Methods and compositions for enhancing cancer treatment effects of bacterial extracellular vesicles

Abstract

The present invention relates to a method and a composition for enhancing the efficacy of cancer treatment of extracellular vesicles of bacteria, and more specifically, to a method and a composition for enhancing cancer treatment efficacy of extracellular vesicles of bacteria. The present invention relates to a method and a composition for enhancing cancer treatment efficacy of extracellular vesicles of bacteria by using a single immune checkpoint inhibitor or various combinations of two or more types of immune checkpoint inhibitors. The composition exhibits a synergistic effect compared to a single dose form of various inhibitors, and exhibits an effect of reducing side effects caused by a high dose of a single dose form, and thus can be effectively used in the development of a cancer drug.

Description

[0001] This application is a divisional application of the patent application filed on June 22, 2021, with application number 202180051621.0 and entitled "Method and Composition for Enhancing the Cancer Treatment Effect of Bacterial Extracellular Vesicles". Technical Field

[0002] This application claims priority to Korean Patent Application No. 10-2020-0076053, filed on June 22, 2020, the entirety of which is incorporated herein by reference.

[0003] This invention relates to a method and composition for enhancing the therapeutic effect of bacterial extracellular vesicles on cancer, and more specifically, to a method and composition for enhancing the therapeutic effect of bacterial extracellular vesicles on cancer using a single immune checkpoint inhibitor or various combinations of two or more types of immune checkpoint inhibitors. Background Technology

[0004] Cancer is one of the leading causes of death, but in some cases it is curable if detected in its early stages. However, cancer becomes very difficult to treat once it has progressed to a certain stage, especially when metastasis is involved. Recently, it has become known and recognized that cancer is not regulated by the immune system due to the presence of an immunosuppressive environment in most cancers. Therefore, various methods have been developed to promote the immune system's regulation of cancer. Cancer immunotherapy is a strategy that treats cancer by utilizing the body's immune function, and it has received considerable attention and expectation since William B. Coley observed tumor regression in the late 19th century by administering a mixture of bacteria known as Coley's toxin. Given the disappointing results of various cancer immunotherapies attempted in the 1980s and 1990s, the development of consistent and reproducible cancer immunotherapies is urgently needed. Recently, cancer immunotherapy has become a reality with the significant therapeutic effects shown by monoclonal antibodies targeting immune checkpoint inhibitors against various cancers. By blocking one or more immune checkpoints, immune checkpoint inhibitors remove a barrier in the immune system and suppress the immunosuppressive tumor microenvironment. Immune checkpoint inhibitors include monoclonal antibodies targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), and programmed death ligand 1 (PDL1).

[0005] Antigen-specific T lymphocyte immune responses are highly complex and meticulously regulated, representing a representative example of specific immune responses, such as the killing of antigen-containing cells, including tumor cells, by cytotoxic T lymphocytes. Antigen recognition requires co-stimulatory signals to activate antigen-specific T lymphocytes. These signals are achieved through the simultaneous binding of CD80 and CD40 expressed on antigen-presenting cells with their corresponding ligands, such as CD28 and CD40L expressed on the surface of T lymphocytes.

[0006] However, activated T lymphocytes are inactivated after a period of time through the activation of co-inhibitory signals, thereby preventing tissue damage caused by excessive immune stimulation. There are various co-inhibitory signals: for example, 1) T lymphocyte CTLA4 binds to its ligands CD80 and CD86 on antigen-presenting cells, inactivating naive or memory T lymphocytes. 2) T lymphocyte PD1 binds to its ligands PDL1 and PDL2 on antigen-presenting cells, inhibiting the function of T lymphocytes in peripheral tissues. The body's immune function regulates overall T lymphocyte function by modulating these co-stimulatory and co-inhibitory signals while recognizing antigens. These signals are called immune checkpoints.

[0007] The body's immune function eliminates tumor cells by detecting tumor-specific neoantigens expressed on tumor cells. Conversely, tumor cells evade these immune attacks by altering the tumor microenvironment to activate inhibitory immune checkpoints, or by employing T-lymphocyte immune tolerance or immune editing to avoid attacks from tumor-specific cytotoxic T lymphocytes. Recently, therapeutic effects have been found on various cancers, such as malignant melanoma, renal cell carcinoma, or non-small cell lung cancer, by activating tumor-specific cytotoxic T lymphocytes, which are suppressed using immune checkpoint inhibitors (monoclonal antibodies targeting CTLA4, PD1, or PDL1). However, immune checkpoint inhibitors are only effective in certain cancers and only in a limited number of patients with these cancers, suggesting that different types of immunosuppressive mechanisms play a role in various cancers.

[0008] Although immune checkpoint inhibitors have shown significant therapeutic effects in cancer treatment, these effects vary from patient to patient. Therefore, numerous studies are actively underway focusing on the specificities of the tumor microenvironment, such as the expression levels of CTLA4, PD1, PDL1, or PDL2 and the number of T lymphocytes in cancer patients, as well as various combination therapies. Recently, it has been found that the therapeutic efficacy of immune checkpoint inhibitors varies depending on the distribution of gut bacteria in patients, and in animal studies, specific gut bacteria can enhance the therapeutic effect of immune checkpoint inhibitors. However, it remains unclear how these specific gut bacteria, and which substances derived from them, enhance the therapeutic effect of immune checkpoint inhibitors.

[0009] Meanwhile, it has been reported that all cells, including Gram-negative and Gram-positive bacteria, naturally release extracellular vesicles. Extracellular vesicles originating from Gram-negative bacteria are also called outer membrane vesicles. Bacterial extracellular vesicles have a diameter of 20-200 nm and contain a variety of bioactive substances, such as proteins, lipids, genetic material (DNA and RNA), and peptidoglycan. Extracellular vesicles released from Gram-negative and Gram-positive bacteria also contain virulence factors, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively. Bacterial extracellular vesicles function as communicators by specifically delivering proteins or genetic material and cellular signals internally. They also help kill competing organisms and improve bacterial survival. In addition, they deliver toxins to host cells to regulate the pathogenesis of bacterial infectious diseases.

[0010] Recently, extracellular vesicles of various bacteria have been shown to have direct therapeutic effects on a variety of diseases, including cancer, and they can be used as drug carriers for treating these diseases. They have already been used clinically or developed as vaccine carriers to prevent or treat various diseases, such as meningitis.

[0011] Furthermore, bacterial extracellular vesicles contain various components that can activate the immune system. Because they do not contain live bacteria, they are safer than the bacteria themselves. It has been reported that intravenous administration of bacterial extracellular vesicles to tumor-bearing mice induced a long-term anti-tumor immune response, effectively eliminating tumor tissue without significant side effects. Nature Communications (8:626, 2017). Furthermore, because bacterial extracellular vesicles are nanoscale entities, they specifically accumulate in tumor tissues through enhanced permeability and retention (EPR) effects. They are thought to induce the release of IFN-γ from natural killer cells and T lymphocytes. To induce an anti-tumor immune response ( Nature Communications .8:626, 2017).

[0012] However, there is no evidence yet to suggest whether the combination of bacterial extracellular vesicles and anticancer agents (including immune checkpoint inhibitors) with different mechanisms of action can enhance the therapeutic effect of anticancer treatment. Summary of the Invention

[0013] Technical issues

[0014] The inventors sought to develop methods for enhancing the efficacy of cancer treatment and combination drugs for treating cancer that effectively inhibit tumor growth and show no side effects. The inventors discovered that the above objectives can be achieved by combining the administration of bacterial extracellular vesicles and immune checkpoint inhibitors, thus completing this invention.

[0015] Technical solution

[0016] To achieve the objectives of this invention, the present invention provides a method for enhancing the cancer therapeutic effect of bacterial extracellular vesicles by combining the administration of bacterial extracellular vesicles with immune checkpoint inhibitors, and a pharmaceutical composition for the prevention or treatment of cancer comprising bacterial extracellular vesicles and immune checkpoint inhibitors as active ingredients.

