KR102601831B1 - Exosomes Derived From Canine For Inhibiting Proliferation of Canine Solid Cancer - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cancer in canines containing exosomes derived from canines. The exosomes derived from canines according to the present invention target breast cancer cells and reduce the size of cancer. Because it can effectively reduce it, it can be useful for preventing or treating cancer in canine animals.

Description

Canine exosomes have an effect of inhibiting the proliferation of solid cancer {Exosomes Derived From Canine For Inhibiting Proliferation of Canine Solid Cancer}

The present invention relates to a composition for preventing or treating cancer in canine animals containing exosomes derived from canine animals.

Currently, adult stem cell therapy is being applied clinically in a variety of ways as a new method to treat incurable/incurable diseases, and many successful treatment cases have been reported. However, the problem of zoonosis due to internalization of xenogenic serum (e.g., fetal bovine/calf serum) that may occur during the process of in vitro culturing to proliferate stem cells extracted and selected from donors; When stem cells are transplanted into the body, there are risk factors such as tumor formation problems and vascular occlusion causing infarcts that may occur due to stem cell characteristics such as vigorous proliferation and relatively large cell size. .

In addition, when applying treatment using autologous stem cells, the regenerative treatment efficacy of stem cells, such as the ability to proliferate in vitro, the ability to migrate to lesions, and the ability to secrete therapeutic factors, varies from patient to patient, making the versatility of stem cell therapy difficult. There are limits.

Therefore, ways to avoid various risk factors or problems that may arise from the treatment directly using living stem cells mentioned above are being actively studied, and as part of this, extracellular vesicles (EVs) derived from specific cells are used in stem cells. Research results are emerging that it can replace cell therapy functions.

EVs are substances with a double lipid membrane structure secreted from cells, and depending on their size, they are classified into exosomes (40-200 nm), microvesicles (200-1,000 nm), and large oncosomes (1- 10 μm), of which exosomes are small endoplasmic reticulum about 100 nanometers in size secreted by cells and play a role in representing the nature and state of the cell.

In other words, an exosome is a nano-sized endoplasmic reticulum naturally produced within a cell. It contains proteins and genetic information and transmits various signals, including genetic information, outside the cell to other cells to promote development, proliferation, and differentiation. , immune regulation, angiogenesis, and the progression of various diseases. Exosomes, which are biological nanovesicles, have recently attracted great attention as drug delivery vehicles due to their advantages such as avoidance of immune response, excellent compatibility with the human body, drug loading function, targeted delivery effect to specific cells, and stability in the blood. is receiving.

In addition, the contents (protein, mRNA, miRNA) that exosomes package vary depending on the characteristics of the origin cell, and the fate of the target cell varies depending on the contents of the exosome after moving to the target cell. . In fact, there are reports from several research groups that surface proteins, internal proteins, and RNAs of exosomes move to target cells and are reprogrammed.

Although it is not clear by what molecular mechanism these exosomes are created, not only red blood cells, but also various types of immune cells including B-lymphocytes, T-lymphocytes, dendritic cells, platelets, macrophages, tumor cells, Stem cells are also known to produce and secrete exosomes while alive. In particular, exosomes derived from stem cells are known to play a role in intercellular communication because they contain nuclear components as well as receptors and proteins.

In addition, exosomes derived from stem cells contain relatively less animal serum than stem cells, so the risk of symptoms (zoonosis) caused by infection with animal serum can also be ruled out.

Accordingly, as an alternative to direct injection/transplantation of living stem cells, treatment using extracellular vesicles derived from specific cells, such as stem cells, has emerged and is showing efficacy in many preclinical tests and is in the clinical stage for the treatment of several diseases. There are also reports of cases of entry into .

However, these studies using exosomes have not been applied to animal tumor research.

Animal models are gaining importance in regenerative medicine for healing and restoring function of damaged or diseased tissues. Among these, more than 370 types of genetic diseases have currently been reported in canines, and most of them resemble diseases and dysfunctions that occur in humans. Therefore, the importance of canines as an animal model for studying the mechanisms and pathogenesis of human genetic diseases, especially complex inherited rare recessive diseases such as X-linked severe combined immunodeficiency and Duchenne muscular dystrophy, which are difficult to study directly in humans, is increasing. there is. In this way, canines are a useful animal model not only for solid organ transplantation, but also for studying human pathological mechanisms such as cancer, cardiovascular disease, bone regeneration, diabetes, leukemia, and spinal cord injury, and for testing new treatments. In addition, it is an ideal large animal model for studying various treatment methods such as stem cell transplantation and gene therapy.

