contains a cryoprotectant such as glycerol, sucrose, or trehalose.

[0048] A bacterial population can be prepared by mixing the desired species in appropriate proportions. Bacterial species are publicly available. For example, bacterial species can be obtained from specialized companies or depositary institutions. The desired bacterial population can also be supplied (in the desired proportions) by specialized commercial suppliers. Companies such as Diater are suitable commercial suppliers. Other possible commercial suppliers of bacterial populations are Roxall, Inmunotek, and Allergy Therapeutics.

[0049] The present invention also provides a kit of parts comprising the above-described bacterial population or bacterial composition. In certain embodiments, the kit also includes instructions for its use in the preparation of the immunogenic composition of the first embodiment. All embodiments described above for the bacterial population or bacterial composition also apply to the kit of parts.

[0050] The present invention also provides the use of the above-mentioned bacterial populations or bacterial compositions or kits of parts for preparing immunogenic compositions as defined in a first aspect of the present invention.

[0051] Antigen determinants An immunogenic composition according to a first aspect of the present invention contains an antigenic determinant derived from a tumor. In most embodiments, the tumor is a malignant tumor. The antigenic determinant may be contained in a pure, isolated antigen or in tumor cells. In some embodiments, the antigenic determinant is in the form of a tumor cell. This has the advantage that the immunogenic composition can contain not just one, but several antigenic determinants characteristic of a particular type of tumor, and thus improve the potential for immune activation targeting that particular type of tumor.

[0052] Tumor cells can be provided by performing a tumor biopsy. Tumor cells may originate from a single subject (a single tumor) or from a pool of biopsy cells from tumors of the same type. Tumor cells can also be obtained from tumor cell lines.

[0053] In some embodiments, the immunogenicity determinant is in the form of whole tumor cells. This is understood as tumor cells that include the entire cell membrane, particularly cells that maintain the structural integrity of the cell membrane. This can have the advantage of enhanced interaction with antigen-presenting cells. In this case, the cells must be inactivated in a manner that does not destroy the structural integrity of the cells (see above). In other embodiments, the immunogenicity determinant is in the form of tumor cell lysates. This has the advantage of ensuring cessation of tumor cell proliferation and ease of use. Non-limiting methods for cell lysis include temperature treatment (i.e., freeze-thaw and / or heat treatment), high-pressure treatment, sonication, and mechanical treatment, all of which are known in the art. For example, tumor lysates can be obtained by subjecting tumor cells to a procedure consisting of three cycles of freezing (-80°C) and thawing (37°C). Other, less limiting methods are based on suspending cells in a hypotonic solution (osmotic dissolution) and / or using chemicals (e.g., antibiotics, chelating agents, detergents, and / or solvents) that can interact with specific components of the cell wall and allow internal components to leak out through the cell wall.

[0054] Antigenic determinants may also be in the form of pure isolated antigens or combinations of pure isolated antigens. This has the advantage of greater versatility and scalability. Relevant tumor-associated antigens are disclosed, for example, in Novellino et al., Cancer Immunol Immunother 2005. A trained immune response can be directed to several types of tumors in which one or more selected antigens are dominant. Immunogenic compositions may also contain cocktails having antigens selected from several tumors, thus enabling the treatment of different tumors with the same immunogenic composition. This requires that the dominant antigens be identified. For example, in one embodiment, the immunogenic composition may contain antigens selected from the group consisting of AFP, CEA, HER-2, p53, WT1, and combinations thereof.

[0055] Adjuvant The immunogenic compositions of the present invention may contain further compounds. In certain embodiments, the immunogenic compositions of the present invention may also contain compounds selected from monosaccharides, amino acids, fatty acids, substrates or metabolites of the Krebs cycle, β-glucans, ketone bodies, lactates, glyceraldehyde, glycerol, glutamates, and combinations thereof. As shown in the following examples, the inventors have found that the addition of the immunogenic compositions of the present invention enhances the immunogenic response, thereby improving the efficacy of the treatment (greater reduction in tumor size / termination of tumors and substantial elimination of metastases). These compounds are hypothesized to be nutrients that enable monocytes and macrophages to be activated and have a sufficient energy source to initiate the innate immune response induced by the immunogenic compositions. These compounds can be considered adjuvants.

[0056] Non-limiting examples of the above compounds include glucose, fructose, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucan, monounsaturated fatty acids and polyunsaturated fatty acids, and combinations thereof. In certain embodiments, the immunogenic composition of the present invention further comprises compounds selected from the group consisting of glucose, fructose, glutamine, arginine, malate, glutamate, and combinations thereof. In another particular embodiment, the immunogenic composition of the present invention further comprises glucose.

[0057] This application also discloses compounds selected from monosaccharides, amino acids, fatty acids, substrates or metabolites of the Krebs cycle, β-glucans, ketone bodies, lactates, glyceraldehyde, glycerol, glutamates, and combinations thereof as immunogenic adjuvants. The use of compounds selected from monosaccharides, amino acids, fatty acids, substrates or metabolites of the Krebs cycle, β-glucans, ketone bodies, lactates, glyceraldehyde, glycerol, glutamates, and combinations thereof in the preparation of vaccines or immunogenic compositions is also disclosed. The use of compounds selected from monosaccharides, amino acids, fatty acids, substrates or metabolites of the Krebs cycle, β-glucans, ketone bodies, lactates, glyceraldehyde, glycerol, glutamates, and combinations thereof for stimulating the activity of antigen-presenting cells is also disclosed. The use of compounds selected from monosaccharides, amino acids, fatty acids, substrates or metabolites of the Krebs cycle, β-glucans, ketone bodies, lactates, glyceraldehyde, glycerol, glutamates, and combinations thereof to enhance a trained immune response in a subject is also disclosed. In particular, glucose, fructose, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucans, monounsaturated fatty acids, polyunsaturated fatty acids, and combinations thereof, for example, compounds selected from glucose, fructose, glutamine, arginine, malate, glutamates, and combinations thereof. In particular, the compound is glucose.

[0058] The amount of the compound defined above, such as glucose, in the immunogenic composition or pharmaceutical composition of the present invention may be 0.5 to 10%. In some embodiments, the amount is 1 to 10%, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. In certain embodiments, the amount of the compound in the immunogenic composition or pharmaceutical composition of the present invention is 5%.

[0059] The immunogenic compositions of the present invention may also contain conventional adjuvants. Non-limiting adjuvants for use in the immunogenic compositions of the present invention include inorganic compounds, such as alum, aluminum hydroxide, aluminum phosphate and calcium hydroxide, mineral oils, such as paraffin oil, toxoids, squalene, detergents, such as Quil A, cytokines, such as IL-1, IL-2, IL-12, Freund's complete adjuvants, and Freund's incomplete adjuvants.