[0017] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, which consists of bacterial extracellular vesicles and immune checkpoint inhibitors.

[0018] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, which is essentially composed of bacterial extracellular vesicles and immune checkpoint inhibitors.

[0019] Typically, Gram-negative bacteria consist of two membranes: an outer membrane and an inner membrane (or cytoplasmic membrane), while Gram-positive bacteria have only one cell membrane (cytoplasmic membrane). In this invention, the term "bacterial extracellular vesicle" generally refers to membrane vesicles naturally released from bacteria (including transformed bacteria) (natural extracellular vesicles), as well as artificially generated membrane vesicles (artificial extracellular vesicles). The natural extracellular vesicles are referred to as extracellular vesicles, outer membrane vesicles, detached vesicles, microvesicles, microparticles, or exosomes; it is understood that all these entities are included in the extracellular vesicles described in this invention.

[0020] The artificial extracellular vesicles are membrane vesicles artificially formed by a method selected from the group consisting of extrusion, sonication, cell lysis, pH changes, temperature changes, homogenization, freeze-thaw cycles, electroporation, mechanical disruption, and treatment of bacteria (or including transformed bacteria) with various chemical materials such as antibiotics or surfactants (detergents) or combinations thereof. Furthermore, it is understood that all these entities are included in the extracellular vesicles described in this invention, and the methods for forming artificial extracellular vesicles are not limited thereto. Furthermore, for Gram-negative bacteria, the artificial extracellular vesicles include outer membrane-derived vesicles (OMDV), inner membrane-derived vesicles (IMDV), and both inner and outer membrane-derived extracellular vesicles (e.g., vesicles having both outer and inner membrane components, vesicles with the inner membrane present within the outer membrane, vesicles with the outer membrane present within the inner membrane, etc.), and cell membrane-derived vesicles (CMDV), including OMDV, IMDV, and both inner and outer membrane-derived vesicles, having a diameter of 20 nm-1000 nm. Furthermore, it is understood that all these entities are included in the artificial extracellular vesicles described in this invention.

[0021] In this invention, the type of bacteria is not limited. Specifically, the bacteria can be Gram-negative or Gram-positive.

[0022] Examples of Gram-negative bacteria may include Escherichia coli (Escherichia coli spp.) Escherichia), Helicobacter spp. Helicobacter Haemophilus spp. Hemophilus ), Neisseria ( Neisseria ), Cyanobacteria ( Cyanobacterium ), Klebsiella spp. Klebsiella Acetobacter spp. Acetobacter Acinetobacter spp. Acinetobacter ), Enterobacteriaceae ( Enterobacter Chlamydia ( ) Chlamydia ), Vibrio genus ( Vibrio ), Pseudomonas spp. Pseudomonas Salmonella ( Salmonella Thiobacillus spp. Thiobacter ), genus *Borrelia* Borrelia Burkholderia spp. Burkholderia ), Serratia ( Serratia ), genus *Treponema* ( Treponema ), genus Wenken ( Rikenella ), genus *Alternaria* ( Alistipes ), genus *Orychophragmus* ( Marinilabilia ), Proteus spp. Proteus ), Aquatic Fungi ( Enhydrobacter ), Methylobacterium ( Methylobacterium Morganella ( ) Morganella ), Copper-loving bacteria ( Cupriavidus Yersinia spp. Yersinia ), Shigella spp. Shigella Legionella ( ) Legionella ), Oligotrophomonas spp. Stenotrophomonas ) and Moraxella spp. Moraxella Examples of Gram-positive bacteria may include Bacillus spp. ( ). Bacillus Nocardia ( ) Nocardia Clostridium ( Clostridium ), Propionibacterium spp. Propionibacterium Actinomycetes ( Actinomyces ), Enterococcus spp. Enterococcus Corynebacterium spp. Corynebacterium Listeria spp. Listeria Lactobacillus ( ) Lactobacillus Gardnerella spp. Gardnerella ), Mycobacterium ( Mycobacterium Mycoplasma genus Mycoplasma Staphylococcus spp. Staphylococcus Streptomyces ( Streptomyces Micrococcus ( Micrococcus Streptococcus ( Streptococcus Bifidobacterium spp. Bifidobacterium ), Anaerobic bacteria (Anaerostipes ), genus *Plasmodium* Coprococcus ), genus *Mirabilis* ( Atopobium ), Proteobacterium spp. Faecalibacterium Lactococcus spp. Lactococcus ) and Bacteroides ( Bacteroides Bacteria, etc.

[0023] In one aspect of the invention, the bacteria may be transformed bacteria. The transformed bacteria include bacteria transformed to attenuate the toxicity of extracellular vesicles, examples of which may include bacteria with deleted or modified endotoxin-producing genes. Preferably, the transformed bacteria may be transformed to… msbB Genes were deleted ( ΔmsbB (bacteria), more preferably transformed by msbB Gram-negative bacteria with deleted genes are preferably transformed. msbB E. coli with deleted genes ( Escherichia coli (but not limited to this).

[0024] In addition, the bacteria include bacteria that have been transformed to efficiently release extracellular vesicles.

[0025] Furthermore, the bacteria include bacteria transformed to target specific cells or tissues, examples of which may include bacteria transformed to target tumor blood vessels, tumor tissue, or tumor cells. Additionally, the bacteria used in this invention include, but are not limited to, bacteria transformed to fuse with the cell membrane of target cells, bacteria transformed to express substances for treating and / or diagnosing diseases, and bacteria transformed to inhibit a specific substance while simultaneously expressing another specific substance.

[0026] Transformation of bacterial cells can include, but is not limited to, methods of stimulating cells to increase or alter the expression of substances such as proteins, and methods of increasing or inhibiting protein expression through gene introduction.

[0027] Methods to increase the expression of a specific protein can use plasmid DNA, RNA, or bacteriophages, and all commonly known methods can be used, such as heat shock, calcium phosphate precipitation, liposome transfection, electroporation, and microinjection. To suppress the expression of a specific protein, a specific gene can be removed from the cell, and all commonly known methods can be used, such as methods using antisense RNA.

[0028] In one aspect of the invention, the bacteria may be bacteria cultured in a chemically defined culture medium.

[0029] In this invention, "chemically defined culture medium" differs from "natural culture medium" which uses naturally derived substances with undefined components (such as serum and tissue extracts), and refers instead to a synthetic culture medium prepared using only substances with defined components and chemical properties. Preferably, bacteria are cultured in a chemically defined culture medium to produce extracellular vesicles exhibiting uniform effects.

[0030] In this invention, the chemically defined culture medium may be selected from the group consisting of M9 medium, DMEM medium (Dulbecco's modified Eagle's medium) and RPMI 1640 medium (Roswell Park Memorial Institute medium 1640), but is not limited thereto.

[0031] In one aspect of the invention, the bacteria can be transformed to express one or more of the following groups of proteins selected from immune checkpoint proteins, cell adhesion molecules, various antibodies including immune checkpoint inhibitors, proteins that enhance therapeutic effects (cytokines, chemokines, growth factors) such as IL-12 or interferon-γ, targeting proteins, cell membrane fusion substances, and their fusion proteins, but are not limited thereto. These substances can be effectively displayed on the surface of extracellular vesicles using fusion proteins of various proteins (e.g., outer membrane proteins or inner membrane proteins) (e.g., a fusion protein of PrsA (an inner membrane protein) and human epidermal growth factor (EGF; a targeting protein)).

[0032] Preferably, the bacteria may be bacteria transformed to express human immune checkpoint proteins (PD1, PDL1, etc.) or antibodies thereof to inhibit the action of immune checkpoint inhibitors on the surface of extracellular vesicles; more preferably, they may be Gram-negative bacteria transformed to express human immune checkpoint proteins (PD1, PDL1, etc.) or antibodies thereof; most preferably, they may be Escherichia coli transformed to express human immune checkpoint proteins (PD1, PDL1, etc.) or antibodies thereof, but are not limited thereto.