In addition, as the incidence of incurable diseases such as cancer in dogs is significantly increasing, the development of new treatments for these incurable diseases is also very necessary.

Because of these biological similarities, cancer research in dogs provides valuable information that differentiates it from cancer research targeting only humans or laboratory animals, and recognition of this value is supported by studies to identify cancer-related genes, environmental risk factors, Research areas such as understanding the biological characteristics of cancer and, in particular, developing and evaluating the efficacy of new cancer treatments, are increasing significantly.

However, there are still no reports related to cancer treatment using this in dogs worldwide.

Therefore, isolating and characterizing specific cells from various tissues of dogs is a major task in the field of cell research. Stem cells derived from humans and mice have been attempted in various ways in the prior art, but in the case of canines, although they are useful large animal models for research on human diseases as described above, research is still underdeveloped. Isolation of stem cells from adipose tissue, bone marrow, umbilical cord blood, etc. is possible, but the yield is limited, and the method of obtaining adipose tissue or bone marrow from an individual is invasive and causes pain.

Meanwhile, hematopoietic stem cells, one of the adult stem cells, are cells that can differentiate into all cells that make up blood (red blood cells, white blood cells, platelets, and lymphocytes), and are mainly derived from hematopoietic stem cells in the bone marrow. The cells that make up the immune system continuously renew themselves. Among the cells that make up the immune system, in particular, natural killer cells (hereinafter referred to as 'NK cells') have been studied extensively as their ability to non-specifically kill cancer cells and virus-infected cells has been revealed. Defects in differentiation and activity are associated with breast cancer (Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001) and melanoma cancer (Ryuke Y, et al., Melanoma Res., 13: 349- 356, 2003) and lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002). Based on these studies, NK cell therapy, which uses NK cells to treat intractable diseases such as cancer, is emerging.

Natural killer cells (NK cells) are one of the innate immune cells and are cells that exhibit selective cytotoxicity against cancer cells. Unlike other immune cells, natural killer cells can immediately detect and immediately eliminate cancer cells. This is because they can distinguish between cancer cells and normal cells through various immune receptors present on the surface of natural killer cells. In many cancer patients, defects in the number or anticancer activity of natural killer cells are found, and abnormalities in the function of natural killer cells are known to be closely related to the occurrence of these cancers.

The process by which natural killer cells kill cells involves the release of cytoplasmic granules containing perforin and granzymes, and processes involving FasL and TRAIL. Natural killer cells secrete various cytokines, such as IFN-γ, INF-α, GM-CSF, and IL-10, and express several receptors on the cell surface. These receptors are responsible for cell adhesion, activation of cell killing ability, or cell death. Involved in suppressing killing ability.

Therefore, “tumor suppression using exosomes,” which has never been applied to animals, can provide important basic information for future development of next-generation cell therapy in the field of animal diseases and various disease-related mediation studies, and the characteristics of these exosomes should be taken into consideration. Cell therapy using exosomes isolated from adult stem cells is expected to become a new paradigm that can not only safely and effectively treat cancer, but also overcome the limitations of existing stem cell therapy.

1. Republic of Korea Patent No. 10-1762833

Under these circumstances, the present inventors have diligently researched effective and safe treatments for treating cancer in canine animals (particularly dogs). As a result, the present inventors isolated exosomes, which are extracellular vesicles, from canine adipose tissue-derived mesenchymal stem cells, which are adult stem cells of dogs; Exosomes, which are extracellular vesicles, were isolated from canine peripheral blood mononuclear cells-derived natural killer cells (NK cells) and their characteristics were analyzed. By demonstrating that when MSC-derived exosomes and NK cell-derived exosomes are applied to a mammary tumor model, an anti-tumor effect on mammary tumors appears without weight loss, the present invention provides the potential as a safe and effective cancer treatment. Completed.

Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer in canines, which contains exosomes derived from canines as an active ingredient.

In addition, another object of the present invention is to provide a feed composition for preventing or improving cancer in canines, which contains exosomes derived from canines as an active ingredient.