[0060] Preparation of immunogenic compositions As described above, the immunogenic compositions of the present invention can be prepared in situ by contacting a bacterial population with tumor-derived antigenic determinants. This may be, for example, an autotherapy case involving tumor cells or cell lysates obtained from a subject being treated with the immunogenic composition. In this case, it is advantageous that the bacterial population is readily available, for example, as a suspension or lyophilized preparation.

[0061] The antigenic determinants may be as disclosed above. For example, in some embodiments, a method for preparing an immunogenic composition provides tumor cells obtained from a patient's tumor, or from tumors of the same type from several patients (a pool of tumor cells), or from a tumor cell line, and intends to inactivate said tumor cells. Thus, in one embodiment, a method for preparing an immunogenic composition of the present invention comprises the following steps: (i) inactivating tumor cells, and (ii) contacting the inactivated tumor cells from step (i) with the bacterial population defined above. In some embodiments, the cells are inactivated by a method that maintains the structural integrity of the cell membrane, such as irradiation or phenolization. In some embodiments, a method for preparing an immunogenic composition intends to provide tumor cell lysates. Suitable methods for lysing cells are disclosed above. For example, tumor lysates can be obtained by subjecting a sample obtained from tumor tissue or a cell line to a procedure consisting of three cycles of freezing (-80°C) and thawing (37°C). In certain embodiments, a method for preparing an immunogenic composition includes adding additional compounds, such as glucose, fructose, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucan, monounsaturated fatty acids, polyunsaturated fatty acids, and combinations thereof, such as compounds selected from glucose, fructose, glutamine, arginine, malate, glutamate, and combinations thereof, particularly glucose.

[0062] In further embodiments, immunogenic compositions may be prepared by contacting pure isolated tumor antigens with a bacterial population defined above, and optionally adding glucose, fructose, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucan, monounsaturated fatty acids, polyunsaturated fatty acids, and combinations thereof, for example, compounds selected from glucose, fructose, glutamine, arginine, malate, glutamate, and combinations thereof, particularly glucose.

[0063] The contact process may be carried out at room temperature and kept on ice until use. Contact can be carried out by gentle mixing, for example, by pipetting. In one particular example, an immunogenic composition can be prepared by pipetting an antigenic determinant (e.g., a tumor lysate obtained by lysing about 0.5 × 10⁶ tumor cells) with about 50 μL of bacterial population (10⁸ / mL) at room temperature.

[0064] Pharmaceutical composition Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the immunogenic composition defined above, together with pharmaceutically acceptable excipients and / or carriers. The application also discloses a pharmaceutical composition comprising a therapeutically effective amount of the bacterial population defined above, together with pharmaceutically acceptable excipients and / or carriers. The pharmaceutical composition of the present invention can be considered a vaccine. Thus, this aspect can also be called a vaccine comprising a therapeutically effective amount of the immunogenic composition defined above, together with pharmaceutically acceptable excipients and / or carriers. A vaccine comprising a therapeutically effective amount of the bacterial population defined above, together with pharmaceutically acceptable excipients and / or carriers is also disclosed.

[0065] The relative amounts of bacterial populations, immunogenicity determinants, pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical composition of the present invention vary depending on the identity, size, and / or condition of the target being treated, and further depending on the route through which the composition is administered.

[0066] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as colorants, coatings, sweeteners, and flavorings may be present in the composition at the discretion of the prescriber.

[0067] The pharmaceutical composition containing the immunological composition of the present invention can be provided in any dosage form, for example, solid or liquid, and can be administered by any suitable route, for example, orally, parenterally, rectally, topically, intranasally, or sublingually, and for that purpose, it contains pharmaceutically acceptable excipients necessary for formulations in the desired dosage form, such as topical formulations (ointments, creams, lipogels, hydrogels, etc.), eye drops, aerosol sprays, injections, osmotic pumps, etc.

[0068] Examples of diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and combinations thereof.

[0069] Examples of granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0070] Exemplary binding excipients include, but are not limited to, starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic rubbers (e.g., acacia, sodium alginate, Chondrus crispus extract, panwar gum, ghatti gum, isapol husk mucus, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone, aluminum magnesium silicate (Veegum), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylate; waxes; water; alcohols; and combinations thereof.

[0071] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, ascorbyl oleate, butylhydroxyanisole, butylhydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate.

[0072] Examples of buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0073] In one embodiment, the pharmaceutical composition of the present invention is an parenteral composition. In another embodiment, the pharmaceutical composition of the present invention is an intradermal composition. In yet another embodiment, the pharmaceutical composition of the present invention is a transdermal composition. In yet another embodiment, the pharmaceutical composition of the present invention is an intravenous composition. In yet another embodiment, the pharmaceutical composition of the present invention is a sublingual composition. In yet another embodiment, the pharmaceutical composition of the present invention is a subcutaneous composition. In yet another embodiment, the pharmaceutical composition of the present invention is an oral composition. In yet another embodiment, the pharmaceutical composition of the present invention is a nasopharyngeal composition.

[0074] In one embodiment, the pharmaceutical composition comprises inactivated bacterial cells / dose 10e7-10e12. In another embodiment, the pharmaceutical composition comprises inactivated bacterial cells / dose 10e8-10e11, for example, 10e8, 10e9, or 10e10.

[0075] In some embodiments, the pharmaceutical composition of the present invention includes an adjuvant. In certain embodiments, the pharmaceutical composition of the present invention includes a compound selected from glucose, fructose, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucan, monounsaturated fatty acids, polyunsaturated fatty acids, and combinations thereof. In other specific embodiments, the pharmaceutical composition of the present invention includes a compound selected from the group consisting of glucose, fructose, glutamine, arginine, malate, glutamate, and combinations thereof. In certain embodiments, the pharmaceutical composition of the present invention includes glucose.

[0076] therapeutic use Eighteenth to eleventh aspects of the present invention relate to the therapeutic use of the compositions of the present invention in the prevention and / or treatment of tumors. In most embodiments, the tumors to be treated are malignant tumors. Therefore, this application essentially refers to the prevention and / or treatment of cancer.

[0077] As described above, it is hypothesized that the activation of leukocytes that mediate innate immunity, triggered by pattern recognition receptors located within bacterial cells, enhances the uptake of antigens from tumors and transforms the initial innate immune response into a tumor-specific adaptive immune response. Examples of leukocytes that mediate innate immunity include antigen-presenting cells such as monocytes, macrophages, and dendritic cells, as well as basophils, eosinophils, Langerhans cells, mast cells, neutrophils, NK cells, and NKT cells.

[0078] Also disclosed are immunogenic compositions as defined in a first aspect of the present invention, bacterial populations as defined in a third aspect of the present invention, or pharmaceutical compositions as defined in a fourth aspect of the present invention, for use in inducing a trained innate immune response against a tumor. This can be rephrased as immunogenic compositions as defined in a first aspect of the present invention, bacterial populations as defined in a third aspect of the present invention, or pharmaceutical compositions as defined in a fourth aspect of the present invention, for use in the preparation of pharmaceuticals for inducing a trained innate immune response against a tumor in a subject requiring induction. Also disclosed are methods for inducing a trained innate immune response against a tumor, comprising administering an immunogenic composition as defined in a first aspect of the present invention, a bacterial population as defined in a third aspect of the present invention, or a pharmaceutical composition as defined in a fourth aspect of the present invention to a subject requiring administration.