[0033] In one exemplary embodiment of the present invention, *E. coli* were transformed with a pHCE-prsA-EGF vector expressing a fusion protein of human EGF and bacterial endometrial protein PrsA. ΔmsbB (where the toxicity of lipopolysaccharide is reduced), in order to produce Escherichia coli. ΔmsbB -prsA-EGF, and isolate extracellular vesicles from it.

[0034] In one aspect of the invention, the pharmaceutical composition may further comprise a drug that inhibits the extracellular vesicle toxicity of bacteria, an immune checkpoint inhibitor, or a combination thereof. The drug includes, but is not limited to, drugs that inhibit toxicity caused by endotoxins (e.g., polymyxin B), anti-inflammatory agents including dexamethasone and aspirin, anticoagulants, and cyclooxygenase (COX) inhibitors.

[0035] In one aspect of the invention, the pharmaceutical composition may further include a medicament for enhancing anticancer effects. The medicament includes, but is not limited to, anticancer agents, interferon gene-stimulating factor (STING) agonists, TGF-β inhibitors, medicaments for inhibiting helper T cell 17 (Th17) immune responses, medicaments for inhibiting the production or activity of interleukin (IL)-6, medicaments for inhibiting the production or activity of vascular endothelial growth factor (VEGF), and medicaments for inhibiting signal transduction of signal transducers and activator of transcription 3 (STAT3). Examples of medicaments for inhibiting Th17 immune responses may include aspirin, and examples of medicaments for inhibiting VEGF production or activity may include medicaments for inhibiting VEGF receptor signal transduction.

[0036] In one aspect of the invention, the membrane of the extracellular vesicles of the bacteria may further include components other than the cell membrane of the bacteria.

[0037] The components outside the cell membrane may include targeting substances, cell membrane fusion substances (fusion agents), cyclodextrins, and polyethylene glycol. Furthermore, these components can be added by various methods, including chemical modification of the cell membrane.

[0038] For example, the membrane components of bacterial extracellular vesicles can be modified using chemical methods employing thiol (-SH) or amino (-NH2) groups. The membrane components of bacterial extracellular vesicles can also be chemically modified with polyethylene glycol through chemical bonding to the bacterial extracellular vesicles.

[0039] Therefore, the extracellular vesicles of bacteria described in this invention can be further prepared by steps including chemical modification of membrane components.

[0040] The extracellular vesicles of bacteria described in this invention can be isolated from bacterial cultures using various methods known in the art. There are no particular limitations on the type of technique used to isolate extracellular vesicles from bacterial cultures; for example, methods such as ultracentrifugation, density gradient ultracentrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidic-based separation, aqueous two-phase systems, or precipitation can be used.

[0041] In this invention, "immune checkpoint" is a superordinate concept of proteins involved in inducing stimulatory or inhibitory signals on the surface of immune cells, manipulating cancer cells to prevent stimulation of immune responses, and inhibiting the smooth progression of cancer cells to evade the surveillance of the immune system. Preferably, immune checkpoint proteins may include programmed cell death-1 (PD1), programmed cell death ligand 1 (PDL1), programmed cell death ligand 2 (PDL2), differentiation cluster 27 (CD27), differentiation cluster 28 (CD28), differentiation cluster 70 (CD70), differentiation cluster 80 (CD80), differentiation cluster 86 (CD86), T cell immune receptor (TIGIT) with Ig and ITIM domains, differentiation cluster 137 (CD137), differentiation cluster 276 (CD276), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), tumor necrosis factor receptor superfamily member 4 (TNFRSF4), and glucocorticoid-induced TNFR. Related protein (GITR), glucocorticoid-induced TNFR-related protein ligand (GITRL), 4-IBB ligand (4-1BBL), cytotoxic T lymphocyte-associated antigen-4 (CTLA4), adenosine A2A receptor (A2aR), T cell activation inhibitory factor 1 containing V-set domain (VTCN1), B and T lymphocyte attenuation factor (BTLA), indoleamine 2,3-dioxygenase (IDO), T cell immunoglobulin domain and mucin-containing domain-3 (TIM3), T cell activation V domain Ig inhibitor (VISTA), cytotoxic cell lectin-like receptor subfamily A (KLRA), preferably PDL1, TIGIT, CD80 or CTLA4.

[0042] In this invention, an "immune checkpoint inhibitor" is an antagonist that targets the immune checkpoint protein. The immune checkpoint inhibitor can enhance proteins that stimulate the immune response or block proteins that inhibit the immune response, thereby exhibiting an anti-cancer effect through the immune response.

[0043] In one aspect of the invention, the immune checkpoint inhibitor may be a protein or peptide, such as a soluble fusion protein. In one aspect, the protein comprises a receptor / ligand binding domain (e.g., an extracellular domain) of PD1, PDL1, PDL2, CD27, CD28, CD70, CD80, CD86, TIGIT, CD137, CD276, KIRs, LAG3, TNFRSF4, GITR, GITRL, 4-1BBL, CTLA4, A2aR, VTCN1, BTLA, IDO, TIM3, VISTA, or KLRA. In one aspect, the receptor / ligand binding domain may be fused with an immunoglobulin Fc domain. Such a fusion protein can be prepared using standard recombinant DNA techniques (e.g., see Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). Furthermore, many expression vectors encoding the fusion motif are already commercially available.

[0044] In another aspect of the invention, the immune checkpoint inhibitor may be an antibody or its antigen-binding fragment that binds to the immune checkpoint protein to be inhibited (e.g., PD1, PDL1, PDL2, CD27, CD28, CD70, CD80, CD86, TIGIT, CD137, CD276, KIRs, LAG3, TNFRSF4, GITR, GITRL, 4-1BBL, CTLA4, A2aR, VTCN1, BTLA, IDO, TIM3, VISTA, or KLRA). In this invention, "antibody" may refer to the complete antibody and its antigen-binding fragment. Antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, and antigen-binding antibody fragments. The antigen-binding fragment of the antibody refers to one or more fragments of an antibody capable of binding an antigen. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, Fv and Fd linked by disulfide bonds, antibody dimers, single-chain antibodies, camel antibodies, isolated CDR-H3, and other antibody fragments containing at least a portion of the intact antibody variable region. These antibody fragments can be obtained using conventional recombinant and / or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.

[0045] In another aspect of the invention, the immune checkpoint inhibitor may be a repressive nucleic acid (e.g., siRNA molecule, shRNA molecule, antisense RNA) that specifically binds to mRNA encoding immune checkpoint proteins (e.g., PD1, PDL1, PDL2, CD27, CD28, CD70, CD80, CD86, TIGIT, CD137, CD276, KIRs, LAG3, TNFRSF4, GITR, GITRL, 4-1BBL, CTLA4, A2aR, VTCN1, BTLA, IDO, TIM3, VISTA, or KLRA). The repressive nucleic acid molecule may be prepared by chemical synthesis, in vitro transcription, or by cleaving long dsRNA with RNase III or Dicer. The repressive nucleic acid molecule may be delivered to tumor or hypoxic tissue in vitro or in mammalian bodies. All conventional delivery methods known in the art may be used. For example, systemic delivery of interfering RNA may use, for example, the methods and compositions described in PCT application number PCT / US2009 / 036223 (each of which is incorporated herein by reference in its entirety). In one specific implementation, the inhibitory nucleic acid is delivered locally. For example, when treating cancer with the inhibitory nucleic acid described herein, delivery to the tumor can be performed by injection into the tumor, as described by Takahashi et al. Journal of Controlled Release 116:90-95 (2006) and Kim et al., Journal of Controlled Release As stated in 129:107-116 (2008) (each of which is incorporated herein by reference in its entirety).