In addition, another object of the present invention is to provide a method for preventing or treating cancer in canines, comprising administering to the canine a composition containing exosomes derived from canines as an active ingredient. There is something to do.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

The terms used herein are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer in canines, comprising exosomes derived from canines as an active ingredient.

The main feature of the present invention is that exosomes derived from canines exert an anti-tumor effect that suppresses cancer size, thereby achieving a therapeutic effect on cancer diseases.

The term "exosome" of the present invention refers to a small vesicle with a membrane structure secreted from various cells, and refers to a vesicle that is released into the extracellular environment after fusion of a multivesicular body and the plasma membrane. The exosome may have a diameter of 40 to 200 nm, but is not limited thereto as long as the exosome of the present invention exerts the intended effect.

In the present invention, cells donating exosomes are not limited as long as they are eukaryotic cells.

In the present invention, the cell donating the exosome is not limited as long as it is a eukaryotic cell, and the exosome may be naturally secreted from a specific cell by culturing the cell, and may be isolated, extracted, and/or concentrated, and the specific cell It may include adult stem cells or natural killer cells (NK cells).

As used herein, the term “stem cell” refers to an undifferentiated cell that has the ability to self-replicate and differentiate into two or more different types of cells. The stem cells of the present invention may be autologous or allogeneic-derived stem cells, and may be derived from any type of animal, including humans and non-human mammals, and are not limited to whether the stem cells are derived from adults or embryos. No.

Stem cells of the present invention may include embryonic stem cells or adult stem cells, and are preferably adult stem cells.

The adult stem cells include canine tissue-derived mesenchymal stem cells (Mesenchymal Stem Cells, MSC), canine tissue-derived hematopoietic stem cells (HSC), and canine tissue-derived mesenchymal stromal cells. , may be multipotent stem cells or amniotic epithelial cells, preferably canine tissue-derived mesenchymal stem cells or canine tissue-derived hematopoietic stem cells, but are not limited thereto.

The mesenchymal stem cells may be mesenchymal stem cells derived from one or more types selected from the group consisting of adipose tissue, blood, brain, umbilical cord, cord blood, bone marrow, muscle, nerve, skin, amniotic membrane, and placenta of canine animals. It is not limited to this.

In the present invention, preferably, the adult stem cells are canine adipose tissue-derived mesenchymal stem cells, which are mesenchymal stem cells (MSCs) isolated from adipose tissue of canine animals. am.

In the present invention, the NK cells can be isolated from one or more types selected from the group consisting of peripheral blood, bone marrow, lymph nodes, spleen, lung, and liver of canine animals, preferably from peripheral blood.

According to one embodiment of the present invention, the NK cells of the present invention are derived from peripheral blood mononuclear cells (PBMC) isolated from the peripheral blood of canine animals.

In the present invention, 'Peripheral Blood Mononuclear Cell (PBMC)' refers to mononuclear cells present in peripheral blood, such as B cells, T cells, Macrophage, Dendritic cells, and NK cells (natural killer cells). It contains granulocytes such as immune cells, basophils, eosinophils, and neutrophils. The PBMC can be separated by conventional manufacturing methods, for example, specific gravity centrifugation using Ficoll-Paque (Blood, 1998, 92: 2989-93, etc.).

As used herein, “canine” refers to an omnivorous animal such as a dog, wolf, fox, coyote, jackal, or wild cat, and refers to a bipedal animal that walks on its toes.

The canids are largely divided into the dog family and the fox family. The dog family includes dogs, maned wolves, striped jackals, black-backed jackals, dingoes, red wolves, Ethiopian wolves, Indian wolves, coyotes, golden jackals, crab-eating foxes, wild dogs, jackals, African wild dogs, small-eared dogs, Falkland wolves, Includes Andean fox, Darwin's fox, Argentine gray fox, Pampas fox, Peruvian desert fox, and white fox.

The fox family includes the Arctic fox, red fox, swift fox, kit fox, Cossack fox, Cape fox, black-tailed sand fox, Bengal fox, Tibetan sand fox, Blandford fox, white-tailed sand fox, Fennec fox, gray fox, and island fox. , Cozumel fox, big-eared fox, and raccoon dog.

As long as the exosome of the present invention is effective, the subject of the canine animal is not limited thereto, but the canine animal in the present invention preferably includes dogs, for example, purebred and/or mixed breed companion dogs ( companion dog, show dog, working dog, herding dog, hunting dog, guard dog, police dog, racing dog and/or It also includes dogs such as laboratory dogs, especially breeding dogs.