[0079] This application also discloses immunogenic compositions as defined in a first aspect of the present invention, bacterial populations as defined in a third aspect of the present invention, or pharmaceutical compositions as defined in a fourth aspect of the present invention, for use in increasing nonspecific effector responses of leukocytes mediating innate immunity. This can be rephrased as immunogenic compositions as defined in a first aspect of the present invention, bacterial populations as defined in a third aspect of the present invention, or pharmaceutical compositions as defined in a fourth aspect of the present invention, for use in the preparation of pharmaceuticals for increasing nonspecific effector responses of leukocytes mediating innate immunity. Also disclosed are methods for increasing nonspecific effector responses of leukocytes mediating innate immunity, comprising administering an immunogenic composition as defined in a first aspect of the present invention, a bacterial population as defined in a third aspect of the present invention, or a pharmaceutical composition as defined in a fourth aspect of the present invention to a subject requiring administration. In some embodiments, the leukocytes mediating innate immunity are selected from antigen-presenting cells such as monocytes, macrophages, and dendritic cells, basophils, eosinophils, Langerhans cells, mast cells, neutrophils, NK and NKT cells, particularly monocytes, macrophages, and dendritic cells.

[0080] This application also discloses a pharmaceutical composition comprising a bacterial population as defined in a third aspect or a bacterial population as defined in a fourth aspect for use in inducing a trained innate immune response against a tumor, wherein inducing a trained innate immune response involves simultaneous, sequential, or separate administration of the bacterial population and one or more tumor antigen determinants within a therapeutic interval. This can be rephrased as the use of a pharmaceutical composition comprising a bacterial population as defined in a third aspect or a bacterial population as defined in a fourth aspect for preparing a pharmaceutical for inducing a trained innate immune response against a tumor, wherein the pharmaceutical is administered simultaneously, sequentially, or separately within a therapeutic interval together with one or more tumor antigen determinants. The present invention also discloses a method for inducing a trained innate immune response against a tumor, the method comprising administering to a subject requiring induction a therapeutically effective amount of a pharmaceutical composition comprising a bacterial population as defined in a third aspect or a bacterial population as defined in a fourth aspect, in combination with one or more tumor antigen determinants, wherein the bacterial population and one or more tumor antigen determinants are administered simultaneously, sequentially, or separately within a therapeutic interval.

[0081] This application also discloses a pharmaceutical composition comprising a bacterial population as defined in a third embodiment or as defined in a fourth embodiment for use in increasing a nonspecific effector response of leukocytes mediating innate immunity, wherein increasing the nonspecific effector response includes simultaneous, sequential, or separate administration of the bacterial population and one or more tumor antigen determinants within a therapeutic interval. This can be rephrased as the use of a pharmaceutical composition comprising a bacterial population as defined in a third embodiment or as defined in a fourth embodiment for preparing a pharmaceutical for increasing a nonspecific effector response of leukocytes mediating innate immunity, wherein the pharmaceutical is administered simultaneously, sequentially, or separately within a therapeutic interval together with one or more tumor antigen determinants. The present invention also discloses a method for increasing a nonspecific effector response of leukocytes that mediates innate immunity, the method comprising administering to a subject requiring the increase a pharmaceutical composition comprising a therapeutically effective amount of a bacterial population defined in a third embodiment or a bacterial population defined in a fourth embodiment, in combination with one or more tumor antigen determinants, the bacterial population and one or more tumor antigen determinants being administered simultaneously, sequentially, or separately within a therapeutic interval.

[0082] The tumors that can be prevented or treated by the use of the immunogenic compositions of the present invention are not particularly limited, as long as tumor cells or tumor-derived antigens are available. Non-limited types of tumors that can be prevented or treated in the sense of the present invention are carcinomas, sarcomas, and hematological malignancies. Carcinomas are common cancers that are cancers of epithelial cells or cells covering the outer or inner surface of organs, glands, or other body structures (e.g., skin, uterus, lungs, breasts, prostate, stomach, intestines) and that tend to metastasize. Carcinomas may be adenocarcinomas, such as breast cancer, lung cancer, colon cancer, prostate cancer, or bladder cancer, and squamous cell carcinomas. Sarcomas originate from connective tissue or supporting tissue (e.g., bone, cartilage, tendons, ligaments, fat, and muscle). Sarcomas may also be osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesosarcoma or mesothelioma (membranous inner surface of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (adipose tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), or mesenchymal or mixed mesodermal tumors (mixed connective tissue type). Hematological malignancies originate from bone marrow and lymphoid tissue. Hematological malignancies include myeloma, which originates from plasma cells in the bone marrow; leukemia, which may be a “liquid tumor” and is a tumor of the bone marrow, and may be myeloid or granulocytic leukemia (myeloid and granulocytic white blood cells), lymphoid, lymphocytic or lymphoblastic leukemia (lymphocytic and lymphocytic blood cells), or polycythemia vera or erythremia (various blood cell products, but predominantly red blood cells); or solid tumors, which may arise in the glands or lymph nodes of the lymphatic system and may be Hodgkin lymphoma or non-Hodgkin lymphoma. Furthermore, there are mixed tumors such as adenosquamous carcinoma, mixed mesodermal tumor, carcinosarcoma or teratoma. All of these types of tumors, as well as other tumors that would be apparent to those skilled in the art, are intended in the sense of the present invention.

[0083] Tumors that can be treated with the immunological compositions of the present invention may also be named based on the organ from which they originate, i.e., the “primary site,” such as the tumor or cancer of the breast, brain, lungs, liver, skin, prostate, testes, bladder, colon and rectum, cervix, uterus, etc. This naming often persists even if the tumor has metastasized to another part of the body different from the primary site. In this sense, tumors intended in the eighth to tenth aspects of the present invention may be, but are not limited to, tumors of the gastrointestinal tract, respiratory system, nervous system, hematopoietic system, epithelium, reproductive system (e.g., cervix, ovaries, uterus, vagina and vulva, etc.), urinary tract, endocrine system, skin, heart, brain, eye, testes, muscle, bone, or breast. In particular embodiments, tumors intended in the eighth to tenth aspects of the present invention are tumors of the gastrointestinal tract, respiratory system, hematopoietic system, epithelium, reproductive system (e.g., cervix, ovaries, uterus, vagina and vulva, etc.), urinary tract, endocrine system, skin, heart, muscle, bone, or breast. In certain embodiments, the tumor is a tumor of the colon, lung, liver, or breast.

[0084] All embodiments of the immunogenic compositions and pharmaceutical compositions containing the same described above also apply to the therapeutic uses described in this section, including embodiments for all of their components. For example, in certain embodiments, the bacteria are the entirety of inactivated bacteria, and the combination includes or comprises Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis.