[0046] In one aspect of the invention, the immune checkpoint inhibitor may be selected from the group consisting of durvalumab, atezolizumab, avelumab, tremelimumab, ipilimumab, pembrolizumab, nivolumab, pitilizumab, BMS986016, cimiplimab, and lirilumab, but is not limited thereto.

[0047] Compared to conventional anticancer cytotoxic drugs, immune checkpoint inhibitors have fewer side effects, such as vomiting and hair loss, and exhibit higher therapeutic efficacy. Because they utilize immune response systems with good memory, the therapeutic effect can persist for a long time after discontinuation of administration. However, it is not yet known whether their anticancer effect is enhanced by combination with bacterial extracellular vesicles. Therefore, the synergistic effect of combining immune checkpoint inhibitors and bacterial extracellular vesicles in cancer treatment may be a technical feature of this invention.

[0048] In one aspect of the invention, bacterial extracellular vesicles and immune checkpoint inhibitors can be administered simultaneously, sequentially, or separately. In this invention, "simultaneously" means that the two drugs are administered at the same time, while "sequentially" means that one drug is administered within 5 minutes, 10 minutes, or several hours after the administration of the other drug. However, the half-life in the cycle of the first administered drug is provided so that the two drugs are present simultaneously at therapeutically effective doses. Furthermore, the methods of simultaneous, sequential, or separate administration are not limited to once, and these methods of administration can be repeated or combined.

[0049] In one respect, bacterial extracellular vesicles and immune checkpoint inhibitors can be included in the same composition along with pharmaceutically acceptable carriers, excipients, and / or diluents.

[0050] On the other hand, bacterial extracellular vesicles and immune checkpoint inhibitors can be provided either as standalone drugs or as kits.

[0051] In one aspect of the invention, the cancer may be selected from gastric cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, colorectal cancer, bladder cancer, colon cancer, cervical cancer, bone cancer, non-small cell bone cancer, hematologic malignancies, skin cancer, head or neck cancer, uterine cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic leukemia. The group comprises, but is not limited to, tumors of the central nervous system (CNS), including, but not limited to, lymphomas of the kidney or ureter, renal cell carcinoma, renal pelvis carcinoma, salivary gland carcinoma, sarcoma, pseudomyxoma, hepatoblastoma, testicular carcinoma, glioblastoma, lip carcinoma, ovarian germ cell tumor, basal cell carcinoma, multiple myeloma, gallbladder carcinoma, choroidal melanoma, ampullary carcinoma of Watt, peritoneal carcinoma, tongue carcinoma, small cell carcinoma, pediatric lymphoma, neuroblastoma, duodenal carcinoma, ureteral carcinoma, astrocytoma, meningioma, renal pelvis carcinoma, vulvar carcinoma, thymic carcinoma, central nervous system (CNS) tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem gliomas, and pituitary adenomas.

[0052] The pharmaceutical compositions described in this invention may comprise only bacterial extracellular vesicles and immune checkpoint inhibitors, or may be formulated in a suitable form with a pharmaceutically acceptable carrier, further comprising excipients or diluents. The carriers include all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microparticles.

[0053] The compositions of the present invention can be administered to mammals, including humans, by any method. For example, the compositions can be administered orally or parenterally. Parenterally administration methods can include, but are not limited to, intravenous, intra-arterial, intramedullary, intrathecal, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intraenteral, local, sublingual, or rectal administration.

[0054] The pharmaceutical compositions of the present invention can be formulated into preparations for oral or parenteral administration according to the administration routes described above.

[0055] The total effective dose of the composition of the present invention can be administered to the patient as a single dose, or, according to a fractionated treatment regimen, as a long-term, multiple-dose administration. In the pharmaceutical composition of the present invention, the content of the active ingredient can vary depending on the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention can be about 0.01. The dosage ranges from 10,000 mg to 10,000 mg, with the optimal dosage being 0.1 mg per day. The effective dose of the pharmaceutical composition ranges from 100 mg to 500 mg per kg of patient body weight. However, the effective dose of the pharmaceutical composition depends on a variety of factors, including the patient's age, weight, health status and sex, disease severity, diet and excretion rate, as well as the formulation method, route of administration and number of treatments. Those skilled in the art can determine the appropriate effective dose of the composition of the present invention by taking these factors into account. The pharmaceutical compositions described in this invention are not particularly limited in formulation, route of administration and method of administration, as long as they exhibit the effects of the present invention.

[0056] In this invention, "target cells" refers to cells that participate in binding to bacterial extracellular vesicles, surface-modified extracellular vesicles, or immune checkpoint inhibitors, or cells that participate in inducing physiological stimulatory or inhibitory signals. Target cells include, but are not limited to, cancer cells, immune cells (T lymphocytes, B lymphocytes, NK cells, NKT cells, macrophages, dendritic cells, monocytes, neutrophils, eosinophils, basophils, mast cells, bone marrow-derived suppressor cells, etc.), endothelial cells, epithelial cells, and fibroblasts.

[0057] This invention provides the use of bacterial extracellular vesicles and immune checkpoint inhibitors in the preparation of cancer therapeutic agents.

[0058] The present invention provides a method for treating cancer, comprising administering an effective amount of a composition to a subject in need, the composition comprising bacterial extracellular vesicles and immune checkpoint inhibitors as active ingredients.

[0059] The "effective dose" of this invention refers to an amount that, when administered to a subject, exhibits effects of improving, treating, detecting and diagnosing cancer, or inhibiting or slowing the progression of cancer, and the "subject" may be an animal, preferably a mammal, particularly an animal including humans, or may be derived from animal cells, tissues, and organs. The subject may be a patient who requires these effects.

[0060] The term "treatment" in this invention refers broadly to improving cancer or symptoms caused by cancer, and may include treating or substantially preventing cancer, or improving its condition, and includes, but is not limited to, alleviating, treating or preventing symptoms or most symptoms caused by cancer.

[0061] As used herein, the term "comprising" has the same meaning as "including" or "characterized in" and does not exclude additional ingredients or method steps not specifically mentioned in the compositions or methods described herein. Furthermore, unless otherwise stated, the term "composed of" means excluding additional elements, steps, or ingredients. The term "essentially composed of" means including substances or steps that do not substantially affect its essential properties, in addition to those described within the scope of the composition or method.

[0062] Beneficial effects

[0063] According to the present invention, compositions comprising bacterial extracellular vesicles and immune checkpoint inhibitors (whether of a single type or a combination of two or more types) as active ingredients exhibit a synergistic effect in cancer treatment compared to monotherapy using each drug. The compositions also exhibit an effect of reducing side effects caused by high-dose administration of single drugs. Therefore, by providing a method for enhancing the efficacy of cancer treatment, the compositions can be used very effectively in the development of cancer therapeutics. Attached Figure Description

[0064] Figure 1 This demonstrates that co-administration of anti-PD1 antibody and natural extracellular vesicles to mice transplanted with colorectal cancer cells (CT26) indicates that... EGF EV M9+ ) experimental scheme.

[0065] Figure 2 ,include Figure 2 A and 2B illustrate the effects of co-administering anti-PD1 antibody and natural extracellular vesicles in mice transplanted with colorectal cancer cells (CT26). EGF EV M9+During this period, the survival rate, tumor volume, body weight, and changes in body temperature of the animals were measured.

[0066] Figure 3, including Figures 3A to 3G This demonstrates the effectiveness of administering natural or artificial extracellular vesicles (EVs) to mice transplanted with bladder cancer cells (MB49). VP-LB CMDV VP-LB or OMDV VP-LB Subsequently, flow cytometry was used to analyze the cells present in the tumor microenvironment to confirm the results of changes in the expression of immune checkpoint proteins (PDL1, CTLA4, and CD80).

