KR20240123764A - Composition for preventing or treating cancer using combination therapy comprising Lactobacillus plantarum strain and immune cells - Google Patents

Abstract

A pharmaceutical composition for treating cancer by administering a Lactobacillus plantarum strain and immune cells in combination, wherein the Lactobacillus plantarum strain or a culture solution of the strain can inhibit the proliferation of cancer cells or induce the death of cancer cells, and when administered in combination with natural killer cells, exhibits a synergistic effect on the inhibition of the proliferation of cancer cells and tumors, and thus can be usefully used for the prevention, treatment or improvement of cancer in the form of a pharmaceutical composition or health functional food.

Description

{Composition for preventing or treating cancer using combination therapy comprising Lactobacillus plantarum strain and immune cells}

The present invention relates to a composition for preventing or treating cancer using combination therapy comprising a Lactobacillus plantarum strain and immune cells.

Conventional cancer treatment involves a combination of chemotherapy, surgery, hormonal therapy, and/or radiation therapy to eliminate the patient's neoplastic cells. Cancer chemotherapy is preferably based on the use of drugs that kill replicating cells faster than the agent kills the patient's normal cells. Surgery is used to remove the tumor mass, but it has little effect once the cancer has metastasized. Radiation is only effective in localized areas. All of these approaches present significant drawbacks and additional risks, such as increased susceptibility to infection.

To improve these problems, combination therapy has recently emerged to enhance the effectiveness of disease treatment. Combination therapy is the use of two or more drugs or methods simultaneously or in relatively rapid succession. The side effects of two or more treatment methods used in combination therapy may be additive or less than additive, while the therapeutic effects may be additive or greater than additive. When each component is administered in combination, the effect that occurs when each component is administered in combination can be expected to have a greater synergistic effect than the sum of the effects that occur when each component is administered alone. Therefore, there is a need to develop an anticancer combination therapy that can synergistically increase the effect of treatment using a single component.

Natural killer cells express several receptors on the cell surface, and these receptors are involved in cell adhesion, activation of cell killing ability, or inhibition of cell killing ability. However, most natural killer cells existing in the body of a normal person exist in an inactive state. Therefore, activated natural killer cells are required to eliminate cancer. In addition, in the case of natural killer cells existing in the body of a cancer patient, functional defects of natural killer cells exist due to the immune evasion mechanism of cancer cells. Therefore, in order to use natural killer cells as a therapeutic agent, it is very important to activate natural killer cells.

Accordingly, the inventors of the present invention developed an anticancer combination therapy that induces the activity of natural killer cells and exhibits a synergistic effect of inhibiting the proliferation of cancer cells and tumors by co-administering a Lactobacillus plantarum strain, or a culture solution of the strain, with natural killer cells.

One aspect is to provide a pharmaceutical composition for preventing or treating cancer, comprising a first active substance including a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof, and a second active substance including immune cells as active ingredients; or comprising the first active substance or the second active substance as active ingredients, wherein the first active substance or the second active substance is administered in combination with the second active substance or the first active substance, respectively.

Another aspect is to provide a health functional food for preventing or improving cancer, comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient, wherein the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof is administered in combination with natural killer cells.

Another aspect is to provide a kit for preventing or treating cancer, comprising a first active substance including a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof, and a second active substance including natural killer cells as active ingredients; or comprising the first active substance or the second active substance as active ingredients, wherein the first active substance or the second active substance is administered in combination with the second active substance or the first active substance, respectively.

Another aspect provides a method for delivering a drug into a subject, the method comprising administering to a subject in need thereof a composition for preventing or improving cancer, the composition comprising a first active agent comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof, and a second active agent comprising natural killer cells as active ingredients; or the composition comprising the first active agent or the second active agent as active ingredients, wherein the first active agent or the second active agent is co-administered with the second active agent or the first active agent, respectively.

Another aspect provides a method for preventing or treating cancer, comprising the steps of: administering to a subject in need thereof a first active agent comprising a strain of Lactobacillus plantarum , a culture of the strain, a lysate of the strain, or a mixture thereof; and administering to a subject in need thereof a second active agent comprising immune cells.

Another aspect provides a use of a pharmaceutical composition for preventing or treating cancer, comprising a first active substance comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof, and a second active substance comprising immune cells as active ingredients; or comprising the first active substance or the second active substance as active ingredients, wherein the first active substance or the second active substance is administered in combination with the second active substance or the first active substance, respectively.

One aspect provides a strain of the genus Lactobacillus, specifically the strain Lactobacillus plantarum GB104, having anticancer activity, for example, anticancer activity against colon cancer.

Lactobacillus is a genus of aerobic or facultative anaerobic gram-positive bacilli widely distributed in nature. Microorganisms belonging to the genus Lactobacillus include Lactobacillus plantarum and Sakei. As a result of research to develop a new strain with excellent anticancer effect, the present inventors selected Lactobacillus plantarum GB104 as an anticancer candidate strain. The strain was deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on January 14, 2020 under the deposit number KCTC14107BP. The strain is a probiotic strain, is harmless to the human body, and can be used without side effects.

The above Lactobacillus has been renamed to Limosilactobacillus or Lactiplantibacillus , and the changed strain names can be used interchangeably in this specification. For example, Lactobacillus plantarum has been renamed to Lactiplantibacillus plantarum .

In this specification, the term " Lactobacillus plantarum GB104" may be described together with L. Plantarum GB104 strain or Lactobacillus plantarum GB104 strain (Accession Number: KCTC14107BP).

In one specific example, the strain may be a strain deposited under the accession number KCTC14107BP.

In one specific example, the strain may be a strain comprising a 16S rRNA gene consisting of a nucleotide sequence of sequence number 1.

In one specific example, the strain may be a strain having 16S rRNA comprising a nucleotide sequence of SEQ ID NO: 1 or a 16S rRNA comprising a nucleotide sequence having 97% or greater nucleotide sequence identity therewith. Specifically, it has at least 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% homology to the nucleotide sequence consisting of SEQ ID NO: 1 of the present specification.

In one specific example, the strain may be a live cell, a dead cell, or a cytoplasmic fraction obtained by disrupting a strain, and preferably, it may be a live cell.

In the present invention, 'probiotics' are understood to mean living microorganisms that improve the intestinal microbial environment of the host in the gastrointestinal tract of animals including humans, thereby exerting beneficial effects on the host's intestines. Probiotics are living microorganisms with probiotic activity, and when fed to humans or animals in the form of dried cells or fermented products in the form of single or complex strains, they can exert beneficial effects on the host's intestinal flora.

One aspect provides a pharmaceutical composition for preventing or treating cancer, comprising a first active substance including a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof, and a second active substance including immune cells as active ingredients; or comprising the first active substance or the second active substance as active ingredients, wherein the first active substance or the second active substance is administered in combination with the second active substance or the first active substance, respectively.

The term "culture" herein may be used interchangeably with "culture supernatant", "culture supernatant", "conditioned culture" or "conditioned medium", and may mean the entire medium including the strain, its metabolites, extra nutrients, etc., obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the Lactobacillus strain can grow and survive in vitro. The culture means a product obtained by culturing a probiotic strain in a known medium, and the product may or may not include the strain itself. The medium may be selected from known liquid media or solid media, and may be, for example, but is not limited to, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, BL agar medium.

The term "lysate" herein may be used interchangeably with "lysate" and means a solution or suspension of an aqueous medium of cells of a microorganism, such as Lactobacillus plantarum, that have been broken apart. A cell lysate comprises macromolecules, such as DNA, RNA, proteins, peptides, carbohydrates, lipids, and/or micromolecules, such as amino acids, sugars, fatty acids, or fractions thereof. The lysate also comprises cell debris, which may be smooth or granular in structure.

The above culture solution may include the culture solution itself, a concentrate thereof, or a lyophilized product obtained by culturing the strain, or a culture supernatant obtained by removing the strain from the culture solution, a concentrate thereof, or a lyophilized product thereof.