[0085] In some embodiments, prevention or treatment is self-administered. This means that the antigenic determinants contained in the immunogenic composition originate from the same subject being treated. In this case, the immunogenic composition, bacterial population, or pharmaceutical composition is intended for use in a method of preventing or treating a tumor, and the method for prevention and / or treatment comprises the following steps: (i) inactivating tumor cells obtained from the subject; (ii) contacting the inactivated tumor cells with a bacterial population to obtain an immunogenic composition; and (iii) administering the immunogenic composition to the same subject from which the tumor cells were obtained. It should be understood that steps (i) and (ii) from the method are in vitro.

[0086] In other embodiments, the treatment is allogeneic rather than autologous. This means that the antigenic determinant originates from one or more subjects that are not the subject being treated. In this case, the immunogenic composition as defined in the first aspect of the present invention, the bacterial population as defined in the third aspect of the present invention, or the pharmaceutical composition as defined in the fourth aspect of the present invention is for use in a method of preventing or treating a tumor, and the method for prevention and / or treatment comprises the following steps: (i) inactivating tumor cells of the same type of tumor obtained from the subject or group of subjects, (ii) contacting the inactivated tumor cells with a bacterial population to obtain an immunogenic composition, and (iii) administering the immunogenic composition to a patient suffering from the same type of tumor as in (i). It should be understood that steps (i) and (ii) from the method are in vitro.

[0087] In one embodiment, the subject from which tumor cells are obtained has an HLA that matches the recipient (allogeneic treatment). In another embodiment, the tumor cells are derived from one or more different subjects whose HLA does not necessarily match that of the recipient (heterogeneous treatment).

[0088] As described above, tumor cells can be extracted from one or more subjects by biopsy. If the tumor cells are not autologous, they can be obtained from a pool of biopsied tumor cells or from a tumor cell line. The tumor cells are then inactivated to obtain whole inactivated cells or cell lysates, which are then contacted with a bacterial population to obtain an immunogenic composition, which is then administered to the patient.

[0089] The route of administration may be parenteral. In one embodiment, the route of administration is intradermal. In another embodiment, the route of administration is subcutaneous. In another embodiment, the route of administration is transdermal. In another embodiment, the route of administration is intravenous. In another embodiment, the route of administration is sublingual. In another embodiment, the route of administration is oral.

[0090] As shown in the following examples, the immunogenic compositions of the present invention can prevent the growth of malignant tumors, reduce the size of malignant tumors, and even eliminate malignant tumors. Therefore, one embodiment of the 8th to 10th aspects of the present invention is for preventing the growth of malignant tumors. Another embodiment of the 8th to 10th aspects of the present invention is for preventing cancer. Another embodiment of the 8th to 10th aspects of the present invention is for treating malignant tumors. Another embodiment of the 8th to 10th aspects of the present invention is for treating cancer.

[0091] Importantly, the following examples also demonstrate that the immunogenic composition effectively inhibits metastasis. Therefore, another embodiment of the 8th to 10th aspects of the present invention is for preventing the growth of secondary tumors. Another embodiment of the 8th to 10th aspects of the present invention is for preventing metastasis. Another embodiment of the 8th to 10th aspects of the present invention is for treating secondary tumors. Another embodiment of the 8th to 10th aspects of the present invention is for preventing metastatic cancer.

[0092] When used in combination therapy, the combination may be in combination with an antitumor agent, radiotherapy, or surgery. More specifically, the immunogenic composition or bacterial population of the present invention is administered separately in any order within an effective therapeutic interval, together with an antitumor agent, radiotherapy, or surgery. In certain embodiments, the combination therapy includes surgery. In other specific embodiments, the combination therapy includes radiation. In certain embodiments, the combination therapy includes an antitumor agent. The antitumor agent may be an antagonist that improves specificity, such as alkylating agents, folic acid antagonists, purines, and pyrimidine analogs, antibiotics and other natural products including anthracyclines and vinca alkaloids, and cytotoxic agents such as antibodies. The antitumor agent may be a hormonal agent or a signaling inhibitor. In certain embodiments, the combination therapy includes chemotherapy. The combination therapy may also include different immunotherapies. For example, in certain embodiments, the combination may be in combination with a monoclonal antibody that blocks immune checkpoint molecules, activated natural killer cells, (CAR) T cells, tumor-infiltrating lymphocytes, or tumor antigen-loaded dendritic cells.

[0093] For completeness, the present invention is also described in the following numbered embodiments.

[0094] 1.a) A bacterial community comprising at least three pathogenic bacterial species selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus viridans, Moraxella catarrhalis, Bordetella pertussis, Neisseria flava, Escherichia coli, and Proteus mirabilis, wherein the bacteria are inactivated bacteria, and (b) An immunogenic composition comprising one or more tumor antigen determinants.

[0095] 2. The immunogenic composition according to Embodiment 1, wherein the bacterial population comprises at least four of the bacterial species.

[0096] 3. The immunogenic composition according to Embodiment 2, wherein the bacterial population comprises at least five of the bacterial species.

[0097] 4. An immunogenic composition according to any one of Embodiments 1 to 3, wherein the bacterial community comprises Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis.

[0098] 5. The immunogenic composition according to claim embodiment 4, wherein the bacterial community consists of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis.

[0099] 6. A bacterial community 5-25% Staphylococcus aureus, 1-20% of pyogenic streptococci (Streptococcus pyogenes), 15-70% of cases are caused by Streptococcus pneumoniae. 1-30% of Haemophilus influenzae, 1-20% of Klebsiella pneumoniae, and Contains 1-25% Moraxella catarrhalis. The immunogenic composition according to Embodiment 5, wherein the total percentage of species in the bacterial population is 100.

[0100] 7. A bacterial community 5-25% Staphylococcus aureus, 1-20% of pyogenic streptococci (Streptococcus pyogenes), 15-50% are Streptococcus pneumoniae. 10-30% of Haemophilus influenzae, 1-20% of Klebsiella pneumoniae, and It contains 5-25% Moraxella catarrhalis. The immunogenic composition according to Embodiment 6, wherein the total percentage of species in the bacterial population is 100.

[0101] 8. A bacterial community 10-20% Staphylococcus aureus, 5-15% of pyogenic streptococci (Streptococcus pyogenes), 20-40% are Streptococcus pneumoniae. 15-25% of Haemophilus influenzae, 5-15% of Klebsiella pneumoniae, and It contains 10-20% Moraxella catarrhalis. The immunogenic composition according to Embodiment 7, wherein the total percentage of species in the bacterial population is 100.

[0102] 9. A bacterial community 15% Staphylococcus aureus, 10% of pyogenic streptococci (Streptococcus pyogenes), 30% of Streptococcus pneumoniae 20% of Haemophilus influenzae, 10% of Klebsiella pneumoniae, and The immunogenic composition according to Embodiment 8, comprising 15% Moraxella catarrhalis.