[0067] Figure 4, including Figures 4A to 4G This demonstrates the effectiveness of administering natural or artificial extracellular vesicles (EVs) to mice transplanted with colorectal cancer cells (CT26). VP-LB CMDV VP-LB or OMDV VP-LB Subsequently, flow cytometry was used to analyze the cells present in the tumor microenvironment to confirm the results of changes in the expression of immune checkpoint proteins (PDL1, CTLA4, and CD80).

[0068] Figure 5 This demonstrates the effectiveness of administering natural or artificial extracellular vesicles (EVs) to mice transplanted with bladder cancer cells (MB49). VP-LB CMDV VP-LB or OMDV VP-LB Subsequently, qRT-PCR analysis of cells present in the tumor microenvironment was used to confirm the results of changes in the expression of the immune checkpoint protein (IDO1).

[0069] Figure 6, including Figures 6A to 6F This demonstrates the use of natural or artificial extracellular vesicles (EVs). VP-LB CMDV VP-LB or OMDV VP-LB After processing spleen cells extracted from mice transplanted with mouse colorectal cancer cells (CT26), flow cytometry analysis was performed to confirm changes in the expression of immune checkpoint proteins (PDL1 and TIGIT). Detailed Implementation

[0070] The present invention provides the following embodiments.

[0071] Implementation Method 1. A pharmaceutical composition for the prevention or treatment of cancer, comprising bacterial extracellular vesicles and immune checkpoint inhibitors as active ingredients.

[0072] Implementation Method 2. The pharmaceutical composition as described in Implementation Method 1, wherein the bacteria are Gram-negative or Gram-positive bacteria.

[0073] Embodiment 3. The pharmaceutical composition as described in Embodiment 2, wherein the Gram-negative bacteria are selected from Escherichia spp. ( Escherichia ), Helicobacter spp. Helicobacter Haemophilus spp. Hemophilus ), Neisseria ( Neisseria ), Cyanobacteria ( Cyanobacterium ), Klebsiella spp. Klebsiella Acetobacter spp. Acetobacter Acinetobacter spp. Acinetobacter ), Enterobacteriaceae ( Enterobacter Chlamydia ( ) Chlamydia ), Vibrio genus ( Vibrio ), Pseudomonas spp. Pseudomonas Salmonella ( Salmonella Thiobacillus spp. Thiobacter ), genus *Borrelia* Borrelia Burkholderia spp. Burkholderia ), Serratia ( Serratia ) and the genus *Treponema* ( Treponema A group consisting of ).

[0074] Embodiment 4. The pharmaceutical composition as described in Embodiment 2, wherein the Gram-positive bacteria are selected from Bacillus spp. ( Bacillus Nocardia ( ) Nocardia Clostridium ( Clostridium ), Propionibacterium spp. Propionibacterium Actinomycetes ( Actinomyces ), Enterococcus spp. Enterococcus Corynebacterium spp. Corynebacterium Listeria ( ) Listeria Lactobacillus ( ) Lactobacillus Gardnerella spp. Gardnerella ), Mycobacterium ( Mycobacterium Mycoplasma genus Mycoplasma Staphylococcus spp. Staphylococcus Streptomyces ( Streptomyces Micrococcus ( Micrococcus ) and Streptococcus spp. Streptococcus A group consisting of ).

[0075] Implementation Method 5. The pharmaceutical composition as described in Implementation Method 1, wherein the bacteria are transformed bacteria.

[0076] Embodiment 6. The pharmaceutical composition as described in Embodiment 5, wherein the bacteria are bacteria that have been transformed to reduce the toxicity of the extracellular vesicles.

[0077] Embodiment 7. The pharmaceutical composition as described in Embodiment 5, wherein the bacteria are bacteria whose endotoxin-producing genes have been deleted or modified.

[0078] Embodiment 8. The pharmaceutical composition as described in Embodiment 5, wherein the bacteria are bacteria transformed to target specific cells or tissues.

[0079] Embodiment 9. The pharmaceutical composition as described in Embodiment 1, wherein the bacteria are bacteria cultured in a chemically defined culture medium.

[0080] Example 10. The pharmaceutical composition of Example 9, wherein the chemically defined culture medium is selected from the group consisting of M9 medium, DMEM medium (Dulbecco's modified Eagle's medium) and RPMI 1640 medium (Roswell Park Memorial Institute medium 1640).

[0081] Embodiment 11. The pharmaceutical composition of Embodiment 5, wherein the bacteria are bacteria transformed to express one or more of the group consisting of cell adhesion molecules, antibodies, targeting proteins, cell membrane fusion materials and their fusion proteins.

[0082] Embodiment 12. The pharmaceutical composition as described in Embodiment 11, wherein the cell membrane fusion material is human epidermal growth factor (EGF).

[0083] Embodiment 13. The pharmaceutical composition of Embodiment 1, wherein the membrane of the extracellular vesicles of the bacteria further comprises components other than the cell membrane of the bacteria.

[0084] Embodiment 14. The pharmaceutical composition of Embodiment 13, wherein the components other than the cell membrane of the bacteria are selected from the group consisting of a targeting material, a cell membrane fusion material, cyclodextrin, and polyethylene glycol.

[0085] Embodiment 15. The pharmaceutical composition as described in Embodiment 1, wherein the membrane component of the extracellular vesicles of the bacteria is chemically modified.

[0086] Embodiment 16. The pharmaceutical composition of Embodiment 15, wherein the membrane component of the bacterial extracellular vesicles is chemically modified with thiol or amine groups, or is polyethylene glycol chemically bound to the bacterial extracellular vesicles.

[0087] Embodiment 17. The pharmaceutical composition of Embodiment 1, wherein the extracellular vesicles of the bacteria are separated by a method selected from the group consisting of ultracentrifugation, density gradient ultracentrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidic-based separation, aqueous two-phase system and polymer-based precipitation.

[0088] Embodiment 18. The pharmaceutical composition of Embodiment 1, wherein the immune checkpoint inhibitor is selected from programmed cell death-1 (PD1), programmed cell death ligand 1 (PDL1), programmed cell death ligand 2 (PDL2), differentiation cluster 27 (CD27), differentiation cluster 28 (CD28), differentiation cluster 70 (CD70), differentiation cluster 80 (CD80), differentiation cluster 86 (CD86), T cell immune receptor (TIGIT) having Ig and ITIM domains, differentiation cluster 137 (CD137), differentiation cluster 276 (CD276), killer cell immunoglobulin-like receptors (KIRs), lymphocyte activation gene 3 (LAG3), tumor necrosis factor receptor superfamily member 4 (TNFRS). The group consists of F4), glucocorticoid-induced TNFR-related protein (GITR), glucocorticoid-induced TNFR-related protein ligand (GITRL), 4-IBB ligand (4-1BBL), cytotoxic T-lymphocyte-associated antigen-4 (CTLA4), adenosine A2A receptor (A2aR), T-cell activation inhibitory factor 1 containing V-set domain (VTCN1), B- and T-lymphocyte attenuation factor (BTLA), indoleamine 2,3-dioxygenase (IDO), T-cell immunoglobulin domain and mucin-containing domain-3 (TIM3), T-cell activation V-domain Ig inhibitor (VISTA), cytotoxic cell lectin-like receptor subfamily A (KLRA), and combinations thereof.

[0089] Embodiment 19. The pharmaceutical composition as described in Embodiment 18, wherein the immune checkpoint inhibitor is a protein, peptide, antibody, antigen-binding fragment thereof, or inhibitory nucleic acid.

[0090] Implementation Method 20. The pharmaceutical composition as described in Implementation Method 19, wherein the inhibitory nucleic acid is siRNA, shRNA, or antisense RNA.

[0091] Implementation Method 21. The pharmaceutical composition as described in Implementation Method 1, wherein the immune checkpoint inhibitor is selected from the group consisting of durvalumab, atezolizumab, avelumab, tremelimumab, ipilimumab, pembrolizumab, nivolumab, pitilizumab, BMS986016, cimiplimab, and lirilumab.