The above culture solution may be obtained by culturing Lactobacillus plantarum in an appropriate medium (e.g., MRS plate medium) at a temperature of more than 10°C or less than 40°C for a certain period of time, for example, 4 to 50 hours.

In one specific example, the first active material comprising at least one selected from the group consisting of the strain, a culture of the strain, a lysate of the strain, or an extract of the mycelia, culture, and lysate may have anticancer activity. Specifically, the anticancer activity may be an activity that delays the occurrence of a tumor or inhibits the growth rate of a tumor.

In one specific example, the anticancer activity may have activity in inhibiting tumor growth, activity in inducing apoptosis of cancer cells, and activity in increasing the degree of activation and number of natural killer cells involved in antitumor immunity.

In one specific example, the anticancer activity includes an effect in which the number of NK cells (specifically, IFN-γ+ NK cells or Granzyme B+ NK cells) significantly increases when the Lactobacillus plantarum GB104 strain is administered to a tumor animal model transplanted with a colon cancer cell line, compared to a control group that was not administered the strain.

In one specific example, the first active substance including at least one selected from the group consisting of the strain, a culture of the strain, a lysate of the strain, or an extract of the mycelia, culture, and lysate may induce the activity of natural killer cells. Specifically, the first active substance may have the activity of increasing the activity and number of NK cells.

The term "immune cell" as used herein refers to a cell that is part of the immune system of a living organism and provides an immune function against infection or other diseases. The immune cell may be, for example, a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a macrophage, a dendritic cell, a natural killer cell, a B cell and/or a T cell, and more preferably, a natural killer cell (NK cell). In the present invention, the immune cell may be naturally derived from an animal, but is not limited thereto, and may include an immune cell manufactured through cell engineering, genetic engineering, etc., for example, a CAR T cell, a CAR NK cell, etc.

The term "natural killer cells (NK cells)" in this specification refers to the main cells that constitute innate immunity, which detect the major histocompatibility complex (MHC) on the surface of infected or abnormal cells and induce the release of cytokines to induce cell lysis or apoptosis. The natural killer cells are a type of lymphocyte and are distributed in the bone marrow, spleen, peripheral lymph nodes, and peripheral blood in the body, accounting for about 10% of peripheral blood lymphocytes, and play an important role in the innate immune response (Ann Rev Immunol., 24: 257-286, 2006). In addition, natural killer cells are positive for CD56 and CD16, but negative for CD3. The way natural killer cells kill cells is through the release of cytoplasmic granules containing perforin and granzyme. Natural killer cells secrete various cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-10. In addition, natural killer cells express several receptors on the cell surface, and these receptors are involved in cell adhesion, activation of cell killing ability, or inhibition of cell killing ability. However, most natural killer cells existing in the body of a normal person exist in an inactive state. Therefore, activated natural killer cells are required to eliminate cancer. In addition, in the case of natural killer cells existing in the body of a cancer patient, functional defects of natural killer cells exist due to the immune evasion mechanism of cancer cells. Therefore, in order to use natural killer cells as a therapeutic agent, it is very important to activate natural killer cells.

In one specific example, the immune cells may be cultured in the presence of a fusion protein comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof.

The term "IL-2" or "interleukin-2", as used herein, unless otherwise stated, means any wild-type IL-2 obtained from any vertebrate source, including mammals, for example, primates (e.g., humans) and rodents (e.g., mice and rats). The IL-2 may be obtained from an animal cell, but also includes that obtained from a recombinant cell capable of producing IL-2. Furthermore, the IL-2 may be wild-type IL-2 or a variant thereof.

In this specification, IL-2 or its variants are collectively referred to as "IL-2 protein" or "IL-2 polypeptide". IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variants specifically bind to, for example, an IL-2 receptor. This specific binding can be confirmed by a method known to those skilled in the art. A specific example of the IL-2 may have the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In addition, at this time, the IL-2 may be in a mature form. Specifically, the mature IL-2 may not include a signal sequence and may have the amino acid sequence of SEQ ID NO: 4. At this time, the IL-2 may be used as a concept including a fragment in which a portion of the N-terminus or C-terminus of the wild-type IL-2 is deleted (truncated). In addition, the fragment of IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids are deleted consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In addition, the fragment of the IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids are deleted sequentially from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

The term "IL-2 variant" as used herein means a form in which a portion of an amino acid is substituted in full-length IL-2 or a fragment of the IL-2 described above. That is, the IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or a fragment thereof. However, the IL-2 variant may have an activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may mean, for example, specific binding to an IL-2 receptor, and this specific binding may be measured by a method known to those skilled in the art.

The term "CD80" in this specification is also called "B7-1" and is a membrane protein present on dendritic cells, activated B cells, and monocytes. CD80 provides a costimulatory signal essential for the activation and survival of T cells. CD80 is known as a ligand for two different proteins, CD28 and CTLA-4, present on the surface of T cells. CD80 is composed of 288 amino acids and may specifically have an amino acid sequence of SEQ ID NO: 5. In addition, "CD80 protein" in this specification means full-length CD80 or a CD80 fragment.

The term "CD80 fragment" used herein refers to a truncated form of CD80. In addition, the CD80 fragment may be an extracellular domain of CD80. A specific example of the CD80 fragment may be one in which the 1st to 34th amino acids from the N-terminus, which is a signal sequence of CD80, are excluded. Specifically, a specific example of the CD80 fragment may be a protein composed of the 35th to 288th amino acids of SEQ ID NO: 5. In addition, a specific example of the CD80 fragment may be a protein composed of the 35th to 242nd amino acids of SEQ ID NO: 5. In addition, a specific example of the CD80 fragment may be a protein composed of the 35th to 232nd amino acids of SEQ ID NO: 5. In addition, a specific example of the CD80 fragment may be a protein composed of the 35th to 139th amino acids of SEQ ID NO: 5. Additionally, a specific example of the CD80 fragment may be a protein consisting of the 142nd to 242nd amino acids of SEQ ID NO: 5.

The above CD80 protein or fragment thereof and the IL-2 protein or variant thereof may be linked by a linker or a carrier. In the present specification, linker and carrier are sometimes used interchangeably.

The above “IL-2”, “IL-2 variant”, “CD80 protein”, “CD80 fragment” or “structure in which IL-2 and CD80 are linked” may include those described in Korean Patent Application No. 10-2020-0155367. The entirety of the document is incorporated herein by reference.

In one specific example, the immune cells are cultured in the presence of a fusion protein comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof, and may have increased expression of CD16 and NKp46 compared to immune cells isolated from bone marrow, cord blood, or peripheral blood mononuclear cells (PBMC).

In one specific example, the immune cells may be cultured in the presence of a fusion protein comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof, and may have increased expression of granzyme B, perforin, and interferon-γ compared to immune cells isolated from bone marrow, cord blood, or peripheral blood mononuclear cells (PBMC).

In one specific example, the immune cells may be natural killer cells (NK cells).

The term "cancer" as used herein refers to a physiological condition in animals that is typically characterized by abnormal or uncontrolled cell growth. Cancer and cancer pathology can be associated with, for example, metastasis, interference with normally functioning surrounding cells, release of cytokines or other secretory products at abnormal levels, suppression or enhancement of inflammatory or immunological responses, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.

The above cancers are known to have significantly reduced natural killer cell activity and suppressed cellular immune mechanisms (Kosin Medical Journal Vol/18 No. 1 pp.36~42, 2003), and in particular, it has been reported that natural killer cell activity is reduced in melanoma, head and neck cancer, lung cancer, ovarian cancer, uterine cancer, bladder cancer, prostate cancer, liver cancer, pancreatic cancer, esophageal cancer, colon cancer, etc. (K.C.P.S. Vol. 14, No. 3 1998). Therefore, when the Lactobacillus platarum strain according to one specific example is used to increase the activity of natural killer cells, it can be effective in the prevention or treatment of cancer.

The cancer may be gastrointestinal cancer or non-gastrointestinal cancer.

The above gastrointestinal cancer is a malignant tumor occurring in the gastrointestinal tract, such as the esophagus, stomach, small intestine or large intestine. The gastrointestinal cancer may be, for example, one or more cancers selected from the group consisting of esophageal cancer, gallbladder cancer, liver cancer, bile duct cancer, pancreatic cancer, stomach cancer, small intestine cancer, large intestine cancer, colon cancer, anal cancer and rectal cancer, but is not limited thereto. In one example, it may be colon cancer.

The above non-gastrointestinal cancer includes, without limitation, malignant tumors occurring in organs other than the gastrointestinal tract or digestive system, and may be, for example, leukemia, acute myeloid leukemia, neuroblastoma, retinoblastoma, lung cancer, head and neck cancer, salivary gland cancer, melanoma, laryngeal cancer, prostate cancer, breast cancer, bladder cancer, kidney cancer, multiple myeloma, cervical cancer, thyroid cancer, ovarian cancer, urethral cancer, skin cancer, osteosarcoma, glioblastoma, brain tumor, or lymphoma, but is not limited thereto.

In one specific example, the cancer may be any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, salivary gland cancer, melanoma, bladder cancer, kidney cancer, blood cancer, esophagus cancer, head and neck cancer, skin cancer, small intestine cancer, anal cancer, colon cancer, rectal cancer, and lymphoma. Preferably, the cancer may be any one selected from the group consisting of liver cancer, colon cancer, breast cancer, kidney cancer, and lung cancer, but is not limited thereto. For example, according to one embodiment of the present invention, the cancer may be colon cancer or liver cancer. The colon cancer includes a malignant tumor occurring in one or more sites selected from the group consisting of the ascending colon, the transverse colon, the descending colon, the sigmoid colon, and the rectal mucosa. The above colon cancer may be one or more types selected from the group consisting of adenocarcinoma, lymphoma, malignant carcinoid tumor, leiomyosarcoma, Kaposi sarcoma, and squamous cell carcinoma, but is not limited thereto. The liver cancer includes both hepatocellular carcinoma originating from hepatocytes and cholangiocarcinoma originating from biliary tract cells. For example, the above may be one or more types selected from the group consisting of hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma, but is not limited thereto. In one specific example, the first active ingredient may contain a Lactobacillus plantarum strain alone as an active ingredient, or may contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

In one specific example, the second active agent may comprise natural killer cells cultured in the presence of a fusion protein comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof.

In one specific example, the first active ingredient and the second active ingredient may be administered simultaneously, sequentially, or in reverse order. For example, the Lactobacillus plantarum strain and the natural killer cells may be administered via independent routes. Each active ingredient may be administered independently by a person skilled in the art with a suitable administration method and dosage.

In one specific example, the first active substance comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof, and the second active substance comprising immune cells may be administered orally, intravenously, intraperitoneally, or subcutaneously.

The Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof may be administered orally, and the immune cells may be administered intravenously or subcutaneously. For example, the first active ingredient may be administered orally, and the second active ingredient may be administered intravenously or subcutaneously, but is not limited thereto.

The above immune cells may be characterized in that they are administered at about 1x10 ¹ cell to about 1x10 ²⁰ cells, preferably about 1x10 ² cells to about 1x 10 ¹³ cells, more preferably about 1x10 ⁴ cells to about 1x 10 ¹¹ cells, and in one embodiment of the present invention, about 1x10 ⁶ cells/head/200μl are administered, but are not limited thereto.

In one specific embodiment, the immune cells may be prepared as a separate formulation for co-administration with the composition of the present invention. The immune cells may be prepared as an injectable formulation containing them, preferably an intravenous formulation, but is not limited thereto. The separate composition containing the immune cells may further contain suitable carriers, excipients and diluents commonly used for administration to humans or animals, and may additionally contain a component capable of maintaining the survival or activity of natural killer cells.

In one specific example, a first oral formulation comprising a first active ingredient and a second oral formulation comprising a second active ingredient are included, wherein the first and second oral formulations can be administered orally.

The term "including as an active ingredient" in this specification means that a Lactobacillus genus strain, a lysate of the strain, a culture medium, or an extract of the culture medium is added, and includes formulation in various forms by adding various components as auxiliary components for drug delivery and stabilization, etc.

In one specific embodiment, the pharmaceutical composition can be administered to a mammal, including a human, by various routes. The route of administration can be any route commonly used, and for example, it can be administered by oral, cutaneous, intravenous, intramuscular or subcutaneous routes, and preferably, it can be administered orally.

As used herein, the term "combination therapy" or "combination administration" or "in combination" refers to any form of simultaneous or concurrent treatment using at least two separate therapeutic agents. The components of the combination therapy may be administered simultaneously, sequentially, or in any order. The components may be administered in different dosages or at different frequencies or by different routes as appropriate.

Specifically, the combination therapy may be administered by administering the Lactobacillus plantarum strain and the immune cells simultaneously, or by administering the Lactobacillus plantarum strain and then the immune cells. The combination therapy according to the present invention may be defined as providing a synergistic effect if the efficacy measured by, for example, the degree of response, the rate of response, the period until disease progression, or the duration of survival is therapeutically superior to the efficacy obtained by administering one or the other of the components of the combination therapy at a conventional dosage. For example, the efficacy of the combination therapy is synergistic if the efficacy is therapeutically superior to the efficacy obtained by using each of the above alone. In particular, a synergistic effect is considered to exist if the conventional dosage of the Lactobacillus plantarum strain and the natural killer cells can be reduced without damaging one or more of the degree of response, the rate of response, the period until disease progression, and the survival data, particularly without damaging the duration of response, and with reduced and/or fewer problematic side effects than when each component is used at a conventional dosage.

The term "administered simultaneously" as used herein is not particularly limited and means that the components of the combination therapy are administered substantially simultaneously, for example, as a mixture or in immediately subsequent sequence.

The term "administered sequentially" as used herein is not particularly limited and means that the components of the combination therapy are not administered simultaneously, but are administered one after the other or in clusters with a specific time interval between administrations. The time intervals can be the same or different between the administrations of each of the components of the combination therapy and can be selected, for example, from the range of 2 minutes to 96 hours, 1 day to 7 days or 1 week, 2 weeks or 3 weeks. Typically, the time intervals between administrations can range from several minutes to several hours, for example, from 2 minutes to 72 hours, from 30 minutes to 24 hours, or from 1 to 12 hours. Additional examples include time intervals from 24 to 96 hours, from 12 to 36 hours, from 8 to 24 hours, and from 6 to 12 hours.

The term “prevention” in this specification may mean any act of suppressing or delaying the onset of a disease state in an individual by administering a pharmaceutical composition according to one aspect.

The term "treatment" as used herein may mean any action by which symptoms of a disease state of a subject are improved or beneficially changed by administration of a pharmaceutical composition according to one aspect.

According to one specific example, the composition may comprise 0.001 wt% to 80 wt% of the Lactobacillus plantarum strain based on the total weight of the composition. In addition, the dosage of the Lactobacillus plantarum strain may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. The strain is included in the composition in a therapeutically effective amount or a nutritionally effective concentration, for example, the strain is present in an amount of 10 ³ to 10 ¹⁶ CFU/g, 10 ³ to 10 ¹⁵ CFU/g, 10 ³ to 10 ¹⁴ CFU/g, 10 ³ to 10 ¹³ CFU/g, 10 ³ to 10 ¹² CFU/g, 10 ⁴ to 10 ¹⁶ CFU/g, 10 ⁴ to 10 ¹⁵ CFU/g, 10 ⁴ to 10 ¹⁴ CFU/g, 10 ⁴ to 10 ¹³ CFU/g, 10 ⁴ to 10 ¹² CFU/g, 10 ⁵ to 10 ¹⁶ CFU/g, 10 ⁵ to 10 ¹⁵ CFU/g, 10 ⁵ to 10 ¹⁴ CFU/g, 10 ⁵ to 10 ¹³ CFU/g, 10 ⁵ to 10 ¹² CFU/g, 10 ⁶ to 10 ¹³ CFU/g, 10 ⁶ to 10 ¹² CFU/g, 10 ⁷ to 10 ¹³ CFU/g, 10 ⁷ to 10 ¹² CFU/g, 10 ⁸ to 10 ¹³ CFU/g or 10 ⁸ to 10 ¹² CFU/g, or can be included in the composition as a culture of an equivalent number of live or dead cells. Specifically, for adult patients, 1X10 ³ to 1X10 ¹⁶ CFU/g of live or dead cells can be administered once or in multiple doses. However, the dosage may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and those skilled in the art can appropriately adjust the dosage by considering these factors. The number of administrations may be once or twice or more within the range of clinically acceptable side effects, and the administration site may be administered to one or more sites. For animals other than humans, the same dosage per kg (body weight) as for humans may be administered, or the dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (heart, etc.) of the target animal and the human. Possible administration routes may include oral, sublingual, parenteral (e.g., subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), rectal, topical (including transdermal), inhalation, and injection, or insertion of an implantable device or substance. As the target animals for treatment according to one specific example, humans and other mammals for the purpose can be exemplified, and specifically, humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, etc. are included. According to one embodiment, the composition comprises a killed dried strain, and can be administered at a dose of 1 g to 10 g, 0.5 g to 1.5 g, 2.5 g to 3.5 g, or 4.5 g to 5.5 g at a time, and can be administered once to three times a day.

The term "therapeutically effective amount," as used herein, means an amount of natural killer cells for the methods and uses of the present invention or a pharmaceutical composition comprising natural killer cells for the methods and uses of the present invention that will elicit a biological or medical response or a desired therapeutic effect in a patient sought by a researcher, physician, or other clinician. The therapeutically effective amount of natural killer cells may vary depending on factors such as the disease state, age, sex, and weight of the subject, and the ability of the natural killer cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.

A pharmaceutical composition according to one embodiment may include a pharmaceutically acceptable carrier and/or additive. For example, it may include sterile water, saline, a conventional buffer (such as phosphoric acid, citric acid, or other organic acids), a stabilizer, a salt, an antioxidant (such as ascorbic acid), a surfactant, a suspending agent, an isotonic agent, or a preservative. For topical administration, it may also include a combination with an organic substance such as a biopolymer, an inorganic substance such as hydroxyapatite, specifically, a collagen matrix, a polylactic acid polymer or copolymer, a polyethylene glycol polymer or copolymer, and chemical derivatives thereof.

When a pharmaceutical composition according to one embodiment is prepared in a dosage form suitable for injection, the Lactobacillus genus cells may be dissolved or dispersed in a pharmaceutically acceptable carrier, or may be frozen in a solution state in which they are dissolved or dispersed.

A pharmaceutical composition according to one specific example may appropriately contain, depending on the administration method or formulation, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc., if necessary. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, 19th ed., 1995]. A pharmaceutical composition according to one specific example may be manufactured in a unit dose form or may be manufactured by introducing into a multi-dose container by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets, or capsules.

The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

In one specific example, the anticancer activity of the first active ingredient may be more effective when administered in combination with the second active ingredient.

In one embodiment, when the Lactobacillus plantarum GB104 strain and immune cells were co-administered to a tumor animal model transplanted with a mouse colon cancer cell line, an enhanced anticancer effect was observed compared to when the Lactobacillus plantarum GB104 strain was administered alone. Specifically, the effect of inhibiting the growth and progression of colon cancer tumors was confirmed. On average, compared to the control group, the experimental group co-administered with GB104 and immune cells showed an effect of reducing tumor volume by 10% to 90%.

Another aspect is to provide a health functional food for preventing or improving cancer, comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient, wherein the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof is administered in combination with immune cells.

The above “strain”, “first active substance”, “immune cell”, “natural killer cell”, “second active substance”, “anticancer activity”, and “combined administration” are as described above.

In one specific embodiment, the food composition can be administered to a mammal, including a human, by various routes. The route of administration can be any route commonly used, and for example, it can be administered by oral, cutaneous, intravenous, intramuscular or subcutaneous routes, and preferably, it can be administered orally.

In one specific example, the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof and immune cells may be administered orally, intravenously, intraperitoneally, or subcutaneously.

In one specific example, the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof may be administered orally, and the immune cells may be administered parenterally. Specifically, the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof may be administered orally, and the immune cells may be administered intravenously or subcutaneously.

In one specific example, the health functional food may additionally contain a food-wise acceptable carrier.

As used herein, the term “food-related acceptable” means that the compound exhibits the property of being non-toxic to cells or humans exposed to it.

The term "improvement" in this specification may mean any action that at least reduces a parameter related to the condition being treated, for example, the degree of symptoms. In this case, the health functional food may be used before or after the onset stage of the condition, simultaneously with or separately from a drug for treatment, for the purpose of preventing or improving cancer.

In the above health functional food, the active ingredient can be added to the food as it is or used together with other food or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to its purpose of use (for prevention or improvement). In general, when manufacturing food or beverage, the health functional food can be added in an amount of about 15 wt% or less, more specifically about 10 wt% or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount can be below the above range.

The above health functional food may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, and may further include one or more of a carrier, a diluent, an excipient, and an additive. Foods to which the compound according to one aspect may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, and health functional foods.

Specific examples of the carrier, excipient, diluent and additive may include at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.

The above health functional food, in addition to containing the effective ingredient, may contain other ingredients as essential ingredients without special limitation. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, like a typical beverage. Examples of the above-mentioned natural carbohydrates may include common sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. In addition to the above-mentioned flavoring agents, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the above-mentioned natural carbohydrates can be appropriately determined by a person skilled in the art.

In addition to the above, health functional foods according to the aspect may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

The above health functional food may be provided mixed with a health functional food for preventing or improving cancer known in the past or with another existing health functional food, and the health functional food for preventing or improving cancer may be a health functional food for preventing or improving metabolic diseases known in the past, an existing health functional food, or a newly developed health functional food.

When the above health functional food contains another health functional food that has a cancer prevention or improvement effect, it is important to mix the amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The above food composition for preventing or improving cancer includes all forms such as functional food, nutritional supplement, health food, and food additives, and the food composition of the above type can be manufactured in various forms according to conventional methods known in the art.

The compositions of the present disclosure may be considered food supplements. Food supplements, also known as dietary supplements or nutritional supplements, may be considered another specific pharmaceutical product. They are intended to supplement the diet and provide nutrients or beneficial ingredients that may not be consumed or may not be consumed in sufficient amounts in a normal diet. Most food supplements are considered foods, but sometimes they are considered drugs, natural health products, or nutraceutical products. In the sense of the present invention, food supplements include nutraceutical products. Food supplements are generally sold over the counter without a prescription. When food supplements take the form of pills or capsules, they contain the same excipients used in pharmaceuticals. However, food supplements may also take the form of foods fortified with certain nutrients (e.g., infant formula). Thus, in certain embodiments, the composition of the present invention is a food supplement.

The composition according to the present invention may be administered as is or mixed with suitable edible liquids or solids or may be freeze-dried in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets, syrups or in the form of unit doses. It may also be in the form of monodoses of the freeze-dried composition which are mixed in a separate liquid container provided together prior to administration.

The composition of the present invention may also be incorporated into various edible foods and foods for infants, such as milk products. The term "edible product" as used herein is intended to encompass in a broad sense any product which can be ingested by an animal in any form (e.g., the product can be perceived by the sense organs). The term "food product" is understood to be an edible product which provides nutritional support to the body. Of particular interest are food supplements and infant formulas. The food preferably comprises a carrier material, such as oatmeal gruel, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycosylated proteins. In certain embodiments, the bacterial cells of the present invention are homogenized with other ingredients, such as cereals or powdered milk, to form an infant formula.

Another aspect is to provide a kit for preventing or treating cancer, comprising a first active substance including at least one selected from the group consisting of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or an extract of the fungus, culture, and lysate, and a second active substance including immune cells as an active ingredient; or comprising the first active substance as an active ingredient and the second active substance being administered together.

Another aspect is a method for delivering a drug into a subject, comprising administering to a subject in need thereof a composition comprising a first active substance comprising at least one selected from the group consisting of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or an extract of the fungus, culture, and lysate, and a second active substance comprising immune cells as active ingredients; or comprising the first active substance as an active ingredient and the second active substance being co-administered.

The above “strain”, “first active substance”, “immune cell”, “natural killer cell”, “second active substance”, “anticancer activity”, and “combined administration” are as described above.

The subject may be a subject suffering from cancer. Additionally, the subject may be a mammal, preferably a human.

The above Lactobacillus plantarum GB104 strain and natural killer cells may be administered to a subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and administration frequency may be selected within an appropriate range by a person skilled in the art. In addition, in addition to the above-mentioned effective ingredient, it may be administered in combination with other drugs known to have a therapeutic effect on cancer (for example, the above-mentioned natural killer cells) or physiologically active substances, or may be formulated in the form of a combination preparation with other drugs.

A cell of a Lactobacillus plantarum strain, or a culture solution of said strain, can inhibit the proliferation of cancer cells or induce the death of cancer cells, and when administered in combination with immune cells, exhibits a synergistic effect on the inhibition of the proliferation of cancer cells and tumors, and can be usefully used for the prevention, treatment or improvement of cancer in the form of a pharmaceutical composition or health functional food.

Figure 1 is a graph analyzing the phenotype of natural killer cells according to one specific example using a flow cytometer.
Figure 2 is an immunohistochemical staining result showing the degree of activation of natural killer cells following administration of the GB104 strain according to one specific example.
Figure 3 is a graph showing the activation of CD8+ anti-tumor immune T cells and natural killer cells following administration of the GB104 strain according to one specific example.
Figure 4 is a graph showing the results of analyzing the synergistic effect by measuring tumor volume after co-administration of the GB104 strain and natural killer cells in a syngeneic MC-38 mouse colon cancer model.
Figure 5 is a graph showing the results of comparative analysis of tumor volume after single and combined administration of the GB104 strain and natural killer cells in a syngeneic MC-38 mouse colon cancer model.
Figure 6 is a graph showing the tumor growth inhibition effect of combined administration of the GB104 strain and natural killer cells in a xenograft HT-29 model.

Hereinafter, preferred embodiments are presented to help understand the present invention. However, the following embodiments are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following embodiments. The embodiments can be modified in various ways, and the embodiments are not limited to the embodiments disclosed below, but can be implemented in various forms.

Example 1: Lactobacillus plantarum ( Lactobacillus plantarum ) Isolation and identification of strain GB104

Isolation and identification of Lactobacillus plantarum GB104 strain was performed according to the methods described in Korean Patent Application No. 10-2020-0186738 and Korean Patent Application No. 10-2022-0080567, which are incorporated herein by reference in their entirety.

Briefly, Lactobacillus plantarum GB104 was isolated from a vaginal sample of a healthy woman who visited a hospital for the purpose of a health checkup. First, a vaginal sample was collected with a cotton swab and inoculated onto a Rogosa SL (MRS) plate medium, and cultured in an anaerobic chamber at 37°C for 48 h. When bacterial colonies grew, single colonies were subcultured onto new MRS plate medium for pure isolation. After pure isolation, strains were cultured using MRS medium. Next, among the cultured strains, Lactobacillus plantarum with an inhibitory effect on fat cell accumulation and low cytotoxicity was isolated. The GB104 strain was finally selected. In order to identify the Lactobacillus plantarum GB104 strain selected above, the 16S rRNA gene sequence obtained through PCR using primers targeting the 16S rRNA gene was analyzed by Sanger sequencing, and Lactobacillus plantarum The 16S rRNA sequence of GB104 is shown as SEQ ID NO: 1. The present inventors named the GB104 strain as " Lactobacillus plantarum GB104" (Accession No.: KCTC 14107BP) and deposited it in the Korean collection for type cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology on January 14, 2020. In addition, the strain name of Lactobacillus plantarum was changed to Lactiplantibacillus plantarum . In the examples below, the changed strain names of existing strains are described interchangeably.

Example 2: Preparation of mGI101 or GI101

To produce a fusion protein comprising a human or mouse CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide including a base sequence encoding a fusion protein comprising a signal peptide, a CD80 fragment, an Ig hinge linked to a linker, an Fc domain, a linker, and an IL-2 variant having two amino acids substituted (R38A, F42A) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific, and loaded into the pcDNA3_4 vector. The vector was introduced into CHO cells (Expi-CHO ^TM ) to express the fusion protein. After introduction of the vector, the culture was collected after culturing for 7 days in an environment of 37°C, 125 RPM, and _CO2 concentration of 8%, and the formed dimeric fusion protein was purified. In one specific example, the monomers of the dimeric fusion protein named “mGI101” (SEQ ID NO: 6) or “GI101” (SEQ ID NO: 7) are as shown in Table 1.

ingredient
(component) Amino acid sequence
(Amino acid sequence) Sequence number mGI101
Monomer VDEQLSKSVKDKVLLPCRYNSPHEDESEDRIYWQKHDKVVLSVIAGKLKVWPEYKNRTLYDNTTYSLIILGLVLSDRGTYSCVVQKKERGTYEVKHLALVKLSIKADFSTPNITESGNPSADTKRITCFASGGFPKPRFSWLENGRELPGINTTISQDPESELYTISSQLDFNTTRNHTIKCLIKYGDAHVSEDFTWEKPPED PPDSGSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGGGGGSAPTSSSTKKTQLQLEHLLLDLQMIL NGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 6 GI101 monomer
(monomer) VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNG SGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGGGGGSAPTSSSTKKTQLQLEHLLLDLQMIL NGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 7

Example 3 Preparation of mouse natural killer cells

Example 3-1. Preparation of mouse bone marrow

To obtain mouse-derived natural killer cells (NK cells), the femur and spleen were extracted from c57BL/6 mice (Orient Bio). The extracted femur was placed in 70% ethanol after removing as much fat and muscle from the surrounding area as possible, and then placed in a 50 mL conical tube containing 5 mL of PBS and stored on ice. The extracted spleen was transferred to a 50 mL conical tube containing PBS and stored on ice. A new 50 mL conical tube was pre-filled with 5 mL of FACS buffer A, which is composed as described in Table 2 below, and 70 μm strainers were placed over it to prepare the femur and spleen, respectively.

For 500 mL Volume Final Concentration FBS 15 ml 3% 500 mM EDTA 10 ml 10 mM 1M HEPES 10 ml 20 mM 25 mg/ml PolymyxinB 200μl 10 μg/ml 10,000 U/ml Penicillin &
10,000 μg/ml Streptomycin 5 ml 100 U/ml Penicillin &100 μg/ml Streptomycin 100 mM Sodium Pyruvate 5 ml 1 mM PBS 454.8 ml

Cut both sides of the femur bone on the 70 μm strainer prepared above, insert a 1 mL needle into the bone hole of a syringe filled with 10 mL of FACS buffer A, flow FACS buffer A, and then flow FACS buffer A into the cut tissue portion to obtain bone marrow. After obtaining bone marrow, centrifuge at 1,300 rpm at 4°C for 10 minutes, remove the supernatant, add 3 mL of ACK lysis buffer (Lonza, # BP10-548E), mix with the cell pellet, and leave at room temperature for 5 minutes. Add 47 mL of FACS buffer A and centrifuge at 1,300 rpm at 4°C for 10 minutes. After that, remove the supernatant, dissolve in 1 mL of FACS buffer A, and count the cells. The spleen was placed on a 70 μm strainer and ground, and FACS buffer A was poured over the tissue to obtain cells. After cell harvesting, centrifuge at 1,300 rpm at 4°C for 10 minutes, remove the supernatant, add 3 mL of ACK lysis buffer (Lonza, # BP10-548E), mix with the cell pellet, and leave at room temperature for 5 minutes. Add 47 mL of FACS buffer A and centrifuge at 1,300 rpm at 4°C for 10 minutes. After that, the supernatant was removed, and the cells were counted after being dissolved in 1 mL of FACS buffer A.

Example 3-2: Isolation and culture of natural killer cells

In Example 3-1, only NK cells (Natural killer cells, NK cells) were isolated from the bone marrow cells obtained using the NK cell isolation Kit, mouse (Miltenyi biotec, #130-115-818), and then centrifuged at 300×g at 4°C and the supernatant was removed.

40 μL of MACS buffer per 10 ⁷ cells was added to loosen the cell pellet, and 10 μL of NK Cell Biotin-Antibody Cocktail per 10 ⁷ cells was added. After incubation at 4°C for 5 minutes, 2 mL of MACS buffer per 10 ⁷ cells was added, centrifuged at 300×g at 4°C, and the supernatant was removed. 2 mL of MACS buffer per 10 ⁷ cells was added to wash, centrifuged at 300×g at 4°C, and the supernatant was removed.

After adding 80 μL of MACS buffer per 10 ⁷ cells, 20 μL of anti-biotin microbeads per 10 ⁷ cells were dispensed and incubated on ice for 10 minutes. 500 μL of cells mixed with anti-biotin microbeads were flowed onto the LS column and the supernatant passing through the column was obtained. The same process as above was repeated, centrifuged at 300×g at 4°C, and the supernatant was removed.

As described in Table 3 below, cells were counted by dissolving them in 1 mL of GC-RPMI medium. According to the measured number of NK cells, 1×10 ⁶ cells/mL were seeded in the GC-RPMI medium, and then mGI101 prepared by the method of Example 2 was treated at a concentration of 100 nM/mL and cultured for 7 days.

For 500 mL Volume Final Concentration FBS 50 ml 10% 1M HEPES 5 ml 20 mM 10,000 U/ml Penicillin &
10,000 μg/ml Streptomycin 5 ml 100 U/ml Penicillin &100 μg/ml Streptomycin 50 mg/ml Gentamicin 0.5 ml 50 μg/ml 100 mM Sodium Pyruvate 5 ml 1 mM 55 mM 2-Mercaptoethanol 0.5 ml 55 μM 100 mM NEAA 5 ml 1 mM 200 mM L-Glutamine 5 ml 2 mM RPMI 424 ml

Example 3-3. Confirmation of purity of natural killer cells

In order to analyze the purity and activation of natural killer cells isolated and cultured in Example 3-2 and the secretion ability of granzyme, perforin, and interferon gamma, flow cytometry was used.

Specifically, natural killer cells were centrifuged at 300×g for 10 minutes, and the supernatant was removed. Thereafter, 1 mL of FACS buffer prepared by adding 3% (v/v) FBS, 10 mM EDTA, 20 mM HEPES, 10 μg/mL PolymyxinB, 100 U/mL Penicillin, 100 μg/mL Streptomycin, and 1 mM Sodium pyruvate to PBS was added, and the cell pellet was resuspended. Thereafter, the cell pellet was diluted with FACS buffer to 2× ¹⁰⁶ cells/mL using a cell counter. 100 ㎕ of the diluted cell solution was added to each 96-well plate, and 100 ㎕ of FACS buffer was added, followed by centrifugation at 300×g for 10 minutes. The supernatant was removed, and 100 ㎕ of FACS buffer was additionally added, and to confirm the purity, FITC-labeled anti-mouse CD3 antibody (FITC mouse anti-CD3 (Clone 17A2)) and APC-labeled anti-mouse NK1.1 antibody (APC mouse anti-NK1.1 (Clone PK136)) described in Table 4 below were treated. After reacting for 20 minutes at 4℃, 100 ㎕ FACS buffer was added, and centrifugation was performed for 5 minutes under 300×g conditions. The supernatant was removed, and 200 ㎕ FACS buffer was added, suspended, and the purity and phenotype of the cells were confirmed using a flow cytometer.

As a result, as shown in Figure 1, the ratio (purity) of the separated natural killer cells was confirmed to be 98.5%.

Target Color Clone Producer Cat# Purity
Marker CD3 FITC 17A2 Biolegend 100204 NK1.1 APC PK136 Biolegend 108710

Example 3-4. Confirmation of natural killer cell activation

In order to confirm whether the natural killer cells isolated and cultured in the above Example 3-2 were activated, the activity marker of the natural killer cells was analyzed. The natural killer cells were centrifuged at 300×g for 10 minutes, and the supernatant was removed. Thereafter, 1 mL of FACS buffer prepared by adding 3% (v/v) FBS, 10 mM EDTA, 20 mM HEPES, 10 μg/mL PolymyxinB, 100 U/mL Penicillin, 100 μg/mL Streptomycin, and 1 mM Sodium pyruvate to PBS was added, and the cell pellet was resuspended. Thereafter, the cell pellet was diluted with FACS buffer to 2× ¹⁰⁶ cells/mL using a cell counter. 100 ㎕ of the diluted cell solution was added to each 96-well plate, and 100 ㎕ of FACS buffer was added, followed by centrifugation at 300×g for 10 minutes. The supernatant was removed, and 100 μl of FACS buffer was additionally added, and for activation analysis, as described in Table 5 below, antibodies BV510-labeled anti-mouse CD16 antibody (BV510 mouse anti-CD16 (Clone Ab9)), PE/cy7-labeled anti-mouse NKp46 antibody (PE/cy7 mouse anti-NKp46 (Clone 27A1.4)), PE-labeled anti-mouse NKG2D antibody (PE mouse anti-NKG2D (Clone CX5)), PerCP/CY5.5-labeled anti-mouse CD244 antibody (PerCP/CY5.5 mouse anti-CD244 (Clone m2B4(B6)458.1)), BV421-labeled anti-mouse CCR5 antibody (BV421 mouse anti-CCR5 (Clone C34-3448)), and BV711-labeled anti-mouse CXCR4 antibody (BV711 mouse anti-CXCR4 (Clone L276F12)) was treated. After reacting for 20 minutes at 4℃, 100 ㎕ FACS buffer was added, and centrifuged for 5 minutes under 300×g conditions. The supernatant was removed, 200 ㎕ FACS buffer was added, and the suspension was performed, and the purity and phenotype of the cells were confirmed using a flow cytometer.

As a result, as shown in Fig. 1, the ratio of natural killer cells expressing CD16 among cultured natural killer cells was 92.3%, the ratio of natural killer cells expressing NKp46 was 96.8%, the ratio of natural killer cells expressing NKG2D was 99.0%, the ratio of natural killer cells expressing CD244 was 98.3%, the ratio of natural killer cells expressing CCR5 was 89.7%, and the ratio of natural killer cells expressing CXCR4 was 95.4%.

Target Color Clone Producer Cat# Activation
Marker CD16/CD32 BV510 Ab9 BD 751688 NKp46 PE/CY7 29A1.4 Biolegend 137618 NKG2D PE CX5 Biolegend 130208 CD244 PerCP/cy5.5 m2B4 (B6)458.1 Biolegend 133513 CCR5 BV421 C34-3448 BD 743695 CXCR4 BV711 L276F12 Biolegend 146517

Example 3-5. Confirmation of granzyme B and perforin secretion ability of natural killer cells

To confirm the secretion ability of granzyme, perforin, and interferon gamma of natural killer cells isolated and cultured in Example 3-2 above, the expression levels of granzyme B, perforin, and interferon gamma in natural killer cells were measured through intracellular staining.

Natural killer cells were centrifuged at 300×g for 10 minutes, and the supernatant was removed. Thereafter, 1 mL of FACS buffer prepared by adding 3% (v/v) FBS, 10 mM EDTA, 20 mM HEPES, 10 μg/mL PolymyxinB, 100 U/mL Penicillin, 100 μg/mL Streptomycin, and 1 mM Sodium pyruvate to PBS was added, and the cell pellet was resuspended. Then, the cell pellet was diluted with FACS buffer to 2× ¹⁰⁶ cells/mL using a cell counter. 100 ㎕ of the diluted cell solution was added to each 96-well plate, and 100 ㎕ of FACS buffer was added, followed by centrifugation at 300×g for 10 minutes. 200 ㎕ BD Cytofix/Cytoperm?? was added for fixation and permeation. After suspension with buffer (perm/fixation buffer), the mixture was reacted at 4°C for 30 minutes. 100 ㎕ of FACS buffer was additionally added, and centrifuged at 300×g for 5 minutes. The PE/cy7-labeled anti-mouse Granzyme B antibody (PE/cy7 anti-mouse Granzyme B (Clone NGZB)), PE-labeled anti-mouse perforin antibody (PE anti-mouse Perforin (Clone S16009A)), and FITC-labeled anti-mouse interferon γ antibody (FITC anti-mouse Interferon γ (Clone XMG1.2)) described in Table 6 below were treated. After reacting at 4°C for 20 minutes, 100 ㎕ FACS buffer was added, and centrifuged at 300×g for 5 minutes. The supernatant was removed, 200 ㎕ of FACS buffer (fixation buffer) was added, and the cell expression level was confirmed using a flow cytometer.

As a result, as shown in Fig. 1, the proportion of natural killer cells containing perforin among the cultured natural killer cells was confirmed to be 96.2%, the proportion of natural killer cells containing granzyme B was confirmed to be 95.7%, and the proportion of natural killer cells containing interferon gamma was confirmed to be 91.3%.

Target Color Clone Producer Cat# Cytotoxicity
marker IFN-γ FITC XMG1.2 Invitrogen 11-7311-82 Perforin PE S16009A Biolegend 154306 GranzymeB PE/cy7 NGZB Invitrogen 25-8898-82

Example 4 Preparation of human natural killer cells

Example 4-1: Obtaining CD3-CD56+ cells

CliniMACS buffer (pH 7.2) was prepared by adding 0.5% (v/v) HSA (human serum albumin) and 1 mM EDTA to CliniMACS PBS/EDTA buffer (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat#: 200-070-029). Leukopak, Reagent (CD3, CD56), infusion solution, rinsing solution, and CliniMACS buffer were connected to the CliniMACS Prodigy device (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat#200-075-301). NK cells were obtained from Leukopak (Ewha Womans University Mokdong Hospital) by running the LP-3-56-separation program.

Example 4-2: Culturing of human natural killer cells

NK cells (CD3-CD56+) isolated in the above Example 4-1 were seeded in a culture bag at ^1x106 cells/mL. NK cell stimulating beads (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat# 130-094-483) were added only at the time of the initial seeding, and the cell number was checked at 2-day intervals from the 6th day after seeding. The medium in Table 4 below was added to maintain a concentration of ^1x106 cells/mL, and the cells were cultured under conditions of 37.0℃ and 5% CO2 until the total cell number became ^12.6x108 cells. The cultured cells were then inoculated into a bioreactor. The medium in Table 7 below was added at a concentration of ^0.7x106 cells/mL, and the culture process was managed by measuring pH, pO2, pCO2, cell metabolites, osmotic pressure, etc. every day. The medium in Table 4 was added so that the concentration was ^0.7x106 cells/mL, and when the culture medium reached 50 L, the culture was performed until the total cell number was ^1.0x1011 cells or more, regardless of the cell concentration.

ingredient
(component) Final content
(final concentration) NK MACS medium - NK MACS supplement 1% Human AB serum 5% GI101 50nM Poloxamer 188 1g/L

Example 4-3: Cell recovery and freezing

The cells cultured in Example 4-2 above were harvested using a Unifuge device (CARR biosystems). 50 L of the culture medium was concentrated to 3 L containing cells and washing buffer. The concentrated cell suspension was concentrated to 500 mL using a LOVO device (Fresenius Kabi). After obtaining the concentrated cells, the freezing formulations in Table 8 below were added according to the composition, and filled into AT vials at a concentration of ^1x108 cells/mL using an M1 device (Aseptic Technologies). The AT vials filled with cells were placed in a controlled rate freezer (CRF, Thermofisher) and frozen. The frozen cells were transferred to a liquid nitrogen tank at -160°C or lower and stored.

ingredient
(component) Final content
(final concentration) Plasma Solution A 72.75% PEG 2.25% 20% Human Serum Albumin 20% DMSO 5%

Experimental Example 1 In a homologous MC-38 colon cancer model L. Plantarum CD8 of GB104 strain ⁺ Confirmation of activation of antitumor immune T cells and natural killer cells

In a colon cancer MC-38 syngeneic transplantation model, we confirmed an increase in immune cells involved in anti-tumor immunity by administration of L. Plantarum GB104 strain.

Specifically, 6-week-old c57BL/6 mice were introduced, and after a 1-week acclimatization period, the right flank area was shaved and the experiment was conducted at 7 weeks of age. The tumor model was established by subcutaneously injecting 2 x 10 ⁵ cells per mouse in 100 μL of the c57BL/6-derived colon cancer cell line MC-38 into the right flank of the mouse. On the 5th day after tumor cell injection, only mice with tumor sizes within a certain range were selected and classified so that each group had the same average tumor size. Then, the L. Plantarum GB104 strain was orally administered to the animal model at 1 x 10 ⁹ CFU per mouse every day from the 6th day until just before the end of the test.

Afterwards, on the 20th day, the activation of CD8 ⁺ antitumor immune T cells and natural killer cells following administration of the GB104 strain was confirmed, and the results are shown in Figures 2 and 3.

Figure 2 is an immunohistochemical staining result showing the degree of activation of natural killer cells according to administration of the GB104 strain according to one specific example.

Figure 3 is a graph showing the activation of CD8+ anti-tumor immune T cells and natural killer cells following administration of the GB104 strain according to one specific example.

As shown in Figures 2 and 3, it was confirmed that the administration of the GB104 strain significantly increased the activation (cell number) of natural killer cells and T cells in tumor cells. These results imply that the GB104 strain can increase anticancer efficacy when used in combination with immune cells (natural killer cells and T cells).

Experimental example 2: In a homologous MC-38 colon cancer model L. Plantarum Confirmation of tumor growth inhibition effect by combined administration of GB104 strain and natural killer cells

The tumor-therapeutic effect of combined administration of L. Plantarum GB104 strain and natural killer cells was confirmed in the MC-38 syngeneic colon cancer model.

Specifically, 5-week-old c57BL/6 mice were introduced, and after a 1-week acclimatization period, the right flank area was shaved and the experiment was conducted at 6 weeks of age. The tumor model was established by subcutaneously injecting the c57BL/6-derived colon cancer cell line MC-38 into the right flank of the mice at 2 x 10 ⁵ cells/100 μL per mouse. The tumor size was measured using a digital calliper and calculated by the formula Tumor volume (mm ³ ) = (width ² x length)/2. On the 5th day after tumor cell injection, only mice with tumor sizes within the range of 10-30 mm ³ were selected, and each group was randomly set up. The L. Plantarum GB104 strain was orally administered to the animal model at 2 x 10 ⁹ CFU per mouse every day from the 5th day until just before the end of the test. Natural killer cells were isolated and cultured from the femur and spleen of c57BL/6 mice using the method of Example 3, and ^5x106 cells/200 μL were intravenously injected twice on days 8 and 15 after tumor cell injection. The results of the co-administration of GB104 and natural killer cells are shown in Figure 4.

Figure 4 is a graph showing the results of analyzing the synergistic effect by measuring tumor volume after co-administration of the GB104 strain and natural killer cells in an MC-38 mouse colon cancer model that was transplanted.

Figure 5 is a graph showing the results of comparative analysis of tumor volume after single and combined administration of the GB104 strain and natural killer cells in an MC-38 mouse colon cancer model that was transplanted.

As shown in Figures 4 and 5, compared to the negative control group, the group administered L. Plantarum GB104 and natural killer cells together showed a significant decrease in tumor size due to synergy compared to the group administered natural killer cells alone, confirming the antitumor enhancement efficacy.

Experimental Example 3: In a xenografted HT-29 colon cancer model L. Plantarum Confirmation of tumor growth inhibition effect by combined administration of GB104 strain and natural killer cells

The tumor treatment effect of combined administration of L. Plantarum GB104 strain and natural killer cells was confirmed in a colorectal cancer HT-29 xenograft model using immunodeficient mice. 6-week-old NOG mice were introduced, and after a 1-week acclimatization period, the hair around the right flank was removed and the experiment was conducted at 7 weeks of age. The tumor model was established by subcutaneously injecting HT-29, a human-derived colorectal cancer cell line, into the right flank of the mouse at 5 x 10 ⁶ cells/100 μL per mouse. The tumor size was measured using a digital calliper and calculated by the formula Tumor volume (mm ³ ) = (width ² x length)/2. On the 4th day after tumor cell injection, mice were randomly selected so that the average tumor size of each group was within the range of 100-110 mm ³ . Then, L. Plantarum GB104 strain was orally administered to the animal model at 1x10 ⁹ CFU per mouse every day from the 4th day until just before the end of the test. Natural killer cells manufactured by the method of Example 4 were intravenously injected six times in total at 1x10 ⁷ cells/200 μL on days 4, 7, 11, 14, 18, and 21 after tumor cell injection.

Figure 6 is a graph showing the tumor growth inhibition effect of combined administration of the GB104 strain and natural killer cells in a xenograft HT-29 model.

As shown in Fig. 6, in a tumor model transplanted with HT-29 cell lines expressing KRAS wild-type, BRAF ^V600E , and p53 ^R273H mutant genes, the tumor size was reduced more significantly in the group administered L. Plantarum GB104 and natural killer cells in combination than in the group administered L. Plantarum GB104 or natural killer cells alone, compared to the negative control group, confirming the enhanced antitumor efficacy of combined administration.

Meanwhile, it was confirmed that the group co-administered with L. Plantarum GB104 and natural killer cells showed an enhanced antitumor effect in the human-derived colon cancer cell line HT-29 and the human-derived colon cancer cell line HCT116, which show different patterns of expression of some genes important for the pathogenesis of colon cancer.

Specifically, HCT116 cells express KRAS ^G13D mutant, BRAF and p53 wild-type genes, while HT-29 cells express KRAS wild-type, BRAF ^V600E and p53 ^R273H mutant genes. Therefore, even if they are the same colon cancer cell lines, the difference in drug response may vary for each cell depending on the difference in the expression pattern of some genes.

Except that the tumor model was an HCT116 cell line transplanted tumor model, the antitumor effect of combined administration of L. Plantarum GB104 and natural killer cells was confirmed in a tumor model transplanted with an HCT116 cell line expressing KRAS ^G13D mutation, BRAF, and p53 wild-type genes using the same method as in Experimental Example 3. As a result, compared to the negative control group, the group administered with L. Plantarum GB104 and natural killer cells in combination showed a more significant reduction in tumor size than the group administered L. Plantarum GB104 or natural killer cells alone, confirming the enhanced antitumor efficacy according to combined administration.

This was confirmed in a xenograft tumor model established in highly immunodeficient mice using human-derived colon cancer cell lines HT-29 and HCT116, which have different expression of some genes, respectively, by co-administering the L. Plantarum GB104 strain, which is a specific example of the present invention, and natural killer cells. This means that when the L. Plantarum GB104 strain, which is a specific example of the present invention, and natural killer cells are co-administered, an effective anticancer treatment effect is shown in colon cancer regardless of the difference in gene expression patterns.

Experimental Example 4: L. Plantarum Confirmation of tumor growth inhibition effect by combined administration of GB104 strain and natural killer cells

The tumor treatment effect of combined administration of L. Plantarum GB104 strain and natural killer cells was confirmed in xenograft models of liver cancer, breast cancer, kidney cancer, and lung cancer using immunodeficient mice.

Specifically, the tumor treatment effect by combined administration of L. Plantarum GB104 strain and NK cells was confirmed in the same manner as Experimental Example 4, except that it was performed in a xenograft model of another cancer type.

As a result, in the tumor model transplanted with the liver cancer cell line, colon cancer cell line, breast cancer cell line, renal cancer cell line, and lung cancer cell line, compared to the negative control group, the group administered L. Plantarum GB104 and natural killer cells together showed a more significant decrease in tumor size than the group administered L. Plantarum GB104 or natural killer cells alone, confirming the enhanced antitumor efficacy of combined administration.

This means that when L. Plantarum GB104 strain, which is one specific example of the present invention, is co-administered with natural killer cells, it exhibits an effective anticancer treatment effect on liver cancer, breast cancer, kidney cancer, and lung cancer.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC14107BP 20200114

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Claims (18)

A first active substance comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof, and a second active substance comprising immune cells as active ingredients; or
A pharmaceutical composition for preventing or treating cancer, comprising the first active substance or the second active substance as an effective ingredient, wherein the first active substance or the second active substance is administered in combination with the second active substance or the first active substance, respectively. A pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the first active ingredient induces the activity of immune cells. A pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the strain is a strain deposited under the accession number KCTC14107BP. A pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the strain comprises a mutation of a naturally occurring Lactobacillus plantarum strain. A pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the immune cells are cultured in the presence of a fusion protein comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof. A pharmaceutical composition for preventing or treating cancer, wherein the immune cells of claim 1 are natural killer cells (NK cells). A pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the first active ingredient and the second active ingredient are administered simultaneously, sequentially or in reverse order. A pharmaceutical composition for preventing or improving cancer according to claim 1, wherein the first active ingredient is administered orally and the second active ingredient is administered parenterally. A pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the first active ingredient is administered orally and the second active ingredient is administered intravenously or subcutaneously. A pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, salivary gland cancer, melanoma, bladder cancer, kidney cancer, blood cancer, esophagus cancer, head and neck cancer, skin cancer, small intestine cancer, anal cancer, colon cancer, rectal cancer, and lymphoma. A pharmaceutical composition for preventing or treating cancer according to claim 10, wherein the colon cancer occurs in any one site selected from the group consisting of the ascending colon, transverse colon, descending colon, sigmoid colon, and rectal mucosa. A health functional food for preventing or improving cancer, comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient, wherein the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof is administered in combination with immune cells. A health functional food for preventing or improving cancer, wherein the strain according to claim 12 is a strain deposited under the deposit number KCTC14107BP. A health functional food for preventing or improving cancer according to claim 12, wherein the immune cells are natural killer cells (NK cells). A health functional food for preventing or improving cancer according to claim 12, wherein the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof and immune cells are administered orally, intravenously, intraperitoneally, or subcutaneously. A pharmaceutical composition for preventing or improving cancer according to claim 12, wherein the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof is administered orally, and the immune cells are administered parenterally. A health functional food for preventing or improving cancer according to claim 12, wherein the Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof is administered orally, and the immune cells are administered intravenously or subcutaneously. A health functional food for preventing or improving cancer according to claim 12, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, salivary gland cancer, melanoma, bladder cancer, kidney cancer, blood cancer, esophagus cancer, head and neck cancer, skin cancer, small intestine cancer, anal cancer, colon cancer, rectal cancer, and lymphoma.