[0103] 10. An immunogenic composition according to any one of Embodiments 1 to 9, wherein the inactivated bacteria include the entire bacterial cell wall.

[0104] 11. An immunogenic composition according to any one of Embodiments 1 to 10, wherein inactivated bacteria maintain the structural integrity of the bacterial cells.

[0105] 12. The immunogenic composition according to any one of Embodiments 1 to 9, wherein the inactivated bacteria are lysing bacteria.

[0106] 13. An immunogenic composition according to any one of Embodiments 1 to 12, wherein the antigenic determinant is in the form of an inactivated tumor cell.

[0107] 14. The immunogenic composition according to Embodiment 13, wherein the inactivated tumor cells include the entire cell membrane.

[0108] 15. The immunogenic composition according to Embodiment 14, wherein inactivated tumor cells maintain the structural integrity of the cell membrane.

[0109] 16. The immunogenic composition according to Embodiment 13, wherein the inactivated tumor cells are lysed cells.

[0110] 17. An immunogenic composition according to any one of Embodiments 1 to 12, wherein the antigenic determinant is in the form of an isolated antigen.

[0111] 18. The immunogenic composition according to Embodiment 17, wherein the isolated antigen is selected from the group consisting of AFP, CEA, HER-2, p53, WT1, and combinations thereof.

[0112] 19. An immunogenic composition according to any one of Embodiments 1 to 18, wherein the antigenic determinant is derived from a malignant tumor.

[0113] 20. An immunogenic composition according to any one of Embodiments 1 to 19, comprising two or more antigenic determinants, wherein two or more antigenic determinants originate from the same tumor.

[0114] 21. An immunogenic composition according to any one of Embodiments 1 to 19, comprising two or more antigenic determinants, wherein two or more antigenic determinants are derived from different tumors.

[0115] 22. The immunogenic composition according to any one of Embodiments 1 to 21, further comprising compounds selected from monosaccharides, amino acids, fatty acids, substrates or metabolites of the Krebs cycle, β-glucans, ketone bodies, lactates, glyceraldehyde, glycerol, glutamates, and combinations thereof.

[0116] 23. The immunogenic composition according to Embodiment 22, wherein the additional compound is selected from the group consisting of glucose, fructose, lactate, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucan, monounsaturated fatty acids and polyunsaturated fatty acids, and combinations thereof.

[0117] 24. The immunogenic composition according to Embodiment 23, wherein the additional compound is selected from the group consisting of glucose, fructose, glutamine, arginine, malate, glutamate, and combinations thereof.

[0118] 25. The immunogenic composition according to Embodiment 24, wherein the additional compound is glucose.

[0119] 26. The immunogenic composition according to any one of Embodiments 22 to 25, wherein the amount of the additional compound is 0.5 to 10%.

[0120] 27. The immunogenic composition according to Embodiment 26, wherein the amount of the additional compound is 2-8%.

[0121] 28. The immunogenic composition according to Embodiment 27, wherein the amount of the additional compound is 5%.

[0122] 29. A bacterial community comprising Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis, wherein the bacteria are inactivated.

[0123] 30. The bacterial community according to Embodiment 29, comprising Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis.

[0124] 31. A bacterial community 5-25% Staphylococcus aureus, 1-20% of pyogenic streptococci (Streptococcus pyogenes), 15-70% of cases are caused by Streptococcus pneumoniae. 1-30% of Haemophilus influenzae, 1-20% of Klebsiella pneumoniae, and Contains 1-25% Moraxella catarrhalis. The bacterial population according to Embodiment 30, wherein the sum of the percentages of species in the bacterial population is 100.

[0125] 32. A bacterial community 5-25% Staphylococcus aureus, 1-20% of pyogenic streptococci (Streptococcus pyogenes), 15-50% are Streptococcus pneumoniae. 10-30% of Haemophilus influenzae, 1-20% of Klebsiella pneumoniae, and It contains 5-25% Moraxella catarrhalis. The bacterial population according to Embodiment 31, wherein the sum of the percentages of species in the bacterial population is 100.

[0126] 33. A bacterial community 10-20% Staphylococcus aureus, 5-15% of pyogenic streptococci (Streptococcus pyogenes), 20-40% are Streptococcus pneumoniae. 15-25% of Haemophilus influenzae, 5-15% of Klebsiella pneumoniae, and It contains 10-20% Moraxella catarrhalis. The bacterial population according to Embodiment 32, wherein the sum of the percentages of species in the bacterial population is 100.

[0127] 34. A bacterial community 15% Staphylococcus aureus, 10% of pyogenic streptococci (Streptococcus pyogenes), 30% of Streptococcus pneumoniae 20% of Haemophilus influenzae, 10% of Klebsiella pneumoniae, and A bacterial population according to Embodiment 33, comprising 15% Moraxella catarrhalis.

[0128] 35. The bacterial population according to any one of embodiments 29 to 34, wherein the inactivated bacteria include the entire bacterial cell wall.

[0129] 36. The bacterial population according to any one of embodiments 29 to 35, wherein the inactivated bacteria maintain the structural integrity of the bacterial cells.

[0130] 37. A method for preparing an immunogenic composition, comprising contacting one or more tumor antigen determinants with a bacterial population defined in any one of embodiments 29 to 36.

[0131] 38. The method involves the following steps: (i) Inactivating tumor cells, and (ii) A method for preparing the immunogenic composition according to Embodiment 37, comprising contacting the inactivated tumor cells of step (i) with the bacterial population defined above.

[0132] 39. A method for preparing the immunogenic composition according to Embodiment 38, wherein tumor cells are inactivated by a method that maintains the structural integrity of the cell membrane.

[0133] 40. A method for preparing the immunogenic composition according to Embodiment 39, wherein tumor cells are inactivated by a method selected from the group consisting of phenolization, irradiation, and treatment with formalin or formaldehyde.

[0134] 41. A method for preparing the immunogenic composition according to Embodiment 40, wherein tumor cells are inactivated by phenolization.

[0135] 42. A method for preparing an immunogenic composition according to any one of embodiments 38 to 41, wherein the tumor cells are derived from tumors of the same type.

[0136] 43. A method for preparing the immunogenic composition according to Embodiment 42, wherein tumor cells are obtained from the target or group of target cells.

[0137] 44. A method for preparing the immunogenic composition according to Embodiment 43, wherein tumor cells are obtained from the same subject.

[0138] 45. A method for preparing the immunogenic composition according to Embodiment 42, wherein the tumor cells are derived from a tumor cell line.

[0139] 46. ​​A method for preparing the immunogenic composition according to Embodiment 38, wherein tumor cells are inactivated by lysis.

[0140] 47. A method for preparing the immunogenic composition according to Embodiment 37, wherein the tumor antigen determinant is in the form of an isolated antigen.

[0141] 48. A method for preparing the immunogenic composition according to Embodiment 47, wherein the isolated antigen is selected from the group consisting of AFP, CEA, HER-2, p53, WT1, and combinations thereof.