[0092] Implementation Method 22. The pharmaceutical composition as described in Implementation Method 1, wherein the extracellular vesicles of the bacteria and the immune checkpoint inhibitor are administered simultaneously, sequentially, or separately.

[0093] Implementation Method 23. The pharmaceutical composition as described in Implementation Method 1, wherein the cancer is selected from gastric cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, colorectal cancer, bladder cancer, colon cancer, cervical cancer, bone cancer, non-small cell bone cancer, hematologic malignancies, skin cancer, head or neck cancer, uterine cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia. The group consists of tumors including lymphocytic lymphoma, renal or ureteral cancer, renal cell carcinoma, renal pelvis cancer, salivary gland cancer, sarcoma, pseudomyxoma, hepatoblastoma, testicular cancer, glioblastoma, lip cancer, ovarian germ cell tumor, basal cell carcinoma, multiple myeloma, gallbladder cancer, choroidal melanoma, Watt's ampullary carcinoma, peritoneal cancer, tongue cancer, small cell carcinoma, pediatric lymphoma, neuroblastoma, duodenal cancer, ureteral cancer, astrocytoma, meningioma, renal pelvis cancer, vulvar cancer, thymic cancer, central nervous system (CNS) tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, and pituitary adenoma.

[0094] Embodiment 24. The pharmaceutical composition as described in Embodiment 1, wherein the pharmaceutical composition is administered to mammals, including humans, by oral or parenteral administration.

[0095] Embodiment 25. The pharmaceutical composition as described in Embodiment 24, wherein the parenteral administration method is selected from intravenous, intramuscular, intraarterial, intramedullary, intrasheath, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intraenteral, local, sublingual, and rectal administration.

[0096] Implementation Method 26. Use of a bacterial extracellular vesicle and immune checkpoint inhibitor in the preparation of a cancer therapeutic agent.

[0097] Implementation 27. A method for treating cancer, the method comprising administering an effective amount of a composition to a subject in need, the composition comprising bacterial extracellular vesicles and immune checkpoint inhibitors as active ingredients.

[0098] The present invention will be described in detail below through the following embodiments. However, the following embodiments are merely illustrative of the invention, and the scope of the invention is not limited to the following embodiments.

[0099] Example 1: The effect of co-administration of bacterial extracellular vesicles (EVs) and immune checkpoint inhibitors in vivo

[0100] Experimental methods

[0101] Preparation of natural extracellular vesicles

[0102] As shown in Table 1 below, a culture medium (M9+) was prepared in which vitamins and trace elements were added to the M9 chemically defined medium. *E. coli* was transformed with the pHCE-prsA-EGF vector expressing a fusion protein of human EGF and bacterial inner membrane protein prsA. ΔmsbB (where the toxicity of lipopolysaccharide is reduced), thus yielding Escherichia coli. ΔmsbB -prsA-EGF. Transformed bacteria were cultured using M9+ medium.

[0103] [Table 1]

[0104] The culture was centrifuged twice at 6,000 × g for 20 minutes at 4 °C to remove bacteria, and the supernatant was filtered through a 0.45 μm pore size filter. The filtrate was then concentrated 50-fold using a membrane capable of removing proteins with a molecular weight less than 100 kDa. After filtering the concentrate through a 0.22 μm pore size filter, the filtrate was centrifuged at 4 °C for 20 minutes. C. Centrifuge at 150,000 × g for 3 hours. Resuspend the particulate matter in 2.5 mL of 50% iodixanol and transfer it to a 5 mL ultracentrifuge tube. Then add 1.5 mL of 40% iodixanol and 1.25 mL of 10% iodixanol sequentially. Subsequently, centrifuge at 200,000 × g for 3 hours. Ultracentrifugation at C for 2 hours yielded pure bacterial extracellular vesicles from the layer between 10% iodixanol and 40% iodixanol.

[0105] Finally, the isolated bacterial extracellular vesicles were identified as natural extracellular vesicles and named... EGF EV M9+ .

[0106] Combination therapy of bacterial extracellular vesicles and immune checkpoint inhibitors

[0107] To evaluate the anticancer activity of the combination of bacterial extracellular vesicles and immune checkpoint inhibitors, mouse colorectal cancer cells (CT26, 1) were subjected to immunotherapy. 10 6 Tumor cells were administered subcutaneously to mice (BALB / c, male, 5 weeks old). Five days after tumor cell administration, the mice were divided into four experimental groups, each consisting of five mice (see Table 2). Purified bacterial extracellular vesicles and immune checkpoint inhibitors were administered to each group, alone or in combination. The immune checkpoint inhibitor used in the experiment was an anti-PD1 antibody (InVivoMAb anti-mouse PD1; Bio X Cell (Lebanon, New Hampshire, USA), catalog number BE0273). First, five days after tumor cell administration, each group was given either PBS or an immune checkpoint inhibitor (Ab).

[0108] Six days after administration of tumor cells, each group was given PBS or EGF EV M9+ (EV). Nine days after tumor cell administration, each group was given PBS or Ab, and then 18 days after tumor cell administration, each group was given PBS or Ab again. Finally, 20 days after tumor cell administration, each group was given PBS or Ab. Figure 1 ).

[0109] Next, for each experimental group, mouse survival was monitored daily from day 6 to day 24 after tumor cell administration, and tumor volume was measured. The tumor volume was calculated using the equation V = lxs by measuring the longest length (l) and the length perpendicular to it (s). 2 / 2 Calculate tumor volume (V).

[0110] In addition, from day 6 to day 24 after the administration of tumor cells, the changes in body weight and body temperature of mice in each experimental group were observed daily.

[0111] [Table 2]

[0112] Experimental results

[0113] Experimental results are as follows Figure 2 As shown.

[0114] like Figure 2 As shown, in the PBS-PBS control group and PBS-EV (0.2... g) and Ab(50) g)-EV(0.2 In the g) experimental group, no mice died, while in Ab(50) In the g)-PBS experimental group, all mice died on day 22. Figure 2 A).

[0115] Compared with the PBS-PBS control group, PBS-EV (0.2) g) and Ab(50) g)-EV(0.2 g) The final tumor volume in the experimental group was significantly reduced, and Ab(50) The g)-PBS experimental group could not be compared because all mice in that group eventually died. Furthermore, it was not comparable to the PBS-EV (0.2) group. g) Compared with the experimental group, Ab(50) g)-EV(0.2 g) The final tumor volume of the experimental group was significantly reduced ( Figure 2 B).

[0116] Therefore, tumor growth was significantly reduced in the group receiving the combined administration of Ab and EV compared to the groups receiving EV or Ab alone, confirming a synergistic effect. Specifically, all mice in the Ab-only group died on day 22, but no mice died in the Ab and EV combined group until the end of the experiment, suggesting that the combination of the two drugs reduces toxicity.

[0117] Throughout the experiment, there were no significant changes in body weight and body temperature in the control group and all experimental groups. Figure 2 C, Figure 2 D).

[0118] Example 2: Preparation and characterization of bacterial extracellular vesicles

[0119] To investigate the scientific basis of the synergistic effect demonstrated in Example 1 above (the synergistic effect of combined bacterial extracellular vesicles and immune checkpoint inhibitors), an additional experiment was conducted. The bacterial extracellular vesicles used in the additional experiment were prepared by the following method.

[0120] Preparation of natural extracellular vesicles

[0121] The natural extracellular vesicles used in the experiment were derived from Escherichia coli BL21(DE3). ΔmsbB Prepared according to Example 4 of KR10-2021-0080108 submitted in Korea on June 21, 2021, the finally isolated natural extracellular vesicles are called EVs. VP-LB .