According to a preferred embodiment of the present invention, the exosome of the present invention is (i) an exosome (MSC-EXO) isolated from mesenchymal stem cells (MSC) derived from canine adipose tissue; or

(ii) It is an exosome (NK-EXO) isolated from natural killer cells (NK cells) derived from dog peripheral blood mononuclear cells (PBMCs).

According to the present invention, the cancers include breast cancer, squamous cell cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, At least one type selected from the group consisting of ovarian cancer, bile duct cancer, and gallbladder cancer, and preferably breast cancer.

In one embodiment of the present invention, the exosome of the present invention reduces the size of cancer while maintaining the target's normal weight, which means that the exosome of the present invention targets only cancer cells and does not affect other normal cells. It suggests that it is safe and has excellent anti-cancer effects.

Additionally, the pharmaceutical composition according to the present invention may additionally include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in preparation and includes, but is limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. If necessary, other common additives such as antioxidants and buffers may be added. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, the formulations can be preferably formulated according to each ingredient. The pharmaceutical composition of the present invention is not particularly limited in formulation, but can be formulated as an injection, inhalation agent, topical skin agent, or oral ingestion agent.

The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, applied to the skin, nasal cavity, or respiratory tract) according to the desired method, and the dosage is determined by the patient's condition, weight, and disease. It varies depending on the degree, drug form, administration route and time, but can be appropriately selected by a person skilled in the art.

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

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the target canine animal, and is generally administered at 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg every day or every other day. Alternatively, it can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The composition of the present invention may further include an appropriate carrier commonly used in the preparation of pharmaceutical compositions. For example, in the case of injections, it may additionally contain a preservative, analgesic agent, solubilizer or stabilizer, and in the case of preparations for topical administration, it may additionally contain a base, excipient, lubricant or preservative.

The composition of the present invention can be administered to an individual by various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection, preferably by direct engraftment or intravenous injection in the subject in need of treatment. Transplantation is possible, but is not limited to this.

The composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers to treat cancer.

In addition, according to another aspect of the present invention, the present invention provides a feed composition for preventing or improving cancer in canines containing exosomes derived from canines as an active ingredient.

Additionally, the feed composition can be used as an additional additive to feed.

As used herein, the term “improvement” means any action that reduces at least the severity of a parameter, such as a symptom, related to the condition being treated.

The feed composition of the present invention can be used by adding the active ingredient directly to the feed or with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use. Generally, when producing food or beverages, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

The feed composition of the present invention has no particular restrictions on other ingredients other than containing the above-mentioned effective ingredients as essential ingredients in the indicated proportions, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the feed composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and It may contain its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients can be used independently or in combination. The proportions of these additives can also be appropriately selected by those skilled in the art.

In addition, according to another aspect of the present invention, a method for preventing or treating cancer in a canine animal, comprising administering to the canine animal a composition containing exosomes derived from canines as an active ingredient. provides.

The composition containing the above-described exosome derived from canine animals as an active ingredient refers to a cell therapy product for canine animals manufactured by isolating exosomes derived from canine animals. .

The term "cell therapy product" in the present invention refers to a medicine (US FDA regulations) used for the purpose of treatment, diagnosis, and prevention using cells and tissues manufactured through isolation, culture, and special manipulation from the subject, and is used to enhance the function of cells or tissues. It refers to a medicine used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro in order to restore them, or changing the biological characteristics of the cells by other methods.

Preferably, the cell therapy agent may be a cell therapy agent for treating cancer in canine animals.

Accordingly, a method of treating a canine animal is provided, comprising administering the cell therapeutic agent of the present invention to an individual in need thereof.

The term "treatment" in the present invention refers to any action in which the symptoms of a canine disease, such as a disease induced by cancer cells, are improved or beneficially changed by administration of the composition of the present invention.

Since the method of the present invention uses a composition containing the exosome of the present invention as an active ingredient, duplicate content is omitted to avoid excessive complexity of the present specification.

Exosomes derived from canines according to the present invention can target cancer cells and effectively reduce the size of cancer, so they can be usefully used for the prevention or treatment of cancer in canines.