[0142] 49. A method for preparing an immunogenic composition according to any one of embodiments 37 to 48, wherein the tumor is a malignant tumor.

[0143] 50. A method for preparing an immunogenic composition according to any one of Embodiments 37 to 49, comprising a further step (iii) of adding a compound defined in any one of Embodiments 22 to 25.

[0144] 51. A method for preparing the immunogenic composition according to Embodiment 50, wherein an additional compound is added in an amount of 0.5 to 10% of the total immunogenic composition.

[0145] 52. A method for preparing the immunogenic composition according to Embodiment 51, wherein an additional compound is added in an amount of 2-8%, for example, 5%, of the total immunogenic composition.

[0146] 53. A pharmaceutical composition comprising, together with pharmaceutically acceptable excipients and / or carriers, an immunogenic composition as defined in any one of Embodiments 1 to 28 or a bacterial population as defined in any one of Embodiments 29 to 36 in a therapeutically effective amount.

[0147] The pharmaceutical composition according to Embodiment 53, comprising 54.10e7~10e12 inactivated bacterial cells / dosage.

[0148] The pharmaceutical composition according to Embodiment 54, comprising inactivated bacterial cells / doses 55.10e9~10e11.

[0149] 56. The pharmaceutical composition according to any one of Embodiments 53 to 55, wherein the pharmaceutical composition is an intradermal, transdermal, subcutaneous, sublingual, intravenous, nasopharyngeal, or oral composition.

[0150] 57. The pharmaceutical composition according to Embodiment 56, wherein the pharmaceutical composition is an intradermal composition.

[0151] 58. The pharmaceutical composition according to Embodiment 56, wherein the pharmaceutical composition is a sublingual composition.

[0152] 59. A pharmaceutical composition according to any one of embodiments 53 to 58, further comprising an adjuvant.

[0153] 60. An immunogenic composition as defined in any one of Embodiments 1 to 28, or a bacterial population as defined in any one of Embodiments 29 to 36, or a pharmaceutical composition as defined in any one of Embodiments 53 to 59, for use as a pharmaceutical.

[0154] 61. An immunogenic composition as defined in any one of Embodiments 1 to 28, a bacterial population as defined in any one of Embodiments 29 to 36, or a pharmaceutical composition as defined in any one of Embodiments 53 to 59, for use in inducing a trained innate immune response against a tumor.

[0155] 62. An immunogenic composition as defined in any one of Embodiments 1 to 28, a bacterial population as defined in any one of Embodiments 29 to 36, or a pharmaceutical composition as defined in any one of Embodiments 53 to 59, for use in increasing the nonspecific effector response of leukocytes that mediates innate immunity.

[0156] 63. An immunogenic composition, bacterial population, or pharmaceutical composition for use according to Embodiment 62, wherein the antigen-presenting cells are selected from the group consisting of monocytes, macrophages, dendritic cells, and combinations thereof.

[0157] 64. An immunogenic composition as defined in any one of Embodiments 1 to 28, or a bacterial population as defined in any one of Embodiments 29 to 36, or a pharmaceutical composition as defined in any one of Embodiments 53 to 59, for use in a method for the prevention and / or treatment of tumors.

[0158] 65. An immunogenic composition, bacterial population, or pharmaceutical composition for use according to any one of embodiments 61 to 64, wherein the tumor is a malignant tumor.

[0159] 66. An immunogenic composition, bacterial population, or pharmaceutical composition for use according to any one of embodiments 61 to 65, wherein the tumor is a secondary tumor.

[0160] 67. An immunogenic composition, bacterial community, or pharmaceutical composition for use according to any one of Embodiments 61 to 66, wherein the tumor originates from the respiratory system, gastrointestinal tract, nervous system, hematopoietic system, reproductive system (e.g., cervix, ovaries, uterus, vagina, and vulva), urinary tract, endocrine system, skin, heart, brain, eye, testis, muscle, bone, or breast, and in particular the tumor originates from the respiratory system, gastrointestinal tract, hematopoietic system, reproductive system, urinary tract, endocrine system, skin, heart, muscle, bone, or breast.

[0161] 68. An immunogenic composition, bacterial population, or pharmaceutical composition for use according to Embodiment 67, wherein the tumor is a mammary tumor.

[0162] 69. An immunogenic composition, bacterial community, or pharmaceutical composition for use according to Embodiment 67, wherein the tumor originates from the gastrointestinal tract.

[0163] 70. An immunogenic composition, bacterial population, or pharmaceutical composition for use according to Embodiment 67, wherein the tumor originates from the colon.

[0164] 71. An immunogenic composition, bacterial population, or pharmaceutical composition for use according to any one of embodiments 60 to 70, wherein the inactivated bacterial cells include the entire bacterial cell wall.

[0165] 72. An immunogenic composition, bacterial community, or pharmaceutical composition for use according to Embodiment 71, wherein the bacterial community comprises Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis.

[0166] 73. An immunogenic composition, bacterial community, or pharmaceutical composition for use according to Embodiment 72, wherein the bacterial community comprises Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis.

[0167] 74. A bacterial community 5-25% Staphylococcus aureus, 1-20% of pyogenic streptococci (Streptococcus pyogenes), 15-50% are Streptococcus pneumoniae. 10-30% of Haemophilus influenzae, 1-20% of Klebsiella pneumoniae, and It contains 5-25% Moraxella catarrhalis. Herein, the total percentage of species in the bacterial population is 100, the immunogenic composition, bacterial population, or pharmaceutical composition for use according to Embodiment 73.

[0168] 75. A bacterial community 10-20% Staphylococcus aureus, 5-15% of pyogenic streptococci (Streptococcus pyogenes), 20-40% are Streptococcus pneumoniae. 15-25% of Haemophilus influenzae, 5-15% of Klebsiella pneumoniae, and It contains 10-20% Moraxella catarrhalis. Herein, the total percentage of species in the bacterial population is 100, the immunogenic composition, bacterial population, or pharmaceutical composition for use according to Embodiment 74.

[0169] 76. A bacterial community 15% Staphylococcus aureus, 10% of pyogenic streptococci (Streptococcus pyogenes), 30% of Streptococcus pneumoniae 20% of Haemophilus influenzae, 10% of Klebsiella pneumoniae, and An immunogenic composition, bacterial population, or pharmaceutical composition for use according to Embodiment 75, comprising 15% Moraxella catarrhalis.

[0170] 77. Methods for prevention and / or treatment involve the following steps: (i) Inactivating tumor cells obtained from the subject, (ii) To obtain an immunogenic composition by contacting inactive tumor cells with a bacterial population, (iii) An immunogenic composition, bacterial population, or pharmaceutical composition for use according to any one of Embodiments 61 to 76, comprising administering the immunogenic composition to the same subject from which tumor cells were obtained.

[0171] 78 The immunogenic composition, bacterial population or pharmaceutical composition for use according to Embodiment 77, wherein the method for prevention and / or treatment comprises a further step of adding a compound defined in any one of Embodiments 22 to 25 to the immunogenic composition.