[0122] Preparation of artificial extracellular vesicles

[0123] The artificial extracellular vesicles used in the experiment included cell membrane-derived vesicles (CMDVs) and outer membrane-derived vesicles (OMDVs). To isolate CMDVs and OMDVs, *E. coli* BL21(DE3) was cultured in plant peptone-based lysozyme broth (VP-LB). ΔmsbB -AmCyan. Centrifuge the culture (6,000×g, 15 min), discard the supernatant to obtain bacterial cell particles, and resuspend them in carrier-2 (10 mM Tris-HCl (pH 8.0), 5% sucrose). Lyse the bacterial cells by passing the suspension through a microfluidic device (10,000 psi, 5 times) and centrifuge (6,000×g, 20 min) to obtain a supernatant containing lysed cell fragments. Concentrate the supernatant 3-fold using a tangential flow filter (tangential flow filtration: >500 kDa filter). Divide the concentrate in half, using one half for CMDV isolation and the other half for OMDV isolation.

[0124] To prepare CMDV, the previously prepared concentrate was treated with Benzonase (final concentration: 50 units / mL) and MgCl2 (final concentration: 2 mM) at 37 °C for 30 min. Subsequently, carrier-2 was added to the concentrate at 4 times its volume, and the concentrate was concentrated 5-fold using a tangential flow filter (>500 kDa). CMDV was then separated by density gradient ultracentrifugation (6.5 mL sample, 3.0 mL 0.8 M sucrose, 3.0 mL 2.5 M sucrose; 200,000 × g, 2 h).

[0125] To prepare OMDV, the previously prepared concentrate was treated with sodium dodecyl sarcosinate (sarkosyl, final concentration: 1%) for 30 minutes at room temperature. Subsequently, carrier-2 was added to the concentrate at 4 times its volume, and the concentrate was concentrated 5-fold using a tangential flow filter (>500 kDa). The concentrate was then treated with Benzonase (final concentration: 50 units / mL) and MgCl2 (final concentration: 2 mM) at 37 °C for 30 minutes. Afterward, carrier-2 was added to the concentrate at 4 times its volume, and the concentrate was concentrated 25-fold using a tangential flow filter (>500 kDa). OMDV was then separated by density gradient ultracentrifugation (6.5 mL sample, 3.0 mL 0.8 M sucrose, 3.0 mL 2.5 M sucrose; 200,000 × g, 2 hours).

[0126] The final separated CMDV and OMDV are referred to as CMDV respectively. VP-LB and OMDV VP-LB .

[0127] Characterization of bacterial extracellular vesicles

[0128] According to previous literature ( BiomaterialsAs described in [113:68-79, 2017], the extracellular vesicles of isolated bacteria were characterized. First, the size distribution was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments). Furthermore, protein concentration and total protein mass were measured using the Bradford assay, and particle concentration and total particle number were measured by nanoparticle tracking analysis using an LM10-HS (Malvern Instruments).

[0129] The characterization results of bacterial extracellular vesicles are shown in Table 3 below.

[0130] [Table 3]

[0131] Example 3: Analysis of changes in the tumor microenvironment (TME) after administration of bacterial extracellular vesicles

[0132] Experimental methods

[0133] Mouse bladder cancer cells (MB49: C57BL / 6; 1×10) were administered. 6 Cells / head) and colon cancer cells (CT26: administered to BALB / c; 1×10 6 Cell / head subcutaneous administration of EVs to mice (C57BL / 6, male, 7 weeks old and BALB / c, female, 7 weeks old) for 7 days of tumor growth. Mice were divided into groups of 5, as shown in Table 2 below. EVs were administered intratumorally to the tumors. VP-LB CMDV VP-LB or OMDV VP-LB When applying EV VP-LB CMDV VP-LB or OMDV VP-LB Twenty-four hours later, tumor tissue was extracted from each group, and changes in the tumor microenvironment were analyzed by flow cytometry or real-time RT-PCR.

[0134] [Table 4]

[0135] Carrier-1: 10 mM L-histidine (pH 7.4), 107 mM NaCl, 2% sucrose

[0136] Carrier-2: 10 mM Tris-HCl (pH 8.0), 5% sucrose

[0137] (1) Flow cytometry analysis of the tumor microenvironment

[0138] Using literature ( Methods Mol Biol.Cells for flow cytometry analysis were prepared using the method described in (1458:95-110, 2016). Anti-PDL1 and anti-CTLA4 antibodies from BD Biosciences Co., Ltd. were used, and an LSR Fortessa (5 laser) model from BD Biosciences was used as the analytical instrument.

[0139] (2) Real-time RT-PCR analysis of the tumor microenvironment

[0140] Using literature (Invitrogen, TRIzol) TM RNA for real-time RT-PCR analysis was prepared using the methods described in the Reagent User Guide, and GoScirpt was used. TM The reverse transcriptase kit (Promega) was used to convert the prepared RNA into cDNA. GAPDH was used as an internal control. Real-time PCR was performed on the StepOnePlus real-time PCR system (Applied Biosystems), and the primers required for real-time PCR were purchased from Bioneer.

[0141] Experimental results

[0142] (1) Flow cytometry analysis of the tumor microenvironment

[0143] The expression changes of immune checkpoint proteins PDL1, CTLA4, and CD80 in cells within the tumor microenvironment were assessed using flow cytometry. Figures 3A to 3G and Figures 4A to 4G ).

[0144] First, the administration of extracellular vesicles of mouse bladder cancer cells (MB49) to mice was analyzed. Compared with the control group, in EVs... VP-LB In the treatment group, PDL1 expression increased by 45.0% (27.7% → 72.7%), CTLA4 expression increased by 43.3% (4.5% → 47.8%), and CD80 expression increased by 14.0% (48.6% → 62.6%). Figures 3A to 3C ). In CMDV VP-LB In the treatment group, PDL1 expression increased by 46.8% (41.5% → 88.3%), and CTLA4 expression increased by 71.9% (24.0% → 95.9%). Figure 3D and 3E Meanwhile, in OMDV VP-LB In the treatment group, PDL1 expression increased by 29.1% (41.5% → 70.6%), and CTLA4 expression increased by 65.3% (24.0% → 89.3%). Figure 3F and 3G ).

[0145] Furthermore, the administration of extracellular vesicles of mouse colon cancer cells (CT26) to mice was analyzed. Compared with the control group, in EVs... VP-LB In the treatment group, PDL1 expression increased by 65.9% (27.9% → 93.8%), CTLA4 expression increased by 61.5% (19.6% → 81.1%), and CD80 expression increased by 16.4% (71.5% → 87.9%). Figures 4A to 4C ). In CMDV VP-LB In the treatment group, PDL1 expression increased by 30.9% (11.5% → 42.4%), and CTLA4 expression increased by 38.8% (26.8% → 65.6%). Figure 4D and 4E Meanwhile, in OMDV VP-LB In the treatment group, PDL1 expression increased by 17.2% (11.5% → 28.7%), and CTLA4 expression increased by 33.9% (26.8% → 60.7%). Figure 4F and 4G ).

[0146] Therefore, when bacterial extracellular vesicles were administered, the proportion of cells expressing the immune checkpoint proteins PDL1, CTLA4, and CD80 in the mouse tumor microenvironment increased.

[0147] The above results can scientifically explain the synergistic effect of bacterial extracellular vesicles and PDL1 in combination with Example 1. Furthermore, in addition to PDL1 inhibitors, when co-administered with substances capable of inhibiting CD80 and CTLA4, an increased anticancer effect of bacterial extracellular vesicles can be expected.

[0148] (2) Real-time RT-PCR analysis results of the tumor microenvironment

[0149] The results of measuring changes in the expression of the immune checkpoint protein IDO1 by real-time RT-PCR are as follows: Figure 5 As shown.

[0150] like Figure 5 As shown, when EV is applied VP-LB CMDV VP-LB or OMDV VP-LB At that time, the expression of the immune checkpoint protein IDO1 in the mouse tumor microenvironment increased. Therefore, when co-administered with an IDO inhibitor, an increased anticancer effect of bacterial extracellular vesicles can be expected.