Figure 1a schematically shows the isolation process of dog tissue-derived exosomes of the present invention. Figure 1b shows the results of electron microscopy and nanosite analysis of dog tissue-derived exosomes of the present invention. Figure 1c shows the results of the nanoparticle tracking system analysis of dog tissue-derived exosomes of the present invention. Figure 1d shows the results of confirming the expression of exosome markers such as Flotillin-1, CD63, CD81, Alix, HSP70, and TSG101 through Western blot in dog tissue-derived exosomes of the present invention.
Figure 2a shows an image of the anti-tumor effect when the mammary tumor cell line REM134 is treated with canine natural killer cell-derived exosomes (NK-EXO) of the present invention, and Figure 2b is a graph of this. Figure 2c is a graph showing the results of treating only the control mammary tumor cell line REM134. Figure 2d shows the results of measuring the weight of the mouse each week.
Figure 3a shows an image of the anti-tumor effect when the mammary tumor cell line REM134 is treated with canine adult stem cell-derived exosomes (MSC-EXO) of the present invention, and Figure 3b is a graph of this. Figure 3c is a graph showing the results of treating only the control mammary tumor cell line REM134. Figure 3d shows the results of measuring the weight of the mouse each week.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Isolation and characterization of canine tissue-derived exosomes

1-1. Isolation of exosomes (NK-EXO) from natural killer cells (NK cells) derived from canine peripheral blood mononuclear cells (PBMCs)

NK cells express various immune receptors on their surface. Immune receptors are divided into activating receptors and inhibitory receptors, and by regulating these receptors, NK cells distinguish between normal cells and cancer cells.

These NK cells make up about 10 to 15% of peripheral blood lymphocytes in humans, but make up a much smaller proportion, about 5%, in dogs.

Therefore, in order to apply it to dog cancer treatment, the present inventors first isolated and proliferated natural killer cells (NK cells) from dog peripheral blood mononuclear cells (PBMCs), and then amplified them. Exosomes (NK-EXO) were isolated and concentrated from natural killer cells (NK cells) derived from canine peripheral blood mononuclear cells (PBMCs) (Figure 1a).

Briefly:

(1) First, to selectively proliferate NK cells from canine blood-derived peripheral blood mononuclear cells (PBMCs), stain with CD5 (MCA1037PE, Bio-Rad, USA) antibody for 30 minutes, then anti-PE Secondary staining was performed with microbeads (Miltenyi Biotec, USA), and only CD5 negative cells were isolated using the MACS system (Miltenyi Biotec). Six 4Х10 CD5 negative cells (PBMCs-derived NK cells) were cultured at ⁷ -day intervals in culture medium supplemented with 500 U/mL human IL-2, 10 ng/mL human IL-15, and 5 ng/mL canine IL-21. PBMCs-derived NK cells were proliferated by passage and cultured at 37°C and 5% CO ₂ conditions for a total of 21 days.

(2) The proliferated PBMCs-derived NK cells were washed with DPBS supplemented with 1% P/S the next day, then replaced with cell culture medium (20 mL) containing exosome-depleted FBS and cultured for 3 days to form PBMCs- Exosomes (NK-EXO) were isolated from derived NK cells.

(3) Cell culture fluid was collected in a 50 mL tube and centrifuged continuously. Continuous centrifugation was performed at 1,500 g for 3 minutes, 2,000 g for 15 minutes, and 3,000 g for 20 minutes. At this time, the supernatant was transferred to a new tube at each step.

(4) After the final centrifugation, the supernatant was filtered through a 0.22 μm filter.

(5) The supernatant of (4) was concentrated using a 10 kDa concentration filter tube.

After centrifugation at 4,000 g for 15 minutes, the liquid under the tube was discarded, and the liquid remaining in the concentration filter was transferred to a new tube.

(6) The process of (5) above was repeated to concentrate the cell culture medium.

(7) PBS filtered through a 0.22 μm filter was added to the concentrated cell culture solution to make a total of 11 mL.

(8) Transfer (7) to a tube dedicated for ultra-high-speed centrifugation, adjust the weight of all tubes to the second decimal place, and then ultra-high-speed centrifuge at 100,000 g for 90 minutes to remove the supernatant.

(9) Add PBS (200-500 μL) filtered through a 0.22 μm filter to dissolve the sediment at the bottom of the tube.

(10) Finally, the concentrated NK-EXO was transferred to a new micro-tube and stored frozen.