[0172] 79. A kit of parts for preparing an immunogenic composition as defined in any one of Embodiments 1 to 28, wherein the kit comprises (a) a bacterial population as defined in any one of Embodiments 29 to 36.

[0173] 80.(b) The kit of parts according to Embodiment 79, further comprising one or more immunogenicity determinants.

[0174] 81.(c) A kit of parts according to any one of embodiments 79 to 80, further comprising a compound as defined in any one of embodiments 22 to 25.

[0175] Throughout this description and in the claims, the word “including” and its variations thereof are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “including” encompasses the case of “consisting of.” Additional objects, advantages, and features of the present invention may become apparent to those skilled in the art by examining the description or can be learned through the practice of the invention. The following examples and drawings are provided for illustrative purposes only and are not intended to limit the invention. Furthermore, the invention covers all possible combinations of the specific preferred embodiments described herein. [Examples]

[0176] Example 1: Combining a bacterial population containing the entire bacterial cell wall with tumor lysates induces a specific antitumor immune response.

[0177] This study aimed to evaluate the ability of specific combinations of inactivated pathogenic bacteria ("bacterial populations") primed with tumor lysates to induce an innate immune response to solid tumors.

[0178] The bacterial population used in this example had the following composition: 15% Staphylococcus aureus, 10% Streptococcus pyogenes, 30% Streptococcus pneumoniae, 20% Haemophilus influenzae, 10% Klebsiella pneumoniae, and 15% Moraxella catarrhalis. The combination was provided by Laboratorios Diater SA. The bacterial population was inactivated by phenolization.

[0179] CT26.CL25 cells were obtained from ATCC® CRL-2639®. These are mouse-derived cells (Mus musculus, Balb / c strain) with fibroblast morphology that proliferate in an adherent state. These cells are derived from Balb / c mouse-induced colon cancer by their ability to transform with the T antigen (SV40 virus). When injected into Balb / c mice, CT-26 cells can induce solid tumors with similar characteristics to the tumors from which they originate. For this reason, CT-26 cells are commonly used as a model to test immunotherapy protocols and studies of host immune response development.

[0180] I obtained glucose from Sigma-Aldrich (G8270).

[0181] CT26.CL25 cell lysates were prepared by subjecting a pellet of 0.5 × 10 e6 CT26.CL25 cells (ATCC® CRL-2639®) to three freezing (-80°C) and thawing (37°C) cycles.

[0182] An immunogenic composition was prepared by pipetting a lysate derived from 0.5 × 10 e6 CT26.CL25 cells with 50 μL (10 e8 / mL) of phenol-inactivated bacterial population at room temperature, and the mixture was stored on ice until use.

[0183] CT-26 cells were subcutaneously inoculated into 12-week-old male Balb / c mice (0.5 × 10 e6 / mouse). After 16 days, the injected cells developed subcutaneous tumors measuring 5 × 5 mm. At this point, the mice were divided into the following four experimental groups. Group 1 - Control: Administration of 50 μL of physiological saline to the opposite side of the tumor. Group 2. Bacterial population: Administration of 50 μL (10 e8 / mL) of bacterial population to the opposite side of the tumor. Group 3. Immunogenic composition: Administration of 50 μL of bacterial population (10 e8 / mL) combined with tumor lysate (obtained from 0.5 × 10 e6 CT-26 cells) to the opposite side of the tumor. Group 4. Immunogenic composition containing glucose: Administration of 50 μL of a bacterial population (10 e8 / mL) combined with tumor lysate (obtained from 0.5 × 10 e6 CT-26 cells) + 5% glucose to the opposite side of the tumor.

[0184] Next, as shown in Figure 1A, the size of subcutaneous tumors induced in mice belonging to different experimental groups was measured weekly.

[0185] The results demonstrated that administration of a bacterial population opposite the tumor (Group 2) failed to induce an immune response against the tumor induced by subcutaneous administration of CT-26 cells, as the growth rate was similar to that of the control group (Group 1, administered with saline). Mice administered with the bacterial population in combination with CT-26 cell oncolysis (Group 3) were able to prevent the growth of the embedded tumor (see details in Figure 1A), and two of the five mice in this group showed complete remission. Administration of the bacterial population in combination with CT-26 cell oncolysis supplemented with 5% glucose (Group 4) was found to be even more effective, as it resulted in smaller tumor sizes than Group 3, and three of the five mice in this group also showed complete remission (see details in Figure 1A).

[0186] On the other hand, to evaluate the development of a specific antitumor response capable of inducing an immunological memory response, experimental groups 3 and 4 were reinjected with the CT-26 tumor cell line (0.5 × 10 e6 cells per mouse) into the opposite side of the tumor injection site. The development of de novo tumors (secondary tumors) was not observed in either of the mice in these two experimental groups, indicating that these mice had developed a specific memory response to the tumor and were therefore protected from further challenges from this type of tumor.

[0187] Example 2: Combining a bacterial population containing the entire bacterial cell wall with tumor lysates prevents tumor development.

[0188] Furthermore, the inventors tested the ability of a combination of CT-26 cell tumor lysate and the phenol-inactivated bacterial population defined in Example 1 to exert a prophylactic immune response (vaccination) against this tumor. For this purpose, male balb / c mice were divided into the following experimental groups. Group 1. Bacterial population: Subcutaneous administration of a bacterial population of 50 μL (10 e8 cfu / mL). Group 2 - Tumor Lysate Control: Subcutaneous administration of tumor lysates obtained from 0.5 × 10 e6 CT-26 cells. Group 3. Immunogenic tumor vaccination: Subcutaneous administration of 50 μL of bacterial population (10 e8 cfu / mL) combined with tumor lysate (obtained from 0.5 × 10 e6 CT-26 cells).

[0189] Two weeks after administration of a bacterial population (group 1), tumor lysate (group 2), or immunogenic composition (group 3), all experimental groups were subcutaneously inoculated with 0.5 × 10 e6 CT-26 cells to induce tumor formation. The results showed that two weeks after inoculation with tumor cells, mice in experimental groups 1 and 2 developed subcutaneous tumors larger than 1 cm in diameter, while group 3 did not show tumor formation. These results suggest that the combination of tumor lysate and inactivated bacteria can protect the subsequent development of tumors generated by the cells used to obtain the tumor lysate, suggesting a tumor vaccination effect and thus enhancing the memory immune response already demonstrated in the initial experiments conducted.

[0190] Example 3: Combining a bacterial population containing the entire bacterial cell wall with tumor lysates induces a specific antitumor immune response in dogs.

[0191] In a third series of experiments, the inventors tested the efficacy of a combination of tumor lysate and a phenol-inactivated bacterial population as defined in Example 1 for the treatment of mammary tumors in dogs. For this purpose, independent biopsies (approximately 2.5 cm in diameter) were obtained from surgically excised mammary cancers in two different dogs. To produce tumor lysate, the biopsies were subjected to three freeze-thaw cycles (-80°C and 37°C). After the final freeze-thaw cycle, 1.5 mL of tumor lysate was obtained, which was frozen in three independent aliquots, each containing 0.5 mL of tumor lysate.