[0151] Example 4: Analysis of the effects of bacterial extracellular vesicles on spleen cells

[0152] The spleen is a secondary lymphoid organ containing various immune cells and influencing the systemic immune response. If a tumor is present, it is assumed that the spleen affects and is influenced by the tumor tissue. Therefore, observing changes in spleen cells in tumor-bearing mice treated with bacterial extracellular vesicles can provide important clues for cancer treatment.

[0153] Experimental methods

[0154] Subcutaneous administration of colorectal cancer cells (CT26, 1×10⁻⁶) to mice (BALB / c, female, 7 weeks old) 6 (Cells / head). The spleen was removed 14 days after tumor cell administration, and individual spleen cells were isolated. EVs were used. VP-LB CMDV VP-LB or OMDV VP-LB Isolated single spleen cells were treated with 1 g / mL of each solution for 24 hours and then subjected to flow cytometry. The method used was as described in the literature (…). Methods Mol Biol. Cells for flow cytometry analysis were prepared using the method described in (1458:95-110, 2016). Anti-PDL1 and anti-CTLA4 antibodies from BD Biosciences Co., Ltd. were used, and an LSR Fortessa (5 laser) model from BD Biosciences was used as the analytical instrument.

[0155] Experimental results

[0156] Mice were administered mouse colorectal cancer cells (CT26), and spleen cells isolated from mice were treated with bacterial extracellular vesicles and analyzed by flow cytometry. Results showed changes in the expression of immune checkpoint proteins PDL1 and TIGIT. Figures 6A to 6F ).

[0157] like Figures 6A to 6F As shown, when using EV VP-LB When splenocytes were treated, PDL1 expression increased by 26.9% (6.0% → 32.9%), and TIGIT expression increased by 10.0% (15.0% → 25.0%). Figure 6A and 6B When using CMDV VP-LB During treatment, PDL1 expression increased by 26.2% (7.0% → 33.2%), and TIGIT expression increased by 14.3% (17.3% → 31.6%). Figure 6C and 6D Meanwhile, when using OMDV VP-LBDuring treatment, PDL1 expression increased by 30.2% (7.0% → 37.2%), and TIGIT expression increased by 10.7% (17.3% → 28.0%). Figure 6E and 6F ).

[0158] Spleen cells isolated from tumor-bearing mice were treated with bacterial extracellular vesicles, and the expression of PDL1 and TIGIT was observed. The results showed that the expression of both PDL1 and TIGIT increased significantly. Therefore, it is reasonable to expect that the anticancer effect may be enhanced when bacterial extracellular vesicles are administered in combination with PDL1 and TIGIT inhibitors.

[0159] Industrial applicability

[0160] According to the present invention, compositions comprising bacterial extracellular vesicles and immune checkpoint inhibitors (whether of a single type or a combination of two or more types) as active ingredients exhibit a synergistic effect in cancer treatment compared to monotherapy using each drug. The compositions also exhibit an effect of reducing side effects caused by high-dose administration of single drugs. Therefore, by providing a method for enhancing the efficacy of cancer treatment, the compositions can be used very effectively to develop cancer therapeutics, thus exhibiting very high industrial applicability.

Claims

1. Use of the composition in the preparation of a cancer therapeutic agent, said composition comprising bacterial extracellular vesicles and immune checkpoint inhibitors as active ingredients. The bacteria mentioned are Escherichia coli (Escherichia coli genus) Escherichia ), The immune checkpoint inhibitors mentioned therein are selected from the group consisting of programmed cell death-1 antagonists, programmed cell death ligand 1 antagonists, programmed cell death ligand 2 antagonists, cytotoxic T lymphocyte-associated antigen-4 antagonists, differentiation cluster 80 antagonists, indoleamine 2,3-dioxygenase antagonists, T cell immune receptor antagonists having Ig and ITIM domains, and combinations thereof.

2. The use as described in claim 1, wherein the bacteria are transformed bacteria.

3. The use as described in claim 2, wherein the bacteria are bacteria that have been transformed to reduce the toxicity of the extracellular vesicles.

4. The use as described in claim 2, wherein the bacteria is a bacterium whose endotoxin-producing gene has been deleted or modified.

5. The use as described in claim 2, wherein the bacteria are bacteria transformed to target specific cells or tissues.

6. The use as claimed in claim 1, wherein the bacteria are bacteria cultured in a chemically defined culture medium.

7. The use as claimed in claim 6, wherein the chemically defined medium is selected from the group consisting of M9 medium, DMEM medium (Dulbecco's modified Eagle's medium) and RPMI 1640 medium (Roswell Park Memorial Institute medium 1640).

8. The use as claimed in claim 2, wherein the bacteria are bacteria transformed to express one or more of the group consisting of cell adhesion molecules, antibodies, targeting proteins, cell membrane fusion materials and their fusion proteins.

9. The use as claimed in claim 8, wherein the cell membrane fusion material is human epidermal growth factor (EGF).

10. The use as claimed in claim 1, wherein the membrane of the extracellular vesicles of the bacteria further comprises components other than the cell membrane of the bacteria.

11. The use as claimed in claim 10, wherein the components other than the cell membrane of the bacteria are selected from the group consisting of targeting materials, cell membrane fusion materials, cyclodextrin, and polyethylene glycol.

12. The use as claimed in claim 1, wherein the membrane components of the extracellular vesicles of the bacteria are chemically modified.

13. The use as claimed in claim 12, wherein the membrane component of the bacterial extracellular vesicles is chemically modified with thiol or amino groups, or is polyethylene glycol chemically bound to the bacterial extracellular vesicles.

14. The use as claimed in claim 1, wherein the extracellular vesicles of the bacteria are separated by a method selected from the group consisting of ultracentrifugation, density gradient ultracentrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidic-based separation, aqueous two-phase systems, and polymer-based precipitation.

15. The use as described in claim 1, wherein the immune checkpoint inhibitor is a protein, peptide, antibody, antigen-binding fragment thereof, or inhibitory nucleic acid.

16. The use as described in claim 15, wherein the inhibitory nucleic acid is siRNA, shRNA, or antisense RNA.

17. The use as claimed in claim 1, wherein the immune checkpoint inhibitor is selected from the group consisting of durvalumab, atezolizumab, avelumab, tremelimumab, ipilimumab, pembrolizumab, nivolumab, pitilizumab, BMS986016, and cimiplimab.

18. The use as claimed in claim 1, wherein the extracellular vesicles of the bacteria and the immune checkpoint inhibitor are administered simultaneously, sequentially, or separately.

19. The use as described in claim 1, wherein the cancer is selected from gastric cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, colorectal cancer, bladder cancer, colon cancer, cervical cancer, bone cancer, non-small cell bone cancer, hematologic malignancies, skin cancer, head or neck cancer, uterine cancer, rectal cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia. The group consists of tumors including lymphocytic lymphoma, renal or ureteral cancer, renal cell carcinoma, renal pelvis cancer, salivary gland cancer, sarcoma, pseudomyxoma, hepatoblastoma, testicular cancer, glioblastoma, lip cancer, ovarian germ cell tumor, basal cell carcinoma, multiple myeloma, gallbladder cancer, choroidal melanoma, Watt's ampullary carcinoma, peritoneal cancer, tongue cancer, small cell carcinoma, pediatric lymphoma, neuroblastoma, duodenal cancer, ureteral cancer, astrocytoma, meningioma, renal pelvis cancer, vulvar cancer, thymic cancer, central nervous system (CNS) tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, and pituitary adenoma.

20. The use as claimed in claim 1, wherein the pharmaceutical composition is administered orally or parenterally to mammals, including humans.

21. The use as described in claim 20, wherein the parenteral administration method is selected from intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intraenteral, local, sublingual, and rectal administration.

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