1-2. Isolation of exosomes (MSC-EXO) from canine adipose tissue-derived mesenchymal stem cells (MSC)

The present inventors isolated and concentrated exosomes (MSC-EXO) from canine adipose tissue-derived mesenchymal stem cells (MSCs), which are adult stem cells (Canine Adult Stem Cells), in order to apply them to the treatment of canine cancer. (Figure 1a).

Briefly, it is as follows:

(1) First, MSCs were obtained from dog adipose tissue.

Abdominal adipose tissue from normal dogs was collected, washed with PBS containing penicillin and streptomycin, and then cut into small pieces using surgical scissors. A 0.1% collagenase type I solution was added and reacted for 1 hour in a shaking water bath at 37°C. The tissue suspension was filtered using a 100 ㎛ cell strainer, centrifuged at 1500 rpm for 5 minutes to remove the supernatant, and then cultured in a T175 flask with DMEM medium containing 10% FBS. It was confirmed that the first MSCs isolated formed colonies within 7 days, attached in a fibroblast-like shape, and grew well. Afterwards, to confirm the adult stem cell performance of the MSCs, the presence or absence of cell surface protein expression such as CD29, CD34, and CD44 and the ability to differentiate into mesodermal cells were confirmed.

4×10 ⁶ MSCs were attached to a 175 T flask and cultured for 1 day.

(2) The MSCs were washed with DPBS supplemented with 1% P/S the next day, then replaced with cell culture medium (20 mL) containing exosome-depleted FBS and cultured for 3 days to extract exosomes from canine adipose tissue-derived MSCs. Exosomes (MSC-EXO) were isolated.

(3) Cell culture fluid was collected in a 50 mL tube and centrifuged continuously. Continuous centrifugation was performed at 1,500 g for 3 minutes, 2,000 g for 15 minutes, and 3,000 g for 20 minutes. At this time, the supernatant was transferred to a new tube at each step.

(4) After the final centrifugation, the supernatant was filtered through a 0.22 μm filter.

(5) The supernatant of (4) was concentrated using a 10 kDa concentration filter tube.

After centrifugation at 4,000 g for 15 minutes, the liquid under the tube was discarded, and the liquid remaining in the concentration filter was transferred to a new tube.

(6) The process of (5) above was repeated to concentrate the cell culture medium.

(7) PBS filtered through a 0.22 μm filter was added to the concentrated cell culture solution to make a total of 11 mL.

(8) Transfer (7) to a tube dedicated for ultra-high-speed centrifugation, adjust the weight of all tubes to the second decimal place, and then ultra-high-speed centrifuge at 100,000 g for 90 minutes to remove the supernatant.

(9) Add PBS (200-500 μL) filtered through a 0.22 μm filter to dissolve the sediment at the bottom of the tube.

(10) Finally, the concentrated MSC-EXO was transferred to a new micro-tube and stored frozen.

Example 2. Characterization of dog tissue-derived exosomes

The present inventors conducted electron microscopy, nanosite, nanoparticle tracking system, and Western blot to analyze the unique characteristics of the dog tissue-derived exosome (MSC-EXO) of the present invention obtained in Example 1. .

As a result, as shown in Figure 1b, through electron microscopy and nanosite analysis, it was confirmed that the dog tissue-derived exosomes of the present invention had an average size of 145 nm.

In addition, as shown in Figure 1c, through Nanoparticle Tracking Analysis (NTA), which can confirm the concentration, size, and size distribution of particles by recording the Brownian motion of individual particles in suspension, The exosomes of the present invention were confirmed to have a diameter of 50 to 200 nm.

Additionally, as shown in Figure 1d, it was confirmed through Western blot that exosome markers such as Flotillin-1, CD63, CD81, Alix, HSP70, and TSG101 were expressed in the exosomes of the present invention.

Example 3. Antitumor effect by canine tissue-derived exosomes

In order to confirm whether the dog tissue-derived exosomes (NK-EXO and MSC-EXO) of the present invention obtained in Example 1 actually exhibit an inhibitory effect on cancer cells, the present inventors treated a mouse mammary tumor model and then observed.

First, 5-6 week old mice (BALB/c-nude, female) were kept in a standard sterile breeding room without specific pathogens (temperature_22 ± 3℃, humidity_50 ± 5%, light/dark cycle_12 hours day, 12 hours night). They were acclimated for 1 week in a breeding environment. In addition, after culturing the canine mammary tumor cell line REM134 in which spares were formed, 1Х10 ³ /60㎕/DPBS was prepared and mixed 1:1 with Matrigel (60㎕). One week later, the mouse was anesthetized and the prepared spare-forming mammary tumor cells were slowly injected subcutaneously into the right mammary fat pad of mice using a 21G syringe to create a mammary tumor model.

Next, tumors were allowed to form in the mammary tumor model mouse for 2 weeks, the formed tumor was confirmed with the naked eye, and then the NK cell-derived exosome NK- of the present invention obtained in Example 1 was injected into the tumor site or blood vessel twice a week. EXO (100 μg) was administered, and tumor size was measured at weekly intervals.

At this time, a group (REM134_1Х10 ³ ) in which mammary tumor model mice were not treated with exosomes was used as a control group (Figure 2c).

As a result, as shown in Figures 2a and 2b, after 8 weeks, the tumor size was significantly reduced by more than 80% when treated with exosome NK-EXO compared to when treated only with the control mammary tumor cell line REM134 (Figure 2c). confirmed.

In addition, when the body weight of the mice was measured weekly, as shown in Figure 2d, it was confirmed that the body weight of the mice in the NK cell-derived exosome administered group was maintained similar to that of the normal control group that was not treated with anything.

In addition, tumors were allowed to form in the mammary tumor model mice for 2 weeks, the formed tumors were confirmed with the naked eye, and then the MSC-derived exosomes of the present invention MSC-EXO obtained in Example 1 were transferred to the tumor site or blood vessels twice a week. (100 μg) was administered, and tumor size and mouse body weight were measured at weekly intervals.

At this time, a group (REM134_1Х10 ³ ) in which mammary tumor model mice were not treated with exosomes was used as a control group (Figure 3c).

As a result, as shown in Figures 3a and 3b, after 8 weeks, the tumor size was significantly reduced by more than 50% when treated with exosomal MSC-EXO compared to when only the control mammary tumor cell line REM134 was treated (Figure 3c). could be confirmed.

In addition, when the body weight of the mice was measured weekly, as shown in Figure 3d, it was confirmed that the body weight of the mice in the MSC-derived exosome administered group was maintained similar to that of the normal control group that was not treated with anything.

Therefore, the above results show that exosomes isolated from canine adipose tissue-derived MSCs (MSC-EXO) and exosomes isolated from NK cells derived from canine PBMCs (NK-EXO) target only cancer cells. This means that it exerts an excellent anticancer effect that safely suppresses solid cancer without affecting other normal cells.

Claims (13)

Exosomes isolated from canine adipose tissue-derived mesenchymal stem cells (MSCs); Or a pharmaceutical composition for preventing or treating breast cancer in dogs containing exosomes isolated from canine peripheral blood mononuclear cells (PBMCs)-derived natural killer cells (NK cells) as an active ingredient. as,
The exosomes have a diameter of 40 to 200 nm; Expresses flotillin-1, CD63, CD81, Alix, HSP70, and TSG101; A pharmaceutical composition for preventing or treating breast cancer in dogs, characterized in that it reduces the size of cancer.
delete delete delete delete delete delete delete delete delete delete Exosomes isolated from canine adipose tissue-derived mesenchymal stem cells (MSCs); Or a feed composition for preventing or improving breast cancer in dogs containing exosomes isolated from natural killer cells (NK cells) derived from dog peripheral blood mononuclear cells (PBMCs) as an active ingredient. ,
The exosomes have a diameter of 40 to 200 nm; Expresses flotillin-1, CD63, CD81, Alix, HSP70, and TSG101; A feed composition for preventing or improving breast cancer in dogs, characterized in that it reduces the size of cancer.
Exosomes isolated from canine adipose tissue-derived mesenchymal stem cells (MSCs); Or, it includes the step of administering to a dog a composition containing exosomes isolated from canine peripheral blood mononuclear cells (PBMCs)-derived natural killer cells (NK cells) as an active ingredient. As a method of treating breast cancer in dogs,
The exosomes have a diameter of 40 to 200 nm; Expresses flotillin-1, CD63, CD81, Alix, HSP70, and TSG101; A method of treating breast cancer in dogs, characterized by reducing the size of the cancer.