[0192] Three doses of the immunogenic composition were administered to recipient animals by subcutaneous injection with a two-week interval between doses. Each dose was prepared before administration by thawing a 500 μl aliquot and combining it with 500 μL (10 e8 cfu / mL) of phenol-inactivated bacterial population. Each animal was administered the immunogenic composition prepared using tumor cells biopsied from its own mammary cancer (self-treatment). No adverse reactions were observed during subcutaneous injection of the tumor lysate and inactivated bacteria combination, or in the days following each administration. After six months, neither animal developed mammary cancer.

[0193] In summary, the results obtained are as follows: 1. Bacterial populations, including the entire bacterial cell wall, were able to generate a specific anti-tumor immune response insofar as they were combined with tumor-derived antigens (see results in groups 3 and 4, Figure 1). Tumor antigens derived from tumor lysates act by directing the nonspecific immune response generated by the bacterial population. 2. Administration of bacterial populations alone does not induce an antitumor response (see Results, Group 2, Figure 1). 3. The addition of glucose (5%) to immunological compositions containing bacterial populations and tumor lysates enhances the specific antitumor response produced by the bacterial population + tumor lysates (see Results, Group 4, Figure 1). It is hypothesized that the addition of glucose in the environment in which the antitumor immune response is initiated aids its development thanks to the energy contribution to the cells involved in this process (primarily antigen-presenting cells, which are highly metabolically active and require a significant energy contribution during the phagocytic process and antigen processing for presentation to specific cells of the immune system). 4. Combinations of bacterial vaccines and tumor lysates (in the presence or absence of glucose) can induce a memory response against tumors (see results in groups 3 and 4, Figure 2). 5. The combination of bacterial populations and tumor lysates induces a prophylactic immune response against tumors. Therefore, this combination is effective as a tumor vaccine. 6. The combination of bacterial populations and tumor lysates elicits a specific antitumor immune response in dogs.

Claims

1. (a) (i) a group including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis, and (ii) a group including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Moraxella catarrhalis. a bacterial population selected from the group consisting of S. pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis; (b) one or more tumor antigenic determinants; and Including, An immunogenic composition, wherein the bacterium is an inactivated whole bacterium that maintains the integrity of its cell wall.

2. The bacterial population is 5-25% Staphylococcus aureus, 1-20% Streptococcus pyogenes; 15-70% Streptococcus pneumoniae; 1-30% of Haemophilus influenzae; 1-20% Klebsiella pneumoniae, and Contains 1-25% Moraxella catarrhalis, 2. The immunogenic composition of claim 1, wherein the sum of the percentages for the species in the bacterial population is 100.

3. The immunogenic composition according to any one of claims 1 to 2, wherein the antigenic determinant is in the form of an inactive tumor cell.

4. An immunogenic composition described in any one of claims 1 to 3, wherein the antigenic determinant is in the form of a tumor cell lysate.

5. 5. The immunogenic composition according to any one of claims 1 to 4, wherein the composition further comprises a compound selected from the group consisting of glucose, fructose, lactate, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucan, monounsaturated and polyunsaturated fatty acids, and combinations thereof, in particular wherein the composition further comprises glucose.

6. 10. A pharmaceutical composition comprising a therapeutically effective amount of an immunogenic composition as defined in any one of claims 1 to 5 together with a pharma- ceutically acceptable excipient and / or carrier.

7. An immunogenic composition as defined in any one of claims 1 to 5 or a pharmaceutical composition as defined in claim 6 for use in the prevention and / or treatment of tumors.

8. 1. A bacterial population for use in the prevention and / or treatment of tumors comprising Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis, the bacterium is an inactivated whole bacterium that maintains cell wall integrity; A bacterial population, wherein said prevention and / or treatment comprises simultaneous, sequential or separate administration within a treatment interval of said bacterial population and one or more tumor antigenic determinants.

9. The bacterial population for use according to claim 8, wherein the tumor antigenic determinant is in the form of inactive tumor cells or tumor cell lysates.

10. 10. The bacterial population for use according to any one of claims 8 to 9, wherein said prevention and / or treatment further comprises the simultaneous, sequential or separate administration within a treatment interval of a compound selected from the group consisting of glucose, fructose, lactate, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucan, monounsaturated and polyunsaturated fatty acids, and combinations thereof.

11. The bacterial population for use according to claim 10, wherein the prevention and / or treatment further comprises simultaneous, sequential or separate administration of glucose within a treatment interval.

12. A bacterial population for use according to any one of claims 8 to 11, wherein said administration is simultaneous.

13. A bacterial population comprising Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis for use in preventing and / or treating a tumor in a subject, comprising: the bacterium is an inactivated whole bacterium that maintains cell wall integrity; The method for prevention and / or treatment includes the steps of: (i) contacting inactive tumor cells or tumor cell lysates obtained from said subject with said bacterial population to provide an immunogenic composition; (ii) administering the immunogenic composition of step (i) to the same subject from which the tumor cells were obtained.

14. The bacterial population for use according to claim 13, further comprising adding to the immunogenic composition of step (i) a compound selected from the group consisting of glucose, fructose, lactate, glyceraldehyde, lactate, pyruvate, oxalate, malate, glutamate, fumarate, α-ketoglutarate, succinate, citrate, glycerol, glutamine, arginine, ketone bodies, β-glucan, monounsaturated fatty acids and polyunsaturated fatty acids, and combinations thereof.

15. 15. The immunogenic composition or pharmaceutical composition for use according to claim 7 or the bacterial population for use according to any one of claims 8 to 14, wherein the administration is systemic.

16. 15. The immunogenic composition or pharmaceutical composition for use according to claim 7 or the bacterial population for use according to any one of claims 8 to 14, wherein the administration is by a route selected from the group consisting of parenteral, intradermal, subcutaneous, transdermal, intravenous, sublingual and oral.

17. An immunogenic composition or pharmaceutical composition for use as described in claim 7, 15 or 16, or a bacterial population for use as described in any one of claims 8 to 16, for use in combination therapy with surgery, radiation or an anti-tumor agent.

18. The immunogenic composition or pharmaceutical composition for use according to any one of claims 7, 15 to 17, or the bacterial population for use according to any one of claims 8 to 17, wherein the tumor is a malignant tumor.

19. The immunogenic composition, bacterial population or pharmaceutical composition of claim 18, wherein the malignant tumor is a tumor of the gastrointestinal tract, nervous system, hematopoietic system, reproductive system, urinary tract, endocrine system, skin, heart, brain, eye, testicle, muscle, bone or breast.

20. The immunogenic composition, bacterial population or pharmaceutical composition of claim 18, wherein the malignant tumor is a colon tumor